To investigate the acute and chronic effects of alcohol application on stem-cell derived human neurons carrying MOR N40D variants, induced neuronal (iN) cell technology (Pang et al., 2011; Yang et al., 2017; Halikere et al., 2019) was used to prepare GABAergic iN cultures from isogenic iPS cell lines homozygous for either MOR N40 or MOR D40 (Halikere et al., 2019) (Fig. 1A). The pluripotency of the source iPS cell lines was confirmed by co-localized immunocytochemistry (ICC) for OCT4 and Tra-1–60 (Fig. 1B). Expression of lentivirus-encoded, GABAergic lineage-specific transcription factors Ascl1 and Dlx2 (Yang et al., 2017) was induced with doxycycline. Lentiviral constructs carrying Ascl1 and Dlx2 contain the selectable antibiotic resistance cassettes puromycin and hygromycin, respectively (Yang et al., 2017). Thus, infected cells were selected for reprogrammed neurons from uninfected iPS. This protocol generated nearly 100% GABAergic neurons with a high degree of neuronal morphology, expression of neuron-specific markers (MAP2 and TuJ 1, not shown), and evidence of synapse formation (Synapsin 1, Fig. 1C). To confirm the identity of the AD-iNs, we performed immunolabeling with antibodies specific for GAD67 (a
