Neuropharmacology 67 (2013) 46e56

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Neuropharmacology

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Linking GABAA receptor subunits to -induced conditioned taste aversion and recovery from acute alcohol intoxication

Y.A. Blednov a,*, J.M. Benavidez a, M. Black a, D. Chandra b, G.E. Homanics b, U. Rudolph c, R.A. Harris a a Waggoner Center for Alcohol and Addiction Research, The University of Texas at Austin, 1 University Station, A4800, Austin, TX 78712, USA b Departments of Anesthesiology and Pharmacology & Chemical Biology, University of Pittsburgh, Room 6060 Biomedical Science Tower-3, 3501 Fifth Avenue, Pittsburgh, PA 15261, USA c Laboratory of Genetic Neuropharmacology, McLean Hospital and Department of Psychiatry, Harvard Medical School, 115 Mill Street, Mailstop #145, Belmont, MA 02478, USA article info abstract

Article history: GABA type A receptors (GABAA-R) are important for ethanol actions and it is of interest to link individual Received 1 September 2012 subunits with specific ethanol behaviors. We studied null mutant mice for six different GABAA-R subunits Received in revised form (a1, a2, a3, a4, a5 and d). Only mice lacking the a2 subunit showed reduction of conditioned taste aversion 12 October 2012 (CTA) to ethanol. These results are in agreement with data from knock-in mice with mutation of the Accepted 28 October 2012 ethanol-sensitive site in the a2-subunit (Blednov et al., 2011). All together, they indicate that aversive property of ethanol is dependent on ethanol action on a2-containing GABAA-R. Deletion of the a2-subunit Keywords: led to faster recovery whereas absence of the a3-subunit slowed recovery from ethanol-induced inco- Gamma-aminobutyric acid Mutant mice ordination (rotarod). Deletion of the other four subunits did not affect this behavior. Similar changes in fl Alcohol this behavior for the a2 and a3 null mutants were found for urazepam motor incoordination. However, Conditioned taste aversion no differences in recovery were found in motor-incoordinating effects of an a1-selective modulator Ataxia () or an a4-selective agonist (). Therefore, recovery of rotarod incoordination is under GABA receptors control of two GABAA-R subunits: a2 and a3. For motor activity, a3 null mice demonstrated higher activation by ethanol (1 g/kg) whereas both a2(/) and a3(/Y) knockout mice were less sensitive to ethanol-induced reduction of motor activity (1.5 g/kg). These studies demonstrate that the effects of ethanol at GABAergic synapses containing a2 subunit are important for specific behavioral effects of ethanol which may be relevant to the genetic linkage of the a2 subunit with human alcoholism. Ó 2012 Elsevier Ltd. All rights reserved.

1. Introduction self-administration in rats. Other work proposes a role for GABAA receptors in the discriminative stimulus effects of ethanol (Hodge g-Aminobutyric acid A receptors (GABAA-Rs) represent the and Cox, 1998) as well as in its motor stimulatory or sedative major inhibitory receptors in the mammalian activities (Hinko and Rozanov, 1990; Koechling et al., 1991). Acute brain. GABAA-Rs mediate a number of pharmacological effects, ethanol treatments can enhance the function of GABAA receptors by including sedation/hypnosis, anxiolysis, and anesthesia for drugs at least three different mechanisms: direct actions on the receptor, such as , , neuroactive steroids, and increased presynaptic release of GABA and increased production of intravenous anesthetics. A number of behavioral effects of ethanol neuroactive steroids (Kumar et al., 2009; Lobo and Harris, 2008). have been also attributed to actions at the GABAA-R (for reviews Increased GABAergic function is sufficiently important for alcohol see: Kumar et al., 2009; Chester and Cunningham, 2002; Enoch, action that activators of GABAA receptors have even been suggested 2008; Lobo and Harris, 2008). Some of the early evidence impli- as ‘substitution’ therapy for treatment of alcohol use disorders cating the GABA receptor system in ethanol’s motivational effects (Chick and Nutt, 2012). came from studies showing that GABAA receptor antagonists Studies relating human allelic variation to alcoholism and other (Hyytia and Koob, 1995) and partial inverse alcohol phenotypes have found linkage with GABAA-R clusters. agonists (Balakleevsky et al., 1990) consistently reduced ethanol Thus, a polymorphism of the g2 subunit of the GABAA receptor has been associated with genetic susceptibility to ethanol-induced motor incoordination and hypothermia, conditioned taste aver- * Corresponding author. Tel.: þ1 512 232 2520; fax: þ1 523 232 2525. sion, and withdrawal (Buck and Hood, 1998). Human genetic E-mail address: [email protected] (Y.A. Blednov). association studies suggest that the GABAA b2, a6, a1 and g2

