Because it has been suggested that microglia interact with neurons through synaptic pruning to shape synapse formation and synaptic transmission (20, 21), we also assayed microglial phagocytosis using isolated synaptosomes of human induced neurons [iNs; induced by ectopic expression of the transcription factor neurogenin 2 (Ngn2)] (57), which contains synaptic proteins such as PSD 95 and SNAP receptor proteins (fig. S8, A and B). These synaptosomes were labeled with pH-sensitive pHrodo dye and were added to both high-PRS and low-PRS microglial cultures after a 7-day intermittent ethanol exposure at 75 mM (fig. S8C). Consistent with the results from the beads phagocytosis assay, we also observed that high-PRS microglial cells exhibited enhanced phagocytic activity toward synaptic proteins after ethanol exