Ethanol Tolerance and Withdrawal Responses in GABAAReceptor Alpha 6 Subunit Null Allele Mice and in Inbred C57BL/6J and Strain 129/SvJ Mice

Abstract

We have been using a genetic strategy to define the contribution of specific candidate genes, such as those encoding subunits of the gamma-aminobutyric acid type A receptor, to various ethanol sensitive responses. We have used the gene knockout approach in mouse embryonic stem cells to create mice in which the gene encoding the alpha6 subunit of the gamma-aminobutyric acid type A receptor is rendered nonfunctional. In the present report, we provide a detailed characterization of several behavioral responses to ethanol in these null allele mice. In a separate series of experiments, behavioral response to ethanol was compared between two inbred strains of mice that are commonly used as background stock in knockout experiments, namely C57BL/6J and Strain 129/SvJ. Wild type (alpha6+/+) and homozygous null allele (alpha6-/-) mice did not differ to the ataxic effects of ethanol on acute functional tolerance (95.8 +/- 8.7 vs. 98.8 +/- 5.7 mg/dl +/- SEM, respectively). Withdrawal hyperexcitability was assessed following chronic exposure to ethanol vapor (EtOH) or air (CONT) in inhalation chambers in a multiple withdrawal treatment paradigm. At the end of the last treatment cycle, mice were scored for handling induced convulsions (HIC). After adjusting for differences in blood ethanol concentration between genotypes 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 < 0.0001) but did not detect an effect of genotype (alpha6+/+/CONT 3.1 +/- 2.0; alpha6-/-/CONT 5.5 +/- 2.5; alpha6+/+/EtOH 30.1 +/- 6.2; alpha6-/-/EtOH 33.0 +/- 5.8 mean units +/- SEM). We also examined these mice for differences in protracted tolerance; at approximately 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 significant effect of treatment (p < 0.001) with ethanol-treated mice demonstrating signs of tolerance as reflected by a reduction in duration of sleep time. However, effect of genotype was not significant (alpha6+/+/CONT 57.4 +/- 7.6; alpha6-/-/CONT 59.0 +/- 7.6; alpha6+/ +/EtOH 34.8 +/- 7.4; alpha6-/-/EtOH 30.8 +/- 5.6 min +/- SEM). From these data we conclude that the alpha6 subunit of the GABA(A)-R exerts little if any influence on acute functional tolerance, withdrawal hyperexcitability, 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, respectively). Withdrawal hyperexcitability was assessed by comparing the area under the 24 hr HIC curves. Strain 129/SvJ mice displayed a much greater basal HIC response compared to C57BL/6J mice (19.8 +/- 4.3 vs. 0.2 +/- 0.2 mean units +/- SEM, respectively); after adjusting for differences 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. ...

Sex Differences in Ethanol’s Anxiolytic Effect and Chronic Ethanol Withdrawal Severity in Mice with a Null Mutation of the 5α-Reductase Type 1 Gene

“…Exposure to air + pyrazole produced a slight enhancement of HIC within the early phase of withdrawal (i.e., 1 st 12 hrs), which subsequently decreased to baseline levels. This finding is consistent with the report by Homanics et al (1998) in which control 129/SvJ mice exhibited marked HIC that peaked approximately 2–3 hr post-removal from the inhalation chamber and returned to baseline at 8 hr. Thus, all of our experiments employed the use of pyrazole in the air controls to offset any potential confound due to a small proconvulsant effect of pyrazole in some genotypes.…”

“…Since the WT mice are a hybrid of C57BL/6J and 129/SvJ background strains, we were unsure whether ethanol withdrawal in this genotype would be mild (similar to C57BL/6J) or moderate to severe (similar to 129/SvJ) and whether the air-controls would exhibit mild HICs as has been reported for 129/SvJ genotypes (Homanics et al, 1998; Metten and Crabbe, 2005). The pattern of hourly HIC scores in the ethanol- and air-exposed mice in the present study (Figure 6A & 6B) was comparable to that in the 129/SvJ male mice (Homanics et al, 1998; Metten and Crabbe, 2005), consistent with the contribution of genes from 129/SvJ strains to the HIC phenotype in the KO and WT mice.…”

Decreased Ethanol Sensitivity and Tolerance Development in γ‐Protein Kinase C Null Mutant Mice Is Dependent on Genetic Background

“…When the null mutants from this same N6 generation were outcrossed to 129/SvEvTac mice, tolerance to the hypothermic, but not the sedative-hypnotic, effects of ethanol was exhibited by the null mutant mice. These results serve to substantiate the relevance of including the effects of genetic background when evaluating the effects of single gene mutations on complex behavioral phenotypes (Gerlai, 1996;Wehner and Bowers, 1995;Banbury Conference, 1997;Homanics et a]., 1998). In addition, initial sensitivity to the hypothermia-inducing effects of ethanol was not decreased in these populations.…”

“…In their study, the phenotype of the parental 129/SvEvTac inbred strain was virtually identical to the D2 receptor-deficient mice, suggesting that cosegregation of 129 alleles around the D2 allele were responsible for the locomotor impairments in the null mutant mice. In studies of ethanol-induced sedation, tolerance, and withdrawal responses in GABA, receptor a6 null mutant mice, Homanics et al (1997Homanics et al ( , 1998 also reported that the background alleles from the parental strains, C57BWGJ and 129/SvJ, appeared to mediate the lack of ethanol responses in the null mutant mice. However, the explanation of differences in ethanol tolerance observed in the present study is more complicated.…”

“…Their results showed that the consumption of the 129 strain was intermediate to the high ethanol-consuming C57BW6J and low-consuming DBN2J inbred strains. Also, Homanics et al (1998) recently tested 129/SvJ mice for ethanol-induced acute tolerance as part of a study of background parental strains for the GABA, receptor a6 subunit null mutant mice. Similar to the results presented herein, they reported that 129/SvJ mice developed acute functional tolerance after a single 1.75 g/kg IP injection of ethanol.…”