Increased ethanol drinking in "humanized" mice expressing the mu opioid receptor A118G polymorphism are mediated through sex-specific mechanisms

Abstract

The A118G single nucleotide polymorphism (SNP) of the mu-opioid receptor gene (Oprm1) has been implicated in mediating the rewarding effects of alcohol. Clinical and preclinical studies suggest that the G allele may confer a genetic vulnerability to alcohol dependence, though it remains unknown whether these effects are sex-specific. We used male and female mice homozygous for the "humanized" 118AA or 118GG alleles to determine whether the A118G SNP potentiates ethanol consumption in a sex-specific manner in both the two-bottle choice and drinking-in-the-dark (DID) paradigms. Mice were also assessed for differences in naltrexone sensitivity, ethanol reward assessed via conditioned place preference (CPP), and sensitivity to the sedative/ataxic effects of ethanol using the rota-rod and loss of righting reflex (LORR) assays. We found that male and female 118GG mice drank significantly more ethanol than 118AA littermates using a continuous access, two-bottle choice paradigm. In the limited-access DID drinking model, (i) female (but not male) 118GG mice consumed more ethanol than 118AA mice and (ii) naltrexone pretreatment was equally efficacious at attenuating ethanol intake in both 118AA and 118GG female mice while having no effect in males. Male and female 118GG and female 118AA mice developed a robust conditioned place preference (CPP) for ethanol. Female 118GG mice displayed less sensitivity to the sedative/ataxic effects of ethanol compared to female 118AA mice on both the rota-rod and the LORR assays while male mice did not differ in their responses on either assay. Our findings suggest that increased ethanol consumption in male 118GG mice may be due to increased ethanol reward, while increased drinking in female 118GG mice might be due to decreased sensitivity to the sedative/ataxic effects of ethanol. Collectively, these data might be used to help identify sex-specific pharmacotherapies to combat alcohol use disorders.

Keywords: A118G; Ethanol; Humanized mice; Mu-opioid receptor; Sex differences.

Figure 1. Male and female 118GG mice show increased ethanol consumption in the two-bottle choice assay

Ethanol intake (g/kg) at each ethanol concentration is shown for both (A) female and (B) male 118GG (GG; open squares) and 118AA (AA; filled circles) mice. Male 118GG (N=5) mice consumed significantly more ethanol (g/kg) than male 118AA (N=10) mice at 6%, 9%, and 12% ethanol. Female 118GG (N=19) mice also drank significantly more ethanol (g/kg) than female 118AA (N=21) control mice at 9%, 12%, and 15%. Data were analyzed using mean ethanol intakes averaged across each of the six days of drinking in two-way mixed ANOVAs. Error bars represent the standard error of the mean (SEM). * = p<0.05; ** = p<0.01.

Figure 2. 118AA and 118GG female mice are equally sensitivity to 1 mg/kg of naltrexone in a two-hour binge-drinking paradigm

Ethanol intake (g/kg) was measured using the two-hour limited-access drinking-in-the-dark (DID) paradigm in female 118AA (AA; N=11) and 118GG (GG; N=10) and in male 118AA (N=8) and 118GG (N=12) mice. White bars represent AA mice and grey bars indicate GG mice. Following 4 days of baseline DID drinking, mice were challenged with 1 mg/kg naltrexone (patterned bars) or saline (open bars). Female GG mice drank significantly more ethanol than AA mice following pretreatment with saline while there was no genotype difference in DID drinking for male mice. Naltrexone significantly attenuated ethanol intake (g/kg) in\\ both female AA and GG mice but not in any of the male mice. Data were analyzed using two-way mixed ANOVAs; error bars represent the standard error of the mean (SEM). # = p<0.05; *** = p<0.001.

Figure 3. Female 118AA and 118GG and Male 118GG mice develop a conditioned place preference (CPP) to the rewarding effects of 2 g/kg of ethanol

The rewarding effects of 2 g/kg of ethanol were assessed in male and female mice using CPP. The amount of time (seconds) mice spent on the ethanol-paired side both prior to (pre; open bars) and after (post; black bars) ethanol conditioning is shown. Female 118AA (AA; N=8) and 118GG (GG; N=8) and male 118GG (N=8) mice spent more time (in seconds) in the ethanol vs. saline-paired conditioning chamber. In contrast, male 118AA (N=8) mice failed to develop an ethanol CPP. Error bars represent the standard error of the mean (SEM). * = p < 0.001 between pre and post conditioning sessions.

Figure 4. Female 118GG mice are less sensitive to the ataxic effects of 2 g/kg of ethanol on the rota-rod than female 118AA mice

The amount of time (in seconds) mice spent on the rota-rod following treatment with 2 g/kg ethanol was assessed in female 118AA (AA; N=9) and 118GG (GG; N=9) and male 118AA (N=6) and 118GG (N=7) mice. Female 118GG mice spent more time on the rota-rod compared to female 118AA mice while there was no difference in time spent on the rota-rod for male 118AA and 118GG mice. Error bars represent the standard error of the mean (SEM). * = p<0.05.

Figure 5. Female 118GG mice are less sensitive to the sedative effects of 4 g/kg of ethanol than female 118AA mice

The amount of time (minutes) to regain the loss of righting reflex (LORR) was assessed following an injection of 4 g/kg of ethanol. Female 118AA (AA; N=9) mice took longer to regain their righting reflex (RR) than female 118GG (GG; N=9) mice. However, there was no difference in the amount of time it took male 118AA (N=5) and male 118GG (N=9) mice to regain their RR. Error bars represent the standard error of the mean (SEM). * = p<0.05.