Abstract and summary Alcohol use disorders (AUD) are complex behaviors accompanied by substantial morbidity, mortality and societal expense. Both genetic and environmental factors contribute to AUD. Despite progress in the human genetics of AUD, especially the identification of genome-wide significant AUD genetic risk variants, the neural basis of AUD in humans is largely unknown. Over the past five years, we have provided compelling evidence that: 1) Human neuronal cells derived from induced pluripotent stem (iPS) cells can be used as a tractable model to study neuropsychiatric disorders including AUD; 2) Ethanol exposure results in an inflammasome response in human neurons; 3) Human neurons carrying OPRM1 A118G minor gene variants showed enhanced sensitivity to opioids and ethanol; 4) Ethanol causes gene expression changes in both human neurons and glial cells; and 5) Microglia-containing 3D neural cultures can be a powerful system to study neuroinflammatory microglia-neuronal interactions. These premises provide a foundation for further mechanistic studies of the genetic and molecular underpinnings of AUD in human iPS cell-derived neural models. We also identified key gaps of knowledge in utilizing human neurons as a model system to study AUD that need to be filled, particularly: 1) how ethanol affects neuro-glial interactions in a human neural context is not known; and 2) how ethanol affects neurogenesis in a 3D context is not known. Moreover, 3) because of the polygenic nature of AUD, the contribution of single gene to AUD risk is likely small and the phenotypical manifestation is strongly influenced by individual’s genetic makeup. To address these outstanding questions, we hypothesize that ethanol impairs neuronal function via affecting neuroglial interactions, which is influenced by individual polygenic risk backgrounds. We have selected 36 subjects of both sexes with either extremely high polygenic risk score (PRS, top 10%ile, n=18 AUD) or low PRS (bottom 10%ile, n=18, no AUD) to test this hypothesis. We will differentiate iPS cells derived from these subjects into both 2D and 3D (i.e. brain organoids) neuronal cells co-cultured with human astrocytes and human microglia. Upon exposure to ethanol, these neural cells are subjected to a combination of morphological, immunocytochemical, electrophysiological, live cell imaging and genomic analyses to unravel the mechanism(s) underlying the impact of ethanol, focusing on neuro-astro-microglial interactions. In a relatively large collection of human iPS cells (n=36 lines) derived from subjects with extreme AUD PRSs, we hope to unravel the convergent phenotype and gene-networks that are linked to extreme high or low AUD PRSs. The results will advance our mechanistic understanding of the pathogenic role of AUD risk gene variants and the influence of polygenic risk background in a human neural system.