0028-3908/$ e see front matter Ó 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.neuropharm.2012.10.016 Y.A. Blednov et al. / Neuropharmacology 67 (2013) 46e56 47 subunit have a role in the development of alcohol depen- 2. Materials and methods dence, although their contributions may vary between ethnic 2.1. Animals group and phenotype (for review see Enoch, 2008). The Collabo- rative Studies on Genetics of Alcoholism (COGA), and other groups, Knockout mice were generated as described previously: a1(/)(Sur et al., have identified a region of 4p associated with alco- 2001), a2(/)(Dixon et al., 2008), a4(/)(Chandra et al., 2006), a5(/) (Collinson et al., 2002), d (/)(Mihalek et al., 1999), a3(/Y) (Yee et al., 2005). All holism, which includes a cluster of four GABAA-R subunits, wherein the strongest linkage lies with a2(Edenberg et al., 2004; Enoch mice used in the experiments were produced from heterozygous breeding, with the exception that due the localization of the Gabra3 on the X chromosome et al., 2006; Soyka et al., 2008). Moreover, single nucleotide poly- a3 null and wild type control mice were produced by breeding female heterozy- morphisms near the human a2 gene modulate the amount of a2 gotes with male wild type mice. a1(/), a4(/), a5(/)andd (/)mutant mRNA and in human brain as well as behavioral sensitivity colonies were maintained on the original mixed genetic background. Mice from a2 to alcohol (Haughey et al., 2008). Two independent studies of gene knockout colony were backcrossed on to C57BL/6J background twice and mice from the a3 knockout colony were backcrossed on to C57Bl/6J background three expression profiles in humans and rodents with high alcohol intake times (starting from a 129X1/SvJ background). After weaning, mice were housed found changes in genes related to GABAergic synaptic function in the conventional facility in University of Texas with ad libitum access to rodent (Tabakoff et al., 2009; Enoch et al., 2012). chow and water with 12-h light/dark cycles (lights on at 7:00 AM). All mice used for behavioral experiments were between 8 and 12 weeks of age. Only male mice GABAA receptor is pentameric in structure, with five subunits were used. Each mouse was used for only one experiment, and all mice were forming an ion pore. Seven classes of GABAA receptor subunits have e e e ε e e ethanol naive at the start of each experiment. All experiments were approved by been described to date (a1 6, b1 3, g1 3, d, , q1 3, p, r1 3), Institutional Animal Care and Use Committees and were conducted in accordance allowing for extensive heterogeneity in receptor subunit compo- with National Institutes of Health guidelines with regard to the use of animals in sition across neuronal cell types and brain regions. However, most research. All efforts were made to minimize animal suffering and reduce the number of animals used. native GABAA receptors are thought to consist of two a, two b, and one g or d subunit. As indicated above, there is also considerable evidence that 2.2. Rationale for the behavioral tests ethanol enhances the function of GABAA receptors, although we are only beginning to elucidate the specific roles of each receptor Published data suggest that the rewarding and aversive effects of EtOH play an important role in determining whether people who drink will continue to consume subtype in ethanol-induced behaviors (Lobo and Harris, 2008; alcohol (Cunningham et al., 2000). Conditioned taste aversion is used as the index of Harris et al., 2008; Boehm et al., 2004; Wallner et al., 2006; Korpi motivational properties of ethanol (mostly aversive in doses higher than 1 g/kg) (Liu et al., 2007). Evidence supporting subunit specificpharmaco- et al., 2009) and the response in this test is negatively correlated with voluntary logical and behavioral roles comes from knock-in mice which ethanol intake (Green and Grahame, 2008). It should be noted that this is one of the possess a point mutation that alters one aspect of protein func- few behaviors which shows stable and substantial correlation with ethanol consumption in animal studies (Blednov et al., 2012). The rotarod test measures an tion (e.g., response to a drug), leaving all other aspects of protein aspect of motor incoordination as well as recovery from acute ethanol intoxication. function intact. These studies show that whereas a1subunit Examples from human research indicate that resistance or low response to the containing receptors mediate the sedative, amnestic, and anti- physiological effects of ethanol may predict the future development of alcoholism convulsant actions of (Rudolph et al., 1999; McKernan (Schuckit, 1986, 1988, 1994). Another reason for the choice of this behavior was its high sensitivity to ethanol intake. Thus, C57Bl/6J mice after one month of ethanol et al., 2000), a2 subunit containing receptors mediate its anxio- consumption with limited access to alcohol showed significantly faster recovery lytic actions (Low et al., 2000; Rudolph and Möhler, 2004)and from acute ethanol intoxication compared with isolated for the same period of time a3/a2 the muscle relaxant actions (Crestani et al., 2001). Poly- (Blednov, unpublished data). Because the ataxia is a complex behavior (Crabbe et al., morphisms in the a2 subunit also implicated this subunit in 2005, 2010), we measured also some behaviors related to ataxia such as missteps human cocaine addiction (Dixon et al., 2010). To evaluate the and grip strength. The behavior in the elevated plus maze serves as an indicator for the level of anxiety. importance of alcohol action on specific GABAA receptor subunits, mutations were introduced with the goal of producing receptors with normal responses to GABA but lacking modulation 2.3. Conditioned taste aversion (CTA) by ethanol. The mutated genes can then be used to replace the Subjects were adapted to a water-restriction schedule (2 h of water per day) over normal GABAA receptor subunits in knock-in mice. This was a 7-day period. At 48-hr intervals over the next 10 days (days 1, 3, 5, 7, 9 and 11), all accomplished for the a1andthea2subunits(Werner et al., mice received 1-hr access to a solution of saccharin (0.15% w/v sodium saccharin in 2006; Blednov et al., 2011). Knock-in mice with mutations tap water). Immediately after 1-hr access to saccharin, mice received injections of (serine 270 to histidine and leucine 277 to alanine) making the saline or ethanol (2.5 g/kg) (days 1, 3, 5, 7 and 9). All mice also received 30-min access to tap water 5 h after each saccharin access period to prevent dehydration a1 subunit of the GABAA receptor resistant to ethanol, showed (days 1, 3, 5, 7 and 9). On intervening days, mice had 2 h continuous access to water quicker recovery from the motor-impairing effects of ethanol and at standard times in the morning (days 2, 4, 6, 8 and 10). Reduced consumption of increased anxiolytic effects of ethanol (Werner et al., 2006). Mice the saccharin solution is used as a measure of CTA. with the same mutations in the a2 subunit of GABAA receptors did not develop conditioned taste aversion in response to ethanol 2.4. Rotarod and showed loss of the motor stimulant effects of ethanol (Blednov et al., 2011). This suggests that specific behavioral Mice were trained on a fixed speed rotarod (Economex; Columbus Instruments effects of ethanol result from direct action of ethanol on these (Columbus, OH); speed of rod, 5.0 rpm), and training was considered complete when mice were able to remain on the rotarod for 60 s. Most mice were able to perform subunit . This interpretation assumes that these muta- this task within first two or three trials. After completion of this training period, tions do not alter other functions of the receptor or produce mice were injected with ethanol (2 g/kg i.p.) and every 15 min after injection each other changes in brain circuitry. However, these mutations are mouse was placed back on the rotarod and latency to fall was measured until the not completely ‘silent’ as they result in changes in gene expres- mouse was able to stay on the rotarod for 60 s. Overall, each experiment was sion in brain, especially in the case of a1subunit(Harris et al., completed within several hours on the same day. 2011). To better define the role of GABAA subunits we studied two 2.5. Elevated plus maze behaviors, conditioned taste aversion to ethanol (CTA) and Mice were evaluated for basal anxiety as well as ethanol-induced anxiolysis recovery from ethanol-induced intoxication using the rotarod, in using the elevated plus maze and a between-groups design as was described knockout mice for six GABAA receptor subunits: a1, a2, a3, a4, a5 (Blednov et al., 2001). Detailed description of experimental procedure is presented and d. in Supplemental materials. 48 Y.A. Blednov et al. / Neuropharmacology 67 (2013) 46e56

2.6. Motor activity testing conditioning). Fig. 1 depicts saccharin intake (as a percentage of trial 0) for each mouse colony over the course of the five Locomotor activity was measured in standard mouse cages in Opto- saccharin access periods. Ethanol administration produced trial- microvarimex (Columbus Instruments, Ohio, USA) after three days of pre- habituation to handling, stress of transfer to experimental cage and to saline dependent reductions in saccharin intake over trials, indicating injection. Detailed description of experimental procedure is presented in Supple- the development of conditioned taste aversion in all groups mental materials. (statistically significant dependence on trial and trial treatment interaction) (Fig. 1; Suppl. Table 2). Two mutant 2.7. Ethanol metabolism colonies e a2(/)andd (/) e showed effects of gene Animals were given a single dose of ethanol (4 g/kg i.p.), and blood samples were deletion but only a2 null mice showed a genotype - taken from the retro-orbital sinus 30, 60, 120, 180 and 240 min after injection. Blood treatment trial interaction indicating different development of ethanol concentration (BEC) values, expressed as mg ethanol per ml blood, were CTA between (/) knockout and wild type mice (Fig. 1 b,f; determined spectrophotometrically by an enzyme assay (Lundquist, 1959). Suppl. Table 2).

