To confirm that both KCNJ6 haplotype and ethanol exposure affect GIRK2 expression, we evaluated GIRK2 immunocytochemistry and KCNJ6 mRNA in neurons using fluorescent in situ hybridization (FISH). GIRK2 increased following IEE (p = 1.0 × 10−20, Fig. 5G, H). By probing KCNJ6 fluorescent puncta colocalized with MAP2 immunocytochemistry, we confirmed that neurons from AF individuals had lower levels of KCNJ6 mRNA compared to UN neurons (Fig. 5J.a, p = 8.2 × 10−3). We also found that the proportion of MAP2+ neurons expressing KCNJ6 was reduced in AF compared with UN (50.1% vs. 64.7%, Fig. 5J.b), and the AF neurons had a greater proportion of cells with low levels of expression (Fig. 5J.c) with no detectable High or Very High (>9 puncta/cell) expression. Differences in expression were found by haplotype in somatic regions of neurons (Fig. 5J.f, p = 5.5 × 10−3) but not in non-somatic processes (Fig. 5J.g, p = 0.23). Ethanol increased KCNJ6 mRNA levels in both groups (Fig. 5J.a–e), eliminating the difference between genotypes. Interestingly, we also observed that the proportions of AF neurons expressing KCNJ6 was increased
