Blednov YA et al. (2014), GABAA receptors containing ρ1 subunits contribu...

XB-ART-49842

PLoS One 2014 Jan 01;91:e85525. doi: 10.1371/journal.pone.0085525.

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GABAA receptors containing ρ1 subunits contribute to in vivo effects of ethanol in mice.

Blednov YA , Benavidez JM , Black M , Leiter CR , Osterndorff-Kahanek E , Johnson D , Borghese CM , Hanrahan JR , Johnston GA , Chebib M , Harris RA .

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GABAA receptors consisting of ρ1, ρ2, or ρ3 subunits in homo- or hetero-pentamers have been studied mainly in retina but are detected in many brain regions. Receptors formed from ρ1 are inhibited by low ethanol concentrations, and family-based association analyses have linked ρ subunit genes with alcohol dependence. We determined if genetic deletion of ρ1 in mice altered in vivo ethanol effects. Null mutant male mice showed reduced ethanol consumption and preference in a two-bottle choice test with no differences in preference for saccharin or quinine. Null mutant mice of both sexes demonstrated longer duration of ethanol-induced loss of righting reflex (LORR), and males were more sensitive to ethanol-induced motor sedation. In contrast, ρ1 null mice showed faster recovery from acute motor incoordination produced by ethanol. Null mutant females were less sensitive to ethanol-induced development of conditioned taste aversion. Measurement of mRNA levels in cerebellum showed that deletion of ρ1 did not change expression of ρ2, α2, or α6 GABAA receptor subunits. (S)-4-amino-cyclopent-1-enyl butylphosphinic acid ("ρ1" antagonist), when administered to wild type mice, mimicked the changes that ethanol induced in ρ1 null mice (LORR and rotarod tests), but the ρ1 antagonist did not produce these effects in ρ1 null mice. In contrast, (R)-4-amino-cyclopent-1-enyl butylphosphinic acid ("ρ2" antagonist) did not change ethanol actions in wild type but produced effects in mice lacking ρ1 that were opposite of the effects of deleting (or inhibiting) ρ1. These results suggest that ρ1 has a predominant role in two in vivo effects of ethanol, and a role for ρ2 may be revealed when ρ1 is deleted. We also found that ethanol produces similar inhibition of function of recombinant ρ1 and ρ2 receptors. These data indicate that ethanol action on GABAA receptors containing ρ1/ρ2 subunits may be important for specific effects of ethanol in vivo.

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Species referenced: Xenopus laevis
Genes referenced: fam110a gabarap grap2 kidins220 pcyt1a

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	Figure 2. Female ρ1 (−/−) mice showed decreased ethanol conditioned taste aversion.Data represent the changes in saccharin consumption produced by injection of saline or ethanol expressed as percent of control trial (Trial 0). A. Development of CTA in males (n = 9–10 for saline injection for both genotypes; n = 20 for ethanol injection for both genotypes). Saline-Ethanol pairings for wild type mice: (F1,28 = 44.9, p<0.001, effect of treatment; F4,112 = 22.4, p<0.001, dependence on trial; F4,112 = 24.9, p<0.001, treatment x trial interaction). Saline-Ethanol pairings for ρ1 null mice: (F1,27 = 28.3, p<0.001, effect of treatment; F4,108 = 19.4, p<0.001, dependence on trial; F4,108 = 19.0, p<0.001, treatment x trial interaction). Genotype-Saline pairings: (F4,68 = 6.6, p<0.001, effect of trial; no dependence on genotype or genotype-trial interaction). Genotype-Ethanol pairings: (F4,152 = 101, p<0.001, effect of trial; no dependence on genotype or genotype-trial interaction). B. Development of CTA in females (n = 9–10 for saline injection for both genotypes; n = 12–20 for ethanol injection for both genotypes). Saline-Ethanol pairings for wild type mice: (F1,128 = 64.4, p<0.001, effect of treatment; F4,112 = 16.5, p<0.001, dependence on trial; F4,112 = 25.8, p<0.001, treatment x trial interaction). Saline-Ethanol pairings for ρ1 null mice: (F1,19 = 14.3, p<0.01, effect of treatment; F4,76 = 26.2, p<0.001, dependence on trial; F4,76 = 25.2, p<0.001, treatment x trial interaction). Genotype-Saline pairings: (F4,68 = 10.5, p<0.001, effect of trial; no dependence on genotype or genotype-trial interaction). Genotype-Ethanol pairings: (F1,30 = 12.3, p<0.001, effect of genotype, F4,120 = 80.9, p<0.001, dependence on trial and no dependence on genotype; F4,120 = 5.9, p<0.001, genotype x trial interaction). Values represent mean ± S.E.M. Data were analyzed by two-way ANOVA. ρ1 (−/−) = ρ1 null mice; (+/+) = wild type mice; EtOH = ethanol.