2.8. Missteps (foot-fault) test 3.2. Ethanol-induced motor incoordination The number of missteps was counted automatically using Columbus Instru- ments’ foot misplacement apparatus (Columbus Instruments, Columbus, OH, USA). Acute administration of ethanol (2 g/kg) produced motor inco- Detailed description of experimental procedure is presented in Supplemental ordination followed by time-dependent recovery in all genotypes materials. (Fig. 2; Suppl. Table 3). However, a2(/) knockout mutant mice 2.9. Grip strength test recovered from this impairment faster than wild type mice (Fig. 2 b; Supplemental Table 3). On the contrary, a3(/Y) knockout mice Grip Strength was assessed using a grip strength meter consisting of horizontal recovered slower than wild type (Fig. 2 c; Supplemental Table 3). forelimb mesh (Columbus Instruments, Columbus, OH). Three successful forelimb No differences in recovery from motor impairment induced by strength measurements within 2 min were recorded and normalized to body weight as previously described (Spurney et al., 2009). ethanol were found for four other mutant mouse colonies (Fig. 2 a,d,e,f; Suppl. Table 3). To determine if deletion of a2ora3 2.10. Hypothermia subunits affected the sensitivity of other GABAA receptors, we tested motor impairment in rotarod by three drugs with different Each mouse was weighed and placed into an individual plastic ventilated GABAAR subunit specificity: flurazepam e specific for g-containing chamber 1 h before the start of testing; no food or water was available. Its core body e temperature was then recorded with a rectal thermometer probe (2 mm ball on GABAAR(Sigel, 2002; Ernst et al., 2003); low doses of zolpidem 2 cm shaft) and digital thermometer (Sensortek TH-8, Suffolk, UK) shortly before an specific for a1 subunit of GABAAR(Sieghart, 1995) and gaboxadol e ethanol injection (2.0 g/kg, i.p.) and then 30 min, 60 min, 90 min and 120 min post- selective for a4 and d containing GABAAR(Chandra et al., 2006; injection. Herd et al., 2009). Because genotype-dependent differences in recovery from motor incoordination induced by ethanol were 2.11. Drug injection found only in two null colonies, a2 and a3, only these mutant mice All ethanol (Aaper Alcohol and Chemical, Shelbyville, KY) solutions were made were used in subsequent experiments. in 0.9% saline (20%, v/v) and injected i.p.)). Flurazepam hydrochloride (Sigma- Administration of gaboxadol (10 mg/kg) produced incoordina- Aldrich, St. Louis, MO; 35.0 mg/kg, i.p), 4,5,6,7-tetrahydroisoxazolo(5,4-c)pyridin-3- tion in wild type as well as in the knockout mice (Fig. 3 e,f; Suppl. ol (THIP hydrochloride or gaboxadol, Sigma-Aldrich, St. Louis, MO; 10 mg/kg i.p.) were dissolved in 0.9% saline and zolpidem (Sigma-Aldrich, St. Louis, MO; 5.0 mg/kg) Table 4). Administration of zolpidem (5 mg/kg) also produced was dissolved in 0.9% saline with few drops of Tween-80 and injected i.p. at incoordination in wild type as well as in knockout mice (Fig. 3 c,d; a volume of 0.1 ml/10 g body weight. Suppl. Table 4). However, no differences between null and wild type mice were found for either drug. Similar to the results from 2.12. Statistical analysis ethanol, a2(/) knockout mice showed faster recovery from fl Mixed (between and within groups) experimental design was used for all the motor impairment induced by urazepam (35 mg/kg) whereas a3 experiments except elevated plus-maze which was been designed as a between- (/Y) knockout mice recovered slower compared with wild type groups comparison. In all other experiments between group design was used for mice (Fig. 3 a,b; Suppl. Table 4). comparison of genotypes and a within group design (i.e., repeated testing of same It is well known that ethanol impairs thermoregulation and mice for each genotype) was used for the calculation effect of trial (CTA experiment), produces hypothermia in mice. Prior studies have shown that body dose (locomotion, grip strength and missteps) or time (rotarod). Different groups of mice were used in all behavioral experiments. temperature during intoxication can affect both the sensitivity of Data were reported as the mean S.E.M. The statistics software program the animals to the behavioral effects of ethanol and the rate of GraphPad Prism (Jandel Scientific, Costa Madre, CA) was used. Analysis of variance ethanol metabolism. To rule out this possibility we studied the (two-way ANOVA or one-way ANOVA with repeated measurements with Bonferroni effect of ethanol (2.0 g/kg) on body temperature in a2(/) and a3 ’ ’ or Dunnett s post hoc tests respectively) and Student s t-test were carried out to evaluate differences between groups. Statistics (three-way ANOVA) for the analysis ( /Y) knockout mice. Ethanol produced clear hypothermic effect in of data obtained in CTA experiments was performed using Statistica version 6 mice of all genotypes. However, no differences in body temperature (StatSoft, Tulsa, Oklahoma). between knockout mice and wild type mice were found during 2 h of observation (Supplemental Fig. 1). 3. Results The differences in recovery from acute ethanol intoxication could be due to differences in effects of ethanol on anxiety, motor 3.1. Conditioned taste aversion activation, sedation or myorelaxation. To test these possibilities, effects of low doses of ethanol were studied on several behaviors Consumption of saccharin during the 1-hr period varied with that might influence rotarod performance. Low doses of ethanol genetic background in both wild type and mutant mice (Suppl. were used because differences in the rotarod motor incoordination Table 1). To correct for these initial differences in tastant were not seen for the initial effect of the 2 g/kg dose, but rather consumption, intake was calculated as a percentage of the trial during recovery when the blood ethanol concentration is lower 0 consumption for each subject by dividing the amount of than the initial value. In addition, the metabolism of ethanol was saccharin solution consumed on subsequent conditioning trials measured for all genotypes to assure that differences in blood by the amount of saccharin solution consumed on trial 0 (before ethanol were not influencing the behavioral results. Y.A. Blednov et al. / Neuropharmacology 67 (2013) 46e56 49

Fig. 1. The development of ethanol-induced conditioned taste aversion was decreased only in mice lacking of a2 subunit. Changes in saccharin consumption produced by injection of saline or ethanol expressed in percent from control trial (Trial 0). A e Development CTA in a1(/) knockout mice (n ¼ 9 for saline injection for both genotypes; n ¼ 9e14 for groups with ethanol injection). B e Development CTA in a2(/) knockout mice (n ¼ 10 for saline injection for both genotypes; n ¼ 11e14 for groups with ethanol injection). C e Development CTA in a3(/Y) knockout mice (n ¼ 7e9 for saline injection for both genotypes; n ¼ 16e19 for groups with ethanol injection). D e Development CTA in a4(/) knockout mice (n ¼ 6 for saline injection for both genotypes; n ¼ 6e7 for groups with ethanol injection). E Development CTA in a5(/) knockout mice (n ¼ 10 for saline injection for both genotypes; n ¼ 10e12 for groups with ethanol injection). F e Development CTA in d (/) knockout mice (n ¼ 11 for saline injection for both genotypes; n ¼ 12e 13 for groups with ethanol injection). Values represent mean SEM.

3.3. Spontaneous locomotion type mice. Additional analyses by one-way ANOVA within each genotype showed that ethanol in doses 1 g/kg and 1.5 g/kg signif- Ethanol dose-dependently reduced motor activity in a2 mutant icantly reduced motor activity in wild type mice whereas no effect colony [F(1,39) ¼ 16.9, p < 0.001 main effect of genotype; of ethanol was found in a2(/) knockout mice. F(2,78) ¼ 24.5, p < 0.001 main effect of dose; F(2,78) ¼ 10.2, p < 0.001 Effects of ethanol on locomotor activity were modest in a3 wild genotype dose interaction] (Fig. 4 a). Post hoc analyses showed type and knockout mice [F(2,60) ¼ 7.9, p < 0.001 main effect of dose; that motor activity of a2(/) knockout mice after administration F(1,30) ¼ 0.18, p > 0.05 main effect of genotype; F(2,60) ¼ 5.5, p < 0.01 of saline or ethanol (1 g/kg) was lower that motor activity of wild genotype dose interaction] (Fig. 4 b). No difference in basal 50 Y.A. Blednov et al. / Neuropharmacology 67 (2013) 46e56

Fig. 2. Faster recovery from motor incoordinating effect of ethanol in a2(/) knockout mice and slower recovery in a3(/Y) knockout mice. Time on the rotarod (sec) after injection of ethanol (2 g/kg). A e rotarod recovery in a1(/) knockout mice (n ¼ 5e7 for each genotype). B e Rotarod recovery in a2(/) knockout mice (n ¼ 7 for each genotype). C e Rotarod recovery in a3(/Y) knockout mice (n ¼ 5e7 for each genotype). D e Rotarod recovery in a4(/) knockout mice (n ¼ 5 for each genotype). E Rotarod recovery in a5(/) knockout mice (n ¼ 5e6 for each genotype). F e Rotarod recovery in d (/) knockout mice (n ¼ 6 each genotype). Data are shown as means S.E.M. and analyzed by two-way ANOVA with Bonferroni post hoc test. **p < 0.01, ***p < 0.01 vs wild type group for the time point.

