ETHANOL TOLERANCE AND WITHDRAWAL RESPONSES IN GABA(A) RECEPTOR-ALPHA-6 SUBUNIT NULL ALLELE MICE AND IN INBRED C57BL 6J AND STRAIN 129/SVJ MICE/

We have been using a genetic strategy to define the contribution of sp ecific candidate genes, such as those encoding subunits of the gamma-a minobutyric acid type A receptor, to various ethanol sensitive respons es. We have used the gene knockout approach in mouse embryonic stem ce lls to create mice in which the gene encoding the alpha 6 subunit of t he gamma-aminobutyric acid type A receptor is rendered nonfunctional. In the present report, we provide a detailed characterization of sever al behavioral responses to ethanol in these null allele mice. In a sep arate series of experiments, behavioral response to ethanol was compar ed between two inbred strains of mice that are commonly used as backgr ound stock in knockout experiments, namely C57BL/6J and Strain 129/SvJ . Wild type (alpha 6(+/+)) and homozygous null allele ((alpha 6(-/-)) mice did not differ to the ataxic effects of ethanol on acute function al tolerance (95.8 +/- 8.7 vs. 98.8 +/- 5.7 mg/dl +/- SEM, respectivel y). Withdrawal hyperexcitability was assessed following chronic exposu re to ethanol vapor (EtOH) or air (CONT) in inhalation chambers in a m ultiple withdrawal treatment paradigm. At the end of the last treatmen t cycle, mice were scored for handling induced convulsions (HIC). Afte r adjusting for differences in blood ethanol concentration between gen otypes at the end of the final treatment cycle, we observed a greater area under the 24-hr HIC curves in mice treated with ethanol (p < 0.00 01) but did not detect an effect of genotype (alpha 6(+/+)/CONT 3.1 +/ - 2.0; alpha 6(-/-)/CONT 5.5 +/- 2.5; alpha 6(+/+)/EtOH 30.1 +/- 6.2; alpha 6(-/-)/EtOH 33.0 +/- 5.8 mean units +/- SEM). We also examined t hese mice for differences in protracted tolerance; at similar to 26 hr into the final withdrawal cycle, each mouse was injected with ethanol (3.5 mg/g body weight) and sleep time was measured. We detected a sig nificant effect of treatment (p < 0.001) with ethanol-treated mice dem onstrating signs of tolerance as reflected by a reduction in duration of sleep time. However, effect of genotype was not significant (alpha 6(+/+)/CONT 57.4 +/- 7.6; alpha 6(-/-)/CONT 59.0 +/- 7.6; alpha 6(+/)/EtOH 34.8 +/- 7.4; alpha 6(-/-)/EtOH 30.8 +/- 5.6 min +/- SEM). From these data we conclude that the alpha 6 subunit of the GABA(A)-R exert s little if any influence an acute functional tolerance, withdrawal hy perexcitability, or protracted tolerance. Strain 129/SvJ and C57BL/6J mice were also compared for acute functional tolerance and were found not to differ (96.3 +/- 4.4 vs. 94.8 +/- 11.3 mg/dl +/- SEM, respectiv ely). Withdrawal hyperexcitability was assessed by comparing the area under the 24 hr HIC curves. Strain 129/SvJ mice displayed a much great er basal HIC response compared to C57BL/6J mice (19.8 +/-: 4.3 vs. 0.2 +/- 0.2 mean units +/- SEM, respectively); after adjusting for differ ences in blood ethanol concentration between strains at the end of the final ethanol treatment cycle, the HIC response was markedly enhanced by ethanol treatment in Strain 129/SvJ mice but not in C57BL/6J mice (50.4 +/- 3.1 vs. 9.5 +/- 5.4 mean units +/- SEM, respectively). The e ffects of treatment (p < 0.0001), strain (p < 0.0001), and the interac tion of strain with treatment (p < 0.01) were significant Since many g ene knockout mice are maintained on a mixed genetic background of Stra in 129/SvJ and C57BL/6J, we conclude that significant differences in t ests of withdrawal hyperexcitability may be confounded by the influenc e of genes that cosegregate with the gene targeted allele.