	Figure 3. Ethanol-induced conditioned place preference in ρ1 (−/−) mice.Data represent the percent of time spent on different types of floor (A, B) or on the grid floor (C, D). A. Males (n = 13–15 per genotype; F1,52 = 14, p<0.001, main effect of floor). B. Females (n = 6 per genotype; F1,20 = 5.7, p<0.05, main effect of floor; F1,20 = 13.7, p<0.01, genotype x floor interaction, p<0.05 vs. another genotype on the same type of floor). C. Males, 1st Preference test (n = 13–15 per genotype and treatment; F1,52 = 43, p<0.001, main effect of treatment, **p<0.001 vs. saline group of corresponding genotype). D. 2nd Preference test (Extinction). Values represent mean ± S.E.M. Data were analyzed by two-way ANOVA with Bonferroni post hoc test. ρ1 (−/−) = ρ1 null mice; (+/+) = wild type mice; EtOH = ethanol.
	Figure 4. Lack of ρ1 increased duration of LORR by ethanol and ketamine but not pentobarbital or flurazepam.A, B, C, D – Males. E, F, G, H – Females. A, E – Ethanol (n = 8–10 per genotype for both sexes; t(16) = 3.3 for males and females, p<0.01 vs. wild type of corresponding genotype). B, F – Pentobarbital (n = 12–15 per genotype for both sexes). C, G – Ketamine (n = 10–14 per genotype for both sexes; t(25) = 2.9 for males and t(22) = 6.3 for females, p<0.01, ***p<0.001 vs. wild type of corresponding genotype). D, H – Flurazepam (n = 7–10 per genotype for both sexes). Values represent mean ± S.E.M. Data were analyzed by Student's t-test. ρ1 (−/−) = ρ1 null mice; (+/+) = wild type mice; EtOH = ethanol; LORR = loss of righting reflex.
	Figure 5. Faster recovery from motor incoordinating effect of ethanol in ρ1 (−/−) mice.Data represent time (sec) on the rotarod after injection of ethanol (2.0 g/kg). A. Males (n = 5–7 per genotype; F1,16 = 10, p<0.01, dependence on genotype; F9, 144 = 163, p<0.001, dependence on time; F9,144 = 6.8, p<0.001, genotype x time interaction). B. Females (n = 6–8 per genotype; F1,12 = 30.5, p<0.001, dependence on genotype; F7,84 = 124, p<0.001, dependence on time; F7,84 = 11.6, p<0.001, genotype x time interaction). Data represent mean ± S.E.M. Data were analyzed by two-way ANOVA with Bonferroni post hoc test (p<0.05, **p<0.001 vs. wild type genotype for each time point). ρ1 (−/−) = ρ1 null mice; (+/+) = wild type mice.
	Figure 6. Ethanol reduced anxiety-related behavior equally in the elevated plus-maze in wild type and ρ1 null mice.A. Percent of time in open arms. B. Percent of entries into the open arms. C. Number of entries into the closed arms. Data from females and males were combined since there were no gender differences. Values represent mean ± S.E.M. Data were analyzed by two-way ANOVA with Bonferroni post hoc test. p<0.05, *p<0.01 vs. saline group of corresponding genotype (n = 13–19 per group). ρ1 (−/−) = ρ1 null mice; (+/+) = wild type mice; EtOH = ethanol.
	Figure 7. Effect of ethanol on motor activity after pre-habituation.A. Males (n = 12–20 per genotype; F1,30 = 4.8, p<0.05 main effect of genotype; F2,60 = 13.8, p<0.001 main effect of treatment; no genotype x treatment interaction). B. Females (n = 13–18 per genotype; F2,56 = 18.4, p<0.001 main effect of treatment; no dependence on genotype or genotype x treatment interaction). Values represent mean ± S.E.M. Data were analyzed by two-way ANOVA with repeated measures with Bonferroni post hoc test (#p<0.05 vs. response of another genotype for the same condition). Effect of ethanol within each genotype was also analyzed by one-way ANOVA with repeated measures with Dunnett's post hoc test (p<0.05, *p<0.01 vs. saline response of corresponding genotype). ρ1 (−/−) = ρ1 null mice; (+/+) = wild type mice; EtOH = ethanol.