(saline injection) motor activity between a3(/Y) knockout mice number of missteps [F(2,32) ¼ 5.4, p < 0.01 main effect of dose] and wild type mice were found. Analyses by one-way ANOVA (Fig. 5 a,c) in a2 mouse colony. However, no differences in effect of within each genotype showed that 1.5 g/kg ethanol reduced motor ethanol on grip strength [F(1,16) ¼ 1.5, p > 0.05 main effect of activity in wild type mice whereas 1 g/kg ethanol increased motor genotype; F(2,32) ¼ 0.1, p > 0.05 genotype dose interaction] or on activity in a3(/Y) knockout mice. number of missteps [ F(1,16) ¼ 0.1, p > 0.05 main effect of genotype; F(2,32) ¼ 1.1, p > 0.05 genotype dose interaction] was found 3.4. Missteps and grip strength between a2(/) knockout and wild type mice (Fig. 5 a,c). Reduction of grip strength [F(2,64) ¼ 7.3, p < 0.001 main effect of Ethanol dose-dependently reduced the grip strength dose; F(1,32) ¼ 0.2, p > 0.05 main effect of genotype; F(2,64) ¼ 0.4, [F(2,32) ¼ 13.7, p < 0.001 main effect of dose] and increased the p > 0.05 genotype treatment interaction] and increase in the Y.A. Blednov et al. / Neuropharmacology 67 (2013) 46e56 51

Fig. 3. Recovery from motor incoordinating effects of flurazepam, gaboxadol and zolpidem in a2(/) and a3(/Y) knockout mice. Time on the rotarod (sec) after injection of flurazepam (35 g/kg), zolpidem (5 mg/kg) and gaboxadol (10 mg/kg). A, C, E Rotarod recovery in a2(/) knockout mice. B, D, F e Rotarod recovery in a3(/Y) knockout mice. A, B e flurazepam, (n ¼ 5e6 per genotype in a2 colony and n ¼ 6e7 per genotype in a3(/Y) colony). C, D e Zolpidem, (n ¼ 6e7 per genotype in a2 colony and n ¼ 6 per genotype in a3(/Y) colony). E, F e Gaboxadol, (n ¼ 7e6 per genotype in a2 colony and n ¼ 5e6 per genotype in a3(/Y) colony). Data are shown as means S.E.M. and analyzed by two-way ANOVA with Bonferroni post hoc test. **p < 0.01, ***p < 0.01 vs wild type group for the time point. number of missteps [F(2,64) ¼ 20.3 p < 0.001 main effect of dose; measured by percentage of time spent in open arm entries and F(1,32) ¼ 1.1, p > 0.05 main effect of genotype; F(2,64) ¼ 1.6, p > 0.05 percentage of open arm entries. In a2 mutant mice and their wild genotype treatment interaction] after ethanol administration type controls, ethanol affected the percent of time spent in open was found in a3 mouse colony (Fig. 5 b,d). No differences between arms [F(1,37) ¼ 4.3, p < 0.05 main effect of dose; F(1,37) ¼ 0.6, p > 0.05 a3(/Y) knockout and wild type mice were found. main effect of genotype; F(1,37) ¼ 2.2, p > 0.05 genotype dose interaction]. However, the percentage of open arm entries showed 3.5. Elevated plus maze only a genotype treatment interaction [F(1,37) ¼ 0.5, p > 0.05 main effect of dose; F(1,37) ¼ 0.1, p > 0.05 main effect of genotype; Locomotor activity was assessed by number of entries into the F(1,37) ¼ 4.6, p < 0.05 genotype dose interaction] (Fig. 6 a,b). closed arms and total numbers of entries, whereas anxiety was Number of entries into the closed arms [F(1,37) ¼ 14.8, p < 0.001 52 Y.A. Blednov et al. / Neuropharmacology 67 (2013) 46e56

Fig. 4. Effect of ethanol on the motor activity of a2(/) and a3(/Y) knockout mice after pre-habituation. A e a2(/) knockout mice (n ¼ 20e21 per genotype). B e a3(/Y) knockout mice (n ¼ 13e19 per genotype). Data are shown as means S.E.M. and analyzed by two-way ANOVA with repeated measures with Bonferroni post hoc test ($p < 0.05, $$$p < 0.001, vs another genotype for the same condition) and within each genotype by one-way ANOVA with repeated measures with Dunnett’s test for Multiple comparisons (*p < 0.05, **p < 0.01, ***p < 0.001 vs saline group). main effect of dose; F(1,37) ¼ 0.1, p > 0.05 main effect of genotype; genotype dose interaction] were increased after treatment with F(1,37) ¼ 0.8, p > 0.05 genotype dose interaction] as well as ethanol in mice of both genotypes (Fig. 6 c,d). In a3 wild type and number of total entries [F(1,37) ¼ 20.2, p < 0.001 main effect of dose; mutant mice, treatment affected the percent of time spent in open F(1,37) ¼ 0.1, p > 0.05 main effect of genotype; F(1,37) ¼ 1.9, p > 0.05 arms [F(1,39) ¼ 13, p < 0.001 main effect of dose; F(1,39) ¼ 0.3,

Fig. 5. Effect of ethanol on the grip strength and number of missteps in a2(/) and a3(/Y) knockout mice. A, C e a2(/) knockout mice. B, D e a3(/Y) knockout mice. A, B e grip strength (n ¼ 8e10 per genotype in a2 colony and n ¼ 15e19 per genotype in a3 colony). C, D e number of missteps (n ¼ 8e10 per genotype in a2 colony and n ¼ 15e19 per genotype in a3 colony). Data are shown as means S.E.M. and analyzed by two-way ANOVA with repeated measures. Y.A. Blednov et al. / Neuropharmacology 67 (2013) 46e56 53

Fig. 6. Anxiolytic effect of ethanol in in a2(/) and a3(/Y) knockout mice in elevated plus maze test. A, B, C, D e a2(/) knockout mice. E, F, G, H e a3(/Y) knockout mice. A, E percent of time in open arms (n ¼ 7e13 per genotype in a2 colony and n ¼ 15e13 per genotype in a3(/Y) colony). B, F e percent of enters into the open arms. C, G e number of enters into the closed arms. D, H e total number of entries. Data from females and males were combined as there were no sex differences. Data are shown as means S.E.M. and analyzed by two-way ANOVA.

