To gain insight into the relationship between AUD PRS and ethanol exposure in microglia, we next investigated whether ethanol exposure causes differential cellular responses in human high-PRS (n = 5) versus low-PRS (n = 6) microglial cells (table S1). We used intermittent ethanol exposure for 7 days, with ethanol replenished daily, as reported previously (46). It has been reported that ethanol activates microglial cells in rodents (47, 48). To investigate if ethanol activates human microglia in culture, we assessed the expression of CD68, a transmembrane protein found in plasma, lysosomal, and endosomal membranes that indicates microglial activation (49). In both high-PRS and low-PRS human microglial cells, we observed elevated expression of CD68 after ethanol exposure (Fig. 3, A and B). The level of ethanol-induced CD68 expression was similar between high-PRS and low-PRS microglial cells (Fig. 3, A and B). Because activated microglia undergo morphological changes from a “ramified” state, a highly branched and elongated appearance, to an “amoeboid” state, with retracted processes and a more rounded shape (50), we used “fractal analysis” in ImageJ at the single-cell level to evaluate