	Figure 8. Effect of ρ1/ρ2 antagonists on ethanol (3.4 g/kg)-induced LORR in wild type and ρ1 (−/−) mice.A. Wild type male mice. (n = 9–11; F2,27 = 17.9, p<0.001). B. Wild type female mice. (n = 9–10; F2,26 = 25, p<0.001). C. ρ1 (−/−) male mice. (n = 7–11; F2,25 = 113, p<0.001). D. ρ1 (−/−) female mice. (n = 9–10; F2,27 = 20.7, p<0.001). p<0.05, p<0.01, **p<0.001 vs. saline; ##p<0.01, ###p<0.001 (S)-ACPBPA vs. (R)-ACPBPA). Values represent mean ± S.E.M. Data were analyzed by one-way ANOVA with Bonferroni post hoc test. ρ1 (−/−) = ρ1 null mice; (+/+) = wild type mice; LORR = loss of righting reflex.
	Figure 9. Effect of ρ1/ρ2 antagonists on ketamine (150 mg/kg)-induced LORR in wild type and ρ1 (−/−) mice.A. Wild type male mice. (n = 9–11; F2,12 = 77.5, p<0.001). B. Wild type female mice. (n = 9–10; F2,12 = 105, p<0.001). C. ρ1 (−/−) male mice. (n = 7–11; F2,12 = 79.3, p<0.001). D. ρ1 (−/−) female mice. (n = 9–10; F2,12 = 27.7, p<0.001). p<0.01, *p<0.001 vs. saline; ###p<0.001 (S)-ACPBPA vs. (R)-ACPBPA). Values represent mean ± S.E.M. Data were analyzed by one-way ANOVA with Bonferroni post hoc test. ρ1 (−/−) = ρ1 null mice; (+/+) = wild type mice; LORR = loss of righting reflex.
	Figure 10. Effect of ρ1/ρ2 antagonists on recovery from ethanol (2.0 g/kg)-induced motor incoordination in wild type and ρ1 (−/−) mice.A. Wild type male mice, n = 6. (S)-ACPBPA (F1,10 = 43.3, p<0.001, effect of treatment; F10,100 = 107, p<0.001, effect of time; F10,100 = 17.1, p<0.001, treatment x time interaction). (R)-ACPBPA (F10,100 = 120, p<0.001, effect of time; no effect of treatment or treatment x time interaction). B. Wild type female mice, n = 6. (S)-ACPBPA (F1,10 = 69, p<0.001, effect of treatment; F9,90 = 196, p<0.001, effect of time; F9,90 = 24, p<0.001, treatment x time interaction). (R)-ACPBPA (F9,90 = 181, p<0.001, effect of time; no effect of treatment or treatment x time interaction). C. ρ1 (−/−) male mice, n = 4–6. (S)-ACPBPA (F10,80 = 67.7, p<0.001, effect of time; no effect of treatment or treatment x time interaction). (R)-ACPBPA (F1,9 = 31.5, p<0.001, effect of treatment; F10,90 = 102, p<0.001, effect of time; F10,90 = 13.4, p<0.001, treatment x time interaction). D. ρ1 (−/−) female mice, n = 6. (S)-ACPBPA (F8,80 = 126, p<0.001, effect of time; no effect of treatment or treatment x time interaction). (R)-ACPBPA (F1,10 = 24.1, p<0.001, effect of treatment; F8,80 = 101, p<0.001, effect of time; F8,80 = 8.1, p<0.001, treatment x time interaction). Values represent mean ± S.E.M. Data were analyzed by two-way ANOVA with repeated measures with Bonferroni post hoc test vs. corresponding saline-injected mice. ρ1 (−/−) = ρ1 null mice; (+/+) = wild type mice.
	Figure 11. GABA sensitivity and ethanol modulation of currents produced by human ρ1 or ρ2 recombinant receptors in oocytes.A. GABA concentration-response curve for ρ1 (n = 5) and ρ2 (n = 6) GABAA receptors. B. Ethanol modulation of EC50 GABA-mediated currents for ρ1 (n = 4–5) and ρ2 (n = 3–9) GABAA receptors (no significant difference, two-way ANOVA).