p > 0.05 main effect of genotype; F(1,39) ¼ 0.4, p > 0.05 a difference in saccharin consumption throughout the CTA test, genotype dose interaction] as well as the percentage of open arm this does not appear to be due to differences in the development entries [F(1,39) ¼ 27, p < 0.001 main effect of dose; F(1,39) ¼ 0.1, of CTA. p > 0.05 main effect of genotype; F(1,39) ¼ 0.1, p > 0.05 Recovery from acute ethanol intoxication (rotarod incoordi- genotype dose interaction] (Fig. 6 e,f). No genotype-dependent nation) appears to be controlled by two GABAA receptor differences were found. Number of entries into the closed arms was subunits: a2anda3. Knock-in mice with mutation of the reduced after ethanol administration [F(1,39) ¼ 7.9, p < 0.01 main ethanol-sensitive site in the a2-subunit (Blednov et al., 2011)did effect of dose] and this effect was dependent on genotype not show the changes in this behavior, suggesting that it is not [F(1,39) ¼ 4.1, p < 0.05 effect of genotype; F(1,39) ¼ 0.1, p > 0.05 due to direct action of ethanol on the receptor, but could be due genotype dose interaction] (Fig. 6 g). In contrast, number of total to increased presynaptic release of GABA (Roberto et al., 2003)or entries were significantly increased [F(1,39) ¼ 20, p < 0.001, effect of compensatory changes from deletion of the a2subunit.The treatment; F(1,39) ¼ 5.1, p < 0.05, effect of genotype; F(1,39) ¼ 0.3, absence of any differences in recovery from motor incoordination p > 0.05 genotype dose interaction] after treatment with ethanol induced by zolpidem and gaboxadol suggest that the function of in mice of both genotypes (Fig. 6 h). a1- or a4/d- containing receptors is not changed in mice lacking a2- or a3-subunits to an extent that could be detected in our 3.6. Ethanol metabolism tests. However, it should be noted that the expression of the a4 subunit is significantly upregulated in a2(/)knockoutmice There were no differences in metabolism of ethanol (4.0 g/kg) (Panzanelli et al., 2011). Further analyses of behavioral effects of between wild type and any knockout mice (Supplemental Fig. 2). low doses of ethanol showed decreased sensitivity to sedative effects of ethanol on pre-habituated motor activity which might 4. Discussion lead to faster recovery from ethanol-induced motor incoordina- tion in a2nullmice.Althougha3(/Y) knockout mice did not Taken together, these results show a link between specific show any sedation at the doses tested, they showed a small GABAA receptor subunits and specific behavioral actions of increase of motor stimulation from ethanol and this may cause ethanol (Table 1)Specifically, CTA induced by ethanol requires the the longer recovery in rotarod behavior. Thus, it is possible that a2 subunit. This is in agreement with data from knock-in mice the behavioral outcome in the rotarod test depends on the with mutation of the ethanol-sensitive site in the a2-subunit balance of signaling from a2- and a3-containing GABAA recep- (Blednov et al., 2011) which also showed a marked reduction in tors. No differences between a2ora3(/Y) knockout mice and development of ethanol-induced CTA. In contrast, the lack of any wild type mice in other types of ataxia-related behaviors such as of five other subunits of GABAAR(a1, a3, a4, a5ord)didnot missteps or grip strength were found (Table 1). This is consistent change ethanol CTA. Although mice lacking the d subunit showed with a study of inbred mouse strains which were screened for 54 Y.A. Blednov et al. / Neuropharmacology 67 (2013) 46e56

Table 1

Summary of the behavioral effects of ethanol in mice lacking different subunits of GABAA receptors.

Behavior Drug a1KO a2KO a3KO a4KO a5KO d KO SHLA a1 SHLA a2 CTA EtOH (2.5 g/kg) ¼ Y ¼¼¼¼YY Rotarod EtOH (2.0 g/kg) ¼ )/¼¼¼) ¼ Flurazepam (35 mg/kg) /)/)¼ )/ ¼ Gaboxadol (10 mg/kg) ¼¼¼) ¼ ) ¼¼ Zolpidem (5 mg/kg) ¼¼ Motor activity pre-habituated (decrease) EtOH (2.0 g/kg) ¼ Y EtOH (1.5 g/kg) YY EtOH (1.0 g/kg) Y ¼ Motor activity pre-habituated (increase) EtOH (1.5 g/kg) ¼¼ EtOH (1.0 g/kg) ¼ [ Open field (decrease) EtOH (1.0 g/kg) Y Open field (increase) EtOH (1.5 g/kg) [ EtOH (1.0 g/kg) [ ¼ EPM (anxiolysis) EtOH (0.75 g/kg) ¼ [ EtOH (1.0 g/kg) [ ¼¼¼ EtOH (1.25 g/kg) [ ¼ EtOH (1.5 g/kg) ¼¼ ¼¼¼ Hypothermia EtOH (2.0 g/kg) ¼¼ Metabolism EtOH (4.0 g/kg) ¼¼¼¼¼¼¼ ¼

CTA e conditioned taste aversion; EPM e elevated plus maze. Y e reduction of behavior in mutant mice compared with correspondent wild type mice; [ e increase of behavior in mutant mice compared with correspondent wild type mice; ¼ e no differences compared with correspondent wild type mice; / e right shift and ) e left shift in mutant mice. The results combined in this table were obtained in this study as well as were published previously (Blednov et al., 2003, 2011; Boehm et al., 2004; Chandra et al., 2008; Kralic et al., 2003; Mihalek et al., 2001; Werner et al., 2006). Note, that motor activity under stress conditions of open field is separated from pre-habituated motor activity. sensitivity to alcohol-induced intoxication using 11 separate GABRA2 haplotypes on the development of addictions is due to an behavioral assays, which found that alcohol sensitivity was interaction with early life stress (Enoch et al., 2010). Human vari- influenced by task-specific sets of genes (Crabbe et al., 2005). The ation within GABRA2 is associated with attenuated negative pattern of results suggested that there is not a single functional responses to alcohol, a known risk factor for vulnerability to alcohol domain that represents ‘balance’ or ‘ataxia’ (Crabbe et al., 2010). use disorders (Uhart et al., 2012) and twin studies found allelic GABAA receptors containing the a3subunitarefoundinseveral associations in GABRA2 for alcohol-induced body sway and motor brain regions (Möhler, 2007), with high levels of expression in incoordination (Lind et al., 2008). There is little information about cholinergic and monoaminergic neurons (Fritschy et al., 1992)in the functional consequences of the human polymorphisms, but several areas of the midbrain and brain stem, including sub- Haughey et al. (2008) suggested that the risk polymorphisms stantia nigra and ventral tegmental area (Fritschy and Möhler, reduced the levels of GABRA2 mRNA (and perhaps protein) in 1995; Pirker et al., 2000; Schwarzer et al., 2001). Thus, the a3 human prefrontal cortex. This appears consistent with our findings subunits are important for GABAergic inhibition on the dopa- that mice lacking GABRA2 show less aversion to alcohol. Overall, minergic, serotoninergic and noradrenergic systems, and there- our results suggest that the effects of ethanol at GABAergic fore may be a pharmacological target for modification of motor synapses containing a2 subunit may be important for behavioral activity. effects of ethanol relevant to the genetic linkage of the a2 subunit Specific behavioral actions of benzodiazepines have also been with human alcohol phenotypes, particularly aversion and motor linked to GABAA receptor subunits using mutant mice and it is of incoordination. In addition, the results of this study indicate that interest to compare data for ethanol and benzodiazepines. Several the a3 subunit of GABAA receptor may have an important role in conclusions from these studies are that receptors containing the sedative effects of ethanol. a2 subunit are critical for the antianxiety and myorelaxant actions of benzodiazepines, with a3 subunits having a role in the anti- Disclosures anxiety and high dose myorelaxant actions, but neither a2ora3 subunits have a role in motor sedation (Rudolph et al., 1999; Authors certify that they have no financial interests or potential Rudolph and Möhler, 2004; Crestani et al., 2001; Morris et al., conflicts of interest. All financial research or project support is 2006; Dixon et al., 2008). There is a remarkable lack of congru- identified in an acknowledgment in the manuscript. Uwe Rudolph ence with ethanol studies in GABAA receptor mutant mice where is a consultant for Sunovion and for Concert Pharmaceuticals. the a2ora3 subunits are not important for the anxiolytic or myorelaxant actions but contribute to the sedative effects Acknowledgments (Table 1). Human genetic studies associate polymorphisms of the GABRA2 We thank Drs. Claire Dixon and David Stephens for helpful gene encoding the GABAA a2-subunit with ethanol dependence in discussions in attempting to resolve our apparently contradictory variety of populations (Edenberg et al., 2004; Enoch et al., 2006; findings. This research was supported by grant from the National Covault et al., 2004; Lappalainen et al., 2005). Interestingly, variants Institute of Alcohol Abuse and Alcoholism, NIH (AA U01 13520 e in GABRA2 genes have also been associated with addictive behav- INIA Project) and NIH grants A06399, AA10422, AA13004 and iors for other drugs such as cocaine (Dixon et al., 2010) and heroin DE14184. (Enoch et al., 2010). It should be noted, that the same variations are also associated with childhood conduct disorder (Dick et al., 2006; Appendix A. Supplementary data Sakai et al., 2010) and with increased impulsivity (Villafuerte et al., 2012) behavioral traits that may contribute to the development of Supplementary data related to this article can be found at http:// addictive behaviors. Some data suggest that the influence of dx.doi.org/10.1016/j.neuropharm.2012.10.016. Y.A. Blednov et al. / Neuropharmacology 67 (2013) 46e56 55

