One technical complication of the CIE experiment is worth noting: for better control of batch effects in vitro cultures, we routinely culture treatment (e.g., CIE) and control neurons in the same plate and in the same incubator. We measured the half-life of ethanol (40 and 75 mM) along a period of 24 h in the cell culture incubator (Fig. 5C). However, we need to be cognizant of the ability of ethanol to evaporate and “contaminate” the untreated control, which we observed (Fig. 5B). In this study, our focus was on the genotype (AA vs. GG OPRM1), so differences in human iNs in responding to CIE, including the evaporation of ethanol into the control condition, were the same between N40 and D40 iN conditions. Therefore, we conclude that it had minimal effect on interpretation in this study.