	Figure 1. Voluntary ethanol consumption was reduced in ρ1 (−/−) male mice in 24-hour two-bottle choice paradigm.A. Ethanol consumption (g/kg/24 hours) in males. (F1,18 = 7.1, p<0.05, main effect of genotype; F4,728 = 36.2, main effect of concentration, p<0.001; no genotype x concentration interaction). B. Ethanol consumption (g/kg/24 hours) in females. (F4,68 = 56.2, p<0.001, main effect of concentration; no main effect of genotype or genotype x concentration interaction). C. Preference for ethanol in males. (F1,18 = 7.1, p<0.05, main effect of genotype; F4,72 = 7.8, p<0.001, main effect of concentration; no genotype x concentration interaction). D. Preference for ethanol in females. (F4,68 = 10.6, p<0.001, main effect of concentration; no main effect of genotype or genotype x concentration interaction). E. Total fluid intake (g/kg/24 hours) in males. (F5,90 = 29.9, p<0.001, main effect of concentration; no main effect of genotype or genotype x concentration interaction). F. Total fluid intake (g/kg/24 hours) in females. (F5,75 = 23.8, p<0.001, main effect of concentration; no main effect of genotype or genotype x concentration interaction; n = 9–10 for both genotypes and sexes). Data were analyzed by two-way ANOVA. ρ1 (−/−) = ρ1 null mice; (+/+) = wild type mice; EtOH = ethanol.

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	Figure 2. Female ρ1 (−/−) mice showed decreased ethanol conditioned taste aversion.Data represent the changes in saccharin consumption produced by injection of saline or ethanol expressed as percent of control trial (Trial 0). A. Development of CTA in males (n = 9–10 for saline injection for both genotypes; n = 20 for ethanol injection for both genotypes). Saline-Ethanol pairings for wild type mice: (F1,28 = 44.9, p<0.001, effect of treatment; F4,112 = 22.4, p<0.001, dependence on trial; F4,112 = 24.9, p<0.001, treatment x trial interaction). Saline-Ethanol pairings for ρ1 null mice: (F1,27 = 28.3, p<0.001, effect of treatment; F4,108 = 19.4, p<0.001, dependence on trial; F4,108 = 19.0, p<0.001, treatment x trial interaction). Genotype-Saline pairings: (F4,68 = 6.6, p<0.001, effect of trial; no dependence on genotype or genotype-trial interaction). Genotype-Ethanol pairings: (F4,152 = 101, p<0.001, effect of trial; no dependence on genotype or genotype-trial interaction). B. Development of CTA in females (n = 9–10 for saline injection for both genotypes; n = 12–20 for ethanol injection for both genotypes). Saline-Ethanol pairings for wild type mice: (F1,128 = 64.4, p<0.001, effect of treatment; F4,112 = 16.5, p<0.001, dependence on trial; F4,112 = 25.8, p<0.001, treatment x trial interaction). Saline-Ethanol pairings for ρ1 null mice: (F1,19 = 14.3, p<0.01, effect of treatment; F4,76 = 26.2, p<0.001, dependence on trial; F4,76 = 25.2, p<0.001, treatment x trial interaction). Genotype-Saline pairings: (F4,68 = 10.5, p<0.001, effect of trial; no dependence on genotype or genotype-trial interaction). Genotype-Ethanol pairings: (F1,30 = 12.3, p<0.001, effect of genotype, F4,120 = 80.9, p<0.001, dependence on trial and no dependence on genotype; F4,120 = 5.9, p<0.001, genotype x trial interaction). Values represent mean ± S.E.M. Data were analyzed by two-way ANOVA. ρ1 (−/−) = ρ1 null mice; (+/+) = wild type mice; EtOH = ethanol.
	Figure 3. Ethanol-induced conditioned place preference in ρ1 (−/−) mice.Data represent the percent of time spent on different types of floor (A, B) or on the grid floor (C, D). A. Males (n = 13–15 per genotype; F1,52 = 14, p<0.001, main effect of floor). B. Females (n = 6 per genotype; F1,20 = 5.7, p<0.05, main effect of floor; F1,20 = 13.7, p<0.01, genotype x floor interaction, p<0.05 vs. another genotype on the same type of floor). C. Males, 1st Preference test (n = 13–15 per genotype and treatment; F1,52 = 43, p<0.001, main effect of treatment, **p<0.001 vs. saline group of corresponding genotype). D. 2nd Preference test (Extinction). Values represent mean ± S.E.M. Data were analyzed by two-way ANOVA with Bonferroni post hoc test. ρ1 (−/−) = ρ1 null mice; (+/+) = wild type mice; EtOH = ethanol.