References Enoch, M.A., Zhou, Z., Kimura, M., Mash, D.C., Yuan, Q., Goldman, D., 2012. GABAergic in postmortem from alcoholics and cocaine addicts; corresponding findings in alcohol-naïve P and NP rats. PLoS Balakleevsky, A., Colombo, G., Fadda, F., Gessa, G.L., 1990. Ro 19-4603, One 7, e29369. a benzodiazepine receptor inverse agonist, attenuates voluntary ethanol Ernst, M., Brauchart, D., Boresch, S., Sieghart, W., 2003. Comparative modeling of consumption in rats selectively bred for high ethanol preference. Alcohol GABA receptors: limits, insights, future developments. Neuroscience 119, 25, 449e452. A 933e943. Blednov, Y.A., Stoffel, M., Chang, S.R., Harris, R.A., 2001. GIRK2 deficient mice. Fritschy, J.M., Möhler, H., 1995. GABAA-receptor heterogeneity in the adult rat brain: Evidence for hyperactivity and reduced anxiety. Physiol. Behav. 74, 109e117. differential regional and cellular distribution of seven major subunits. J. Comp. Blednov, Y.A., Walker, D., Alva, H., Creech, K., Findlay, G., Harris, R.A., 2003. GABAA Neurol. 359, 154e194. receptor alpha 1 and beta 2 subunit null mutant mice: behavioral responses to Fritschy, J.M., Benke, D., Mertens, S., Oertel, W.H., Bachi, T., Mohler, H., 1992. Five ethanol. J. Pharmacol. Exp. Ther. 305, 854e863. subtypes of type A gamma-aminobutyric acid receptors identified in neurons Blednov, Y.A., Borghese, C.M., McCracken, M.L., Benavidez, J.M., Geil, C.R., Ostern- by double and triple immunofluorescence staining with subunit-specific anti- dorff-Kahanek, E., Werner, D.F., Iyer, S., Swihart, A., Harrison, N.L., bodies. Proc. Natl. Acad. Sci. U. S. A. 89, 6726e6730. Homanics, G.E., Harris, R.A., 2011. Loss of ethanol conditioned taste aversion and Green, A.S., Grahame, N.J., 2008. Ethanol drinking in rodents: is free-choice drinking motor stimulation in knockin mice with ethanol-insensitive a2-containing related to the reinforcing effects of ethanol? Alcohol 42, 1e11. GABA(A) receptors. J. Pharmacol. Exp. Ther. 336, 145e154. Harris, R.A., Trudell, J.R., Mihic, S.J., 2008. Ethanol’s molecular targets. Sci. Signal. Blednov, Y.A., Mayfield, R.D., Belknap, J., Harris, R.A., 2012. Behavioral actions of 1, re7. alcohol: phenotypic relations from multivariate analysis of mutant mouse data. Harris, R.A., Osterndorff-Kahanek, E., Ponomarev, I., Homanics, G.E., Blednov, Y.A., Genes Brain Behav. 11, 424e435. 2011. Testing the silence of mutations: transcriptomic and behavioral studies of Boehm 2nd, S.L., Ponomarev, I., Jennings, A.W., Whiting, P.J., Rosahl, T.W., GABA(A) receptor a1 and a2 subunit knock-in mice. Neurosci. Lett. 488, 31e35. Garrett, E.M., Blednov, Y.A., Harris, R.A., 2004. gamma-Aminobutyric acid A Haughey, H.M., Ray, L.A., Finan, P., Villanueva, R., Niculescu, M., Hutchison, K.E., receptor subunit mutant mice: new perspectives on alcohol actions. Biochem. 2008. The human GABA(A) receptor alpha2 gene moderates the acute effects of Pharmacol. 68, 1581e1602. alcohol and brain mRNA expression. Genes Brain Behav. 7, 447e454. Buck, K.J., Hood, H.M., 1998. Genetic association of a GABAA receptor g2 subunit Herd, M.B., Foister, N., Chandra, D., Peden, D.R., Homanics, G.E., Brown, V.J., variant with severity of acute physiological dependence on alcohol. Mamm. Balfour, D.J., Lambert, J.J., Belelli, D., 2009. Inhibition of thalamic excitability by Genome 9, 975e978. 4,5,6,7-tetrahydroisoxazolo[4,5-c]pyridine-3-ol: a selective role for delta- Chandra, D., Jia, F., Liang, J., Peng, Z., Suryanarayanan, A., Werner, D.F., GABA(A) receptors. Eur. J. Neurosci. 29, 1177e1187. Spigelman, I., Houser, C.R., Olsen, R.W., Harrison, N.L., Homanics, G.E., 2006. Hinko, C.N., Rozanov, C., 1990. The role of , aminooxyacetic acid and GABAA receptor alpha 4 subunits mediate extrasynaptic inhibition in thal- gabaculine in the modulation of ethanol-induced motor impairment. Eur. J. amus and dentate gyrus and the action of gaboxadol. Proc. Natl. Acad. Sci. U. S. Pharmacol. 182, 261e271. A. 103, 15230e15235. Hodge, C.W., Cox, A.A., 1998. The discriminative stimulus effects of ethanol are Chandra, D., Werner, D.F., Liang, J., Suryanarayanan, A., Harrison, N.L., Spigelman, I., mediated by NMDA and GABAA receptors in specific limbic brain regions. Olsen, R.W., Homanics, G.E., 2008. Normal acute behavioral responses to Psychopharmacology 139, 95e107. moderate/high dose ethanol in GABAA receptor alpha 4 subunit knockout mice. Hyytia, P., Koob, G.F., 1995. GABA receptor antagonism in the extended amygdala Alcohol. Clin. Exp. Res. 32, 10e18. A decreases ethanol self-administration in rats. Eur. J. Pharmacol. 283, 151e159. Chester, J.A., Cunningham, C.L., 2002. GABA(A) receptor modulation of the Koechling, U.M., Smith, B.R., Amit, Z., 1991. Effects of GABA antagonists and habit- rewarding and aversive effects of ethanol. Alcohol 26, 131e143. uation to novelty on ethanol-induced locomotor activity in mice. Alcohol Chick, J., Nutt, D.J., 2012. Substitution therapy for alcoholism: time for a reappraisal? Alcohol 26, 315e322. J. Psychopharmacol. 26, 205e212. Korpi, E.R., Debus, F., Linden, A.M., Malécot, C., Leppä, E., Vekovischeva, O., Rabe, H., Collinson, N., Kuenzi, F.M., Jarolimek, W., Maubach, K.A., Cothliff, R., Sur, C., Böhme, I., Aller, M.I., Wisden, W., Lüddens, H., 2007. Does ethanol act prefer- Smith, A., Out, F.M., Howell, O., Atack, J.R., McKernan, R.M., Seabrook, G.R., entially via selected brain GABAA receptor subtypes? The current evidence is Dawson, G.R., Whiting, P.J., Rosahl, T.W., 2002. Enhanced learning and memory ambiguous. Alcohol 41, 163e176. and altered GABAergic synaptic transmission in mice lacking the alpha 5 Kralic, J.E., Wheeler, M., Renzi, K., Ferguson, C., O’Buckley, T.K., Grobin, A.C., subunit of the GABAA receptor. J. Neurosci. 