A genetic determinant of the striatal dopamine response to alcohol in men

Abstract

Excessive alcohol use, a major cause of morbidity and mortality, is less well understood than other addictive disorders. Dopamine release in ventral striatum is a common element of drug reward, but alcohol has an unusually complex pharmacology, and humans vary greatly in their alcohol responses. This variation is related to genetic susceptibility for alcoholism, which contributes more than half of alcoholism risk. Here, we report that a functional OPRM1 A118G polymorphism is a major determinant of striatal dopamine responses to alcohol. Social drinkers recruited based on OPRM1 genotype were challenged in separate sessions with alcohol and placebo under pharmacokinetically controlled conditions, and examined for striatal dopamine release using positron emission tomography and [(11)C]-raclopride displacement. A striatal dopamine response to alcohol was restricted to carriers of the minor 118G allele. To directly establish the causal role of OPRM1 A118G variation, we generated two humanized mouse lines, carrying the respective human sequence variant. Brain microdialysis showed a fourfold greater peak dopamine response to an alcohol challenge in h/mOPRM1-118GG than in h/mOPRM1-118AA mice. OPRM1 A118G variation is a genetic determinant of dopamine responses to alcohol, a mechanism by which it likely modulates alcohol reward.

Fig. 1

Human PET study. Axial view of group maps showing change of [¹¹C]-raclopride binding potential (ΔBP; nCi/cc) between placebo and alcohol sessions in (A) AA individuals and (B) AG individuals. Color bars indicate corresponding ΔBP values. Reduction in raclopride binding is attributed to competition with dopamine released by the alcohol challenge; thus, a negative ΔBP indicates an increase in endogenous dopamine release. (C) Relative change in binding potential (%ΔBP) for [¹¹C]-raclopride between alcohol and placebo sessions in four striatal regions of interest. Data are least square means (±SEM). Main genotype effect: p=0.006; *p<0.05 on post hoc tests within individual regions. AVS = anterior ventral striatum, PVS = posterior ventral striatum. (D) Schematic of PET sessions, and blood alcohol concentration profiles over time during the alcohol session (mean±SEM). There was no significant difference between genotypes (F[1,24]=0.51, p=0.48).

Fig. 2

Targeting strategy and functional confirmation for humanized OPRM1 mouse lines. (A) Alignment of human (hOPRM1), mouse (mOPRM1) and humanized (h/mOPRM1) protein sequences. Black triangles indicate exon boundaries. Red triangle points to site of the N40D substitution, corresponding to A118G allelic variation. Color-coding of background indicates degree of conservation, with red indicating full human – mouse homology, and blue non-conservative substitution. As can be seen, replacement of mouse exon 1 with the corresponding human sequence yields a receptor that is highly homologous to the human protein. (B) Exon 1 of the murine OPRM1 locus including additional 400 bp of intron 1 was replaced by the corresponding human sequence, containing either A or G in position 118. Blue and red colors represent mouse and human sequences, respectively. Boxes: respective exons of the OPRM1 coding region. Hatched region: 5’-UTR. Triangle: frt site for flox recombinase. Neo: neomycin selection marker. Arrows point to position of the human and mouse specific PCR primers. (C) PCR based identification of the human (Hu), mouse (Mu) and heterozygote (Het) genotypes. M: Marker lane. (D) [³H]-DAMGO binding to respective humanized receptor transiently expressed in CHO cells. Data are means (±SEM) of triplicates, and mean (±SEM) IC₅₀ values for the respective genotypes are indicated. Non-linear curve-fitting established that no significant genotypes differences were present (E – F) Concentration-response curves for DAMGO and β-endorphin (β-End) in trigeminal ganglion (TG) neurons isolated from h/mOPRM1 118AA or 118GG mice. Data points represent the mean (±SEM) of the agonist-mediated Ca²⁺ current inhibition. Mean EC₅₀ values (±SEM) for the respective genotypes are indicated. Similar to the binding experiment, no genotype differences were found.

Fig. 3

Striatal DA response to alcohol (2g/kg) in the humanized mouse lines, determined by brain microdialysis. (A) Individual probe placements in ventral striatum / Nucleus Accumbens. (B) Dialysate DA levels in h/mOPRM1-118AA and h/mOPRM1-118GG mice. After confirmation of no group differences in baseline concentrations, the data were converted to the percent change from the average baseline concentration obtained prior to pretreatment. Alcohol was given at time point 0. Differential response was indicated by a genotype x time interaction (p<0.005); differences at individual data points as determined by post hoc test are indicated by *p<0.05 (C) In contrast to the DA response, the 5-HT response to the alcohol dose did not differ between genotypes.