	Figure 4. Lack of ρ1 increased duration of LORR by ethanol and ketamine but not pentobarbital or flurazepam.A, B, C, D – Males. E, F, G, H – Females. A, E – Ethanol (n = 8–10 per genotype for both sexes; t(16) = 3.3 for males and females, p<0.01 vs. wild type of corresponding genotype). B, F – Pentobarbital (n = 12–15 per genotype for both sexes). C, G – Ketamine (n = 10–14 per genotype for both sexes; t(25) = 2.9 for males and t(22) = 6.3 for females, p<0.01, ***p<0.001 vs. wild type of corresponding genotype). D, H – Flurazepam (n = 7–10 per genotype for both sexes). Values represent mean ± S.E.M. Data were analyzed by Student's t-test. ρ1 (−/−) = ρ1 null mice; (+/+) = wild type mice; EtOH = ethanol; LORR = loss of righting reflex.
	Figure 5. Faster recovery from motor incoordinating effect of ethanol in ρ1 (−/−) mice.Data represent time (sec) on the rotarod after injection of ethanol (2.0 g/kg). A. Males (n = 5–7 per genotype; F1,16 = 10, p<0.01, dependence on genotype; F9, 144 = 163, p<0.001, dependence on time; F9,144 = 6.8, p<0.001, genotype x time interaction). B. Females (n = 6–8 per genotype; F1,12 = 30.5, p<0.001, dependence on genotype; F7,84 = 124, p<0.001, dependence on time; F7,84 = 11.6, p<0.001, genotype x time interaction). Data represent mean ± S.E.M. Data were analyzed by two-way ANOVA with Bonferroni post hoc test (p<0.05, **p<0.001 vs. wild type genotype for each time point). ρ1 (−/−) = ρ1 null mice; (+/+) = wild type mice.
	Figure 6. Ethanol reduced anxiety-related behavior equally in the elevated plus-maze in wild type and ρ1 null mice.A. Percent of time in open arms. B. Percent of entries into the open arms. C. Number of entries into the closed arms. Data from females and males were combined since there were no gender differences. Values represent mean ± S.E.M. Data were analyzed by two-way ANOVA with Bonferroni post hoc test. p<0.05, *p<0.01 vs. saline group of corresponding genotype (n = 13–19 per group). ρ1 (−/−) = ρ1 null mice; (+/+) = wild type mice; EtOH = ethanol.
	Figure 7. Effect of ethanol on motor activity after pre-habituation.A. Males (n = 12–20 per genotype; F1,30 = 4.8, p<0.05 main effect of genotype; F2,60 = 13.8, p<0.001 main effect of treatment; no genotype x treatment interaction). B. Females (n = 13–18 per genotype; F2,56 = 18.4, p<0.001 main effect of treatment; no dependence on genotype or genotype x treatment interaction). Values represent mean ± S.E.M. Data were analyzed by two-way ANOVA with repeated measures with Bonferroni post hoc test (#p<0.05 vs. response of another genotype for the same condition). Effect of ethanol within each genotype was also analyzed by one-way ANOVA with repeated measures with Dunnett's post hoc test (p<0.05, *p<0.01 vs. saline response of corresponding genotype). ρ1 (−/−) = ρ1 null mice; (+/+) = wild type mice; EtOH = ethanol.
	Figure 8. Effect of ρ1/ρ2 antagonists on ethanol (3.4 g/kg)-induced LORR in wild type and ρ1 (−/−) mice.A. Wild type male mice. (n = 9–11; F2,27 = 17.9, p<0.001). B. Wild type female mice. (n = 9–10; F2,26 = 25, p<0.001). C. ρ1 (−/−) male mice. (n = 7–11; F2,25 = 113, p<0.001). D. ρ1 (−/−) female mice. (n = 9–10; F2,27 = 20.7, p<0.001). p<0.05, p<0.01, **p<0.001 vs. saline; ##p<0.01, ###p<0.001 (S)-ACPBPA vs. (R)-ACPBPA). Values represent mean ± S.E.M. Data were analyzed by one-way ANOVA with Bonferroni post hoc test. ρ1 (−/−) = ρ1 null mice; (+/+) = wild type mice; LORR = loss of righting reflex.