22, 5572e5580. Morrow, A.L., Homanics, G.E., 2003. Deletion of GABAA receptor alpha 1 Covault, J., Gelernter, J., Hesselbrock, V., Nellissery, M., Kranzler, H.R., 2004. Allelic subunit-containing receptors alters responses to ethanol and other anesthetics. and haplotypic association of GABRA2 with alcohol dependence. Am. J. Med. J. Pharmacol. Exp. Ther. 305, 600e607. Genet. B Neuropsychiatr. Genet. 129B, 104e109. Kumar, S., Porcu, P., Werner, D.F., Matthews, D.B., Diaz-Granados, J.L., Crabbe, J.C., Metten, P., Cameron, A.J., Wahlsten, D., 2005. An analysis of the genetics Helfand, R.S., Morrow, A.L., 2009. The role of GABA(A) receptors in the acute of alcohol intoxication in inbred mice. Neurosci. Biobehav. Rev. 28, 785e802. and chronic effects of ethanol: a decade of progress. Psychopharmacology(- Crabbe, J.C., Bell, R.L., Ehlers, C.L., 2010. Human and laboratory rodent low response Berl) 205, 529e564. to alcohol: is better consilience possible? Addict. Biol. 15, 125e144. Lappalainen, J., Krupitsky, E., Remizov, M., Pchelina, S., Taraskina, A., Zvartau, E., Crestani, F., Löw, K., Keist, R., Mandelli, M., Möhler, H., Rudolph, U., 2001. Molecular Somberg, L.K., Covault, J., Kranzler, H.R., Krystal, J.H., Gelernter, J., 2005. Asso- targets for the myorelaxant action of diazepam. Mol. Pharmacol. 59, 442e445. ciation between alcoholism and gamma-amino butyric acid alpha2 receptor Cunningham, C.L., Fidler, T.L., Hill, K.G., 2000. Animal models of alcohol’s motiva- subtype in a Russian population. Alcohol. Clin. Exp. Res. 29, 493e498. tional effects. Alcohol. Res. Health 24, 85e92. Lind,P.A.,MacGregor,S.,Montgomery,G.W.,Heath,A.C.,Martin,N.G., Dick, D.M., Bierut, L., Hinrichs, A., Fox, L., Bucholz, K.K., Kramer, J., Kuperman, S., Whitfield, J.B., 2008. Effects of GABRA2 variation on physiological, psycho- Hesselbrock, V., Schuckit, M., Almasy, L., Tischfield, J., Porjesz, B., Begleiter, H., motor and subjective responses in the alcohol challenge twin study. Twin Res. Nurnberger Jr., J., Xuei, X., Edenberg, H.J., Foroud, T., 2006. The role of GABRA2 in Hum. Genet. 11, 174e182. risk for conduct disorder and alcohol and drug dependence across develop- Liu, C., Showalter, J., Grigson, P.S., 2009. Ethanol-induced conditioned taste avoid- mental stages. Behav. Genet. 36, 577e590. ance: reward or aversion? Alcohol. Clin. Exp. Res. 33, 522e530. Dixon, C.I., Rosahl, T.W., Stephens, D.N., 2008. Targeted deletion of the GABRA2 gene Lobo, I.A., Harris, R.A., 2008. GABA(A) receptors and alcohol. Pharmacol. Biochem. encoding alpha2-subunits of GABA(A) receptors facilitates performance of Behav. 90, 90e94. a conditioned emotional response, and abolishes anxiolytic effects of benzo- Low, K., Crestani, F., Keist, R., Benke, D., Brunig, I., Benson, J.A., Fritschy, J.M., diazepines and barbiturates. Pharmacol. Biochem. Behav. 90, 1e8. Rülicke, T., Bluethmann, H., Möhler, H., Rudolph, U., 2000. Molecular and Dixon, C.I., Morris, H.V., Breen, G., Desrivieres, S., Jugurnauth, S., Steiner, R.C., neuronal substrate for selective attenuation of anxiety. Science 290, 131e134. Vallada, H., Guindalini, C., Laranjeira, R., Messas, G., Rosahl, T.W., Atack, J.R., Lundquist, F., 1959. The determination of ethyl alcohol in blood and tissue. Methods Peden, D.R., Belelli, D., Lambert, J.J., King, S.L., Schumann, G., Stephens, D.N., Biochem. Anal. 7, 217e251. 2010. Cocaine effects on mouse incentive-learning and human addiction are McKernan, R.M., Rosahl, T.W., Reynolds, D.S., Sur, C., Wafford, K.A., Atack, J.R., linked to alpha2 subunit-containing GABAA receptors. Proc. Natl. Acad. Sci. Farrar, S., Myers, J., Cook, G., Ferris, P., Garrett, L., Bristow, L., Marshall, G., U.S.A. 107, 2289e2294. Macaulay, A., Brown, N., Howell, O., Moore, K.W., Carling, R.W., Street, L.J., Edenberg, H.J., Dick, D.M., Xuei, X., Tian, H., Almasy, L., Bauer, L.O., Crowe, R.R., Castro, J.L., Ragan, C.I., Dawson, G.R., Whiting, P.J., 2000. Sedative but not Goate, A., Hesselbrock, V., Jones, K., Kwon, J., Li, T.K., Nurnberger Jr., J.I., anxiolytic properties of benzodiazepines are mediated by the GABA(A) receptor O’Connor, S.J., Reich, T., Rice, J., Schuckit, M.A., Porjesz, B., Foroud, T., alpha1 subtype. Nat. Neurosci. 3, 587e592. Begleiter, H., 2004. Variations in GABRA2, encoding the alpha 2 subunit of the Mihalek, R.M., Banerjee, P.K., Korpi, E.R., Quinlan, J.J., Firestone, L.L., Mi, Z.P., GABA(A) receptor, are associated with alcohol dependence and with brain Lagenaur, C., Tretter, V., Sieghart, W., Anagnostaras, S.G., Sage, J.R., oscillations. Am. J. Hum. Genet. 74, 705e714. Fanselow, M.S., Guidotti, A., Spigelman, I., Li, Z., DeLorey, T.M., Olsen, R.W., Enoch, M.A., 2008. The role of GABA(A) receptors in the development of alcoholism. Homanics, G.E., 1999. Attenuated sensitivity to neuroactive steroids in gamma- Pharmacol. Biochem. Behav. 90, 95e104. aminobutyrate type A receptor delta subunit knockout mice. Proc. Natl. Acad. Enoch, M.A., Schwartz, L., Albaugh, B., Virkkunen, M., Goldman, D., 2006. Dimen- Sci. U. S. A. 96, 12905e12910. sional anxiety mediates linkage of GABRA2 haplotypes with alcoholism. Am. J. Mihalek, R.M., Bowers, B.J., Wehner, J.M., Kralic, J.E., VanDoren, M.J., Morrow, A.L., Med. Genet. B Neuropsychiatr. Genet. 141B, 599e607. Homanics, G.E., 2001. GABA(A)-receptor delta subunit knockout mice have Enoch, M.A., Hodgkinson, C.A., Yuan, Q., Shen, P.H., Goldman, D., Roy, A., 2010. The multiple defects in behavioral responses to ethanol. Alcohol. Clin. Exp. Res. 25, influence of GABRA2, childhood trauma, and their interaction on alcohol, 1708e1718. heroin, and cocaine dependence. Biol. Psychiatry 67, 20e27. 56 Y.A. Blednov et al. / Neuropharmacology 67 (2013) 46e56