	Figure 9. Effect of ρ1/ρ2 antagonists on ketamine (150 mg/kg)-induced LORR in wild type and ρ1 (−/−) mice.A. Wild type male mice. (n = 9–11; F2,12 = 77.5, p<0.001). B. Wild type female mice. (n = 9–10; F2,12 = 105, p<0.001). C. ρ1 (−/−) male mice. (n = 7–11; F2,12 = 79.3, p<0.001). D. ρ1 (−/−) female mice. (n = 9–10; F2,12 = 27.7, p<0.001). p<0.01, *p<0.001 vs. saline; ###p<0.001 (S)-ACPBPA vs. (R)-ACPBPA). Values represent mean ± S.E.M. Data were analyzed by one-way ANOVA with Bonferroni post hoc test. ρ1 (−/−) = ρ1 null mice; (+/+) = wild type mice; LORR = loss of righting reflex.
	Figure 10. Effect of ρ1/ρ2 antagonists on recovery from ethanol (2.0 g/kg)-induced motor incoordination in wild type and ρ1 (−/−) mice.A. Wild type male mice, n = 6. (S)-ACPBPA (F1,10 = 43.3, p<0.001, effect of treatment; F10,100 = 107, p<0.001, effect of time; F10,100 = 17.1, p<0.001, treatment x time interaction). (R)-ACPBPA (F10,100 = 120, p<0.001, effect of time; no effect of treatment or treatment x time interaction). B. Wild type female mice, n = 6. (S)-ACPBPA (F1,10 = 69, p<0.001, effect of treatment; F9,90 = 196, p<0.001, effect of time; F9,90 = 24, p<0.001, treatment x time interaction). (R)-ACPBPA (F9,90 = 181, p<0.001, effect of time; no effect of treatment or treatment x time interaction). C. ρ1 (−/−) male mice, n = 4–6. (S)-ACPBPA (F10,80 = 67.7, p<0.001, effect of time; no effect of treatment or treatment x time interaction). (R)-ACPBPA (F1,9 = 31.5, p<0.001, effect of treatment; F10,90 = 102, p<0.001, effect of time; F10,90 = 13.4, p<0.001, treatment x time interaction). D. ρ1 (−/−) female mice, n = 6. (S)-ACPBPA (F8,80 = 126, p<0.001, effect of time; no effect of treatment or treatment x time interaction). (R)-ACPBPA (F1,10 = 24.1, p<0.001, effect of treatment; F8,80 = 101, p<0.001, effect of time; F8,80 = 8.1, p<0.001, treatment x time interaction). Values represent mean ± S.E.M. Data were analyzed by two-way ANOVA with repeated measures with Bonferroni post hoc test vs. corresponding saline-injected mice. ρ1 (−/−) = ρ1 null mice; (+/+) = wild type mice.
	Figure 11. GABA sensitivity and ethanol modulation of currents produced by human ρ1 or ρ2 recombinant receptors in oocytes.A. GABA concentration-response curve for ρ1 (n = 5) and ρ2 (n = 6) GABAA receptors. B. Ethanol modulation of EC50 GABA-mediated currents for ρ1 (n = 4–5) and ρ2 (n = 3–9) GABAA receptors (no significant difference, two-way ANOVA).
	Figure 1. Voluntary ethanol consumption was reduced in ρ1 (−/−) male mice in 24-hour two-bottle choice paradigm.A. Ethanol consumption (g/kg/24 hours) in males. (F1,18 = 7.1, p<0.05, main effect of genotype; F4,728 = 36.2, main effect of concentration, p<0.001; no genotype x concentration interaction). B. Ethanol consumption (g/kg/24 hours) in females. (F4,68 = 56.2, p<0.001, main effect of concentration; no main effect of genotype or genotype x concentration interaction). C. Preference for ethanol in males. (F1,18 = 7.1, p<0.05, main effect of genotype; F4,72 = 7.8, p<0.001, main effect of concentration; no genotype x concentration interaction). D. Preference for ethanol in females. (F4,68 = 10.6, p<0.001, main effect of concentration; no main effect of genotype or genotype x concentration interaction). E. Total fluid intake (g/kg/24 hours) in males. (F5,90 = 29.9, p<0.001, main effect of concentration; no main effect of genotype or genotype x concentration interaction). F. Total fluid intake (g/kg/24 hours) in females. (F5,75 = 23.8, p<0.001, main effect of concentration; no main effect of genotype or genotype x concentration interaction; n = 9–10 for both genotypes and sexes). Data were analyzed by two-way ANOVA. ρ1 (−/−) = ρ1 null mice; (+/+) = wild type mice; EtOH = ethanol.