Möhler, H., 2007. Molecular regulation of cognitive functions and developmental Sigel, E., 2002. Mapping of the benzodiazepine recognition site on GABAA receptors. plasticity: impact of GABA-A receptors. J. Neurochem. 102, 1e12. Curr. Top. Med. Chem. 2, 833e839. Morris, H.V., Dawson, G.R., Reynolds, D.S., Atack, J.R., Stephens, D.N., 2006. Both Soyka, M., Preuss, U.W., Hesselbrock, V., Zill, P., Koller, G., Bondy, B., 2008. GABA-A2 alpha2 and alpha3 GABAA receptor subtypes mediate the anxiolytic properties receptor subunit gene (GABRA2) polymorphisms and risk for alcohol depen- of benzodiazepine site ligands in the conditioned emotional response para- dence. J. Psychiatr. Res. 42, 184e191. digm. Eur. J. Neurosci. 23, 2495e2504. Spurney, C.F., Gordish-Dressman, H., Guerron, A.D., Sali, A., Pandey, G.S., Rawat, R., Panzanelli, P., Gunn, B.G., Schlatter, M.C., Benke, D., Tyagarajan, S.K., Scheiffele, P., Van Der Meulen, J.H., Cha, H.J., Pistilli, E.E., Partridge, T.A., Hoffman, E.P., Belelli, D., Lambert, J.J., Rudolph, U., Fritschy, J.M., 2011. Distinct mechanisms Nagaraju, K., 2009. Preclinical drug trials in the mdx mouse: assessment of regulate GABAA receptor and gephyrin clustering at perisomatic and axo-axonic reliable and sensitive outcome measures. Muscle Nerve 39, 591e602. synapses on CA1 pyramidal cells. J. Physiol. 589, 4959e4980. Sur, C., Wafford, K.A., Reynolds, D.S., Hadingham, K.L., Bromidge, F., Macaulay, A., Pirker, S., Schwarzer, C., Wieselthaler, A., Sieghart, W., Sperk, G., 2000. GABA(A) Collinson, N., O’Meara, G., Howell, O., Newman, R., Myers, J., Atack, J.R., receptors: immunocytochemical distribution of 13 subunits in the adult rat Dawson, G.R., McKernan, R.M., Whiting, P.J., Rosahl, T.W., 2001. Loss of the major brain. Neuroscience 101, 815e850. GABA(A) receptor subtype in the brain is not lethal in mice. J. Neurosci. 21, Roberto, M., Madamba, S.G., Moore, S.D., Tallent, M.K., Siggins, G.R., 2003. Ethanol 3409e3418. increases GABAergic transmission at both pre- and postsynaptic sites in rat Tabakoff, B., Saba, L., Printz, M., Flodman, P., Hodgkinson, C., Goldman, D., Koob, G., central amygdala neurons. Proc. Natl. Acad. Sci. U. S. A. 100, 2053e2058. Richardson, H.N., Kechris, K., Bell, R.L., Hübner, N., Heinig, M., Pravenec, M., Rudolph, U., Möhler, H., 2004. Analysis of GABAA receptor function and dissection Mangion, J., Legault, L., Dongier, M., Conigrave, K.M., Whitfield, J.B., Saunders, J., of the pharmacology of benzodiazepines and general anesthetics through Grant, B., Hoffman, P.L., 2009. Genetical genomic determinants of alcohol mouse genetics. Annu. Rev. Pharmacol. Toxicol. 44, 475e498. consumption in rats and humans. WHO/ISBRA Study on State and Trait Markers Rudolph, U., Crestani, F., Benke, D., Brunig, I., Benson, J.A., Fritschy, J.M., Martin, J.R., of Alcoholism. BMC Biol. 7, 70. Bluethmann, H., Möhler, H., 1999. Benzodiazepine actions mediated by specific Uhart, M., Weerts, E.M., McCaul, M.E., Guo, X., Yan, X., Kranzler, H.R., Li, N., g-aminobutyric acid A receptor subtypes. Nature 401, 796e800. Wand, G.S., 2012. GABRA2 markers moderate the subjective effects of alcohol. Sakai, J.T., Stallings, M.C., Crowley, T.J., Gelhorn, H.L., McQueen, M.B., Ehringer, M.A., Addict. Biol.. http://dx.doi.org/10.1111/j.1369e1600.2012.00457.x 2010. Test of association between GABRA2 (SNP rs279871) and adolescent Villafuerte, S., Heitzeg, M.M., Foley, S., Yau, W.Y., Majczenko, K., Zubieta, J.K., conduct/alcohol use disorders utilizing a sample of clinic referred youth with Zucker, R.A., Burmeister, M., 2012. Impulsiveness and insula activation during serious substance and conduct problems, controls and available first degree reward anticipation are associated with genetic variants in GABRA2 in a family relatives. Drug Alcohol Depend 106, 199e203. sample enriched for alcoholism. Mol. Psychiatry 17, 511e519. Schuckit, M.A., 1986. Biological markers in alcoholism. Prog. Neuro- Wallner, M., Hanchar, H.J., Olsen, R.W., 2006. Low dose acute alcohol effects on psychopharmacol. Biol. Psychiatry 10, 191e199. GABA A receptor subtypes. Pharmacol. Ther. 112, 513e528. Schuckit, M.A., 1988. Reactions to alcohol in sons of alcoholics and controls. Alcohol. Werner, D.F., Blednov, Y.A., Ariwodola, O.J., Silberman, Y., Logan, E., Berry, R.B., Clin. Exp. Res. 12, 465e470. Borghese, C.M., Matthews, D.B., Weiner, J.L., Harrison, N.L., Harris, R.A., Schuckit, M.A., 1994. Low level of response to alcohol as a predictor of future Homanics, G.E., 2006. Knockin mice with ethanol-insensitive alpha1-containing alcoholism. Am. J. Psychiatry 151, 184e189. gamma-aminobutyric acid type A receptors display selective alterations in Schwarzer, C., Berresheim, U., Pirker, S., Wieselthaler, A., Fuchs, K., Sieghart, W., behavioral responses to ethanol. J. Pharmacol. Exp. Ther. 319, 219e227. Sperk, G., 2001. Distribution of the major gamma-aminobutyric acid(A) receptor Yee, B.K., Keist, R., von Boehmer, L., Studer, R., Benke, D., Hagenbuch, N., Dong, Y., subunits in the basal ganglia and associated limbic brain areas of the adult rat. Malenka, R.C., Fritschy, J.M., Bluethmann, H., Feldon, J., Möhler, H., Rudolph, U., J. Comp. Neurol. 433, 526e549. 2005. A schizophrenia-related sensorimotor deficit links alpha 3-containing Sieghart, W., 1995. Structure and pharmacology of g-aminobutyric acidA receptor GABAA receptors to a dopamine hyperfunction. Proc. Natl. Acad. Sci. U. S. A. 102, subtypes. Am. Soc. Pharmacol. Exp. Ther. 47, 181e234. 17154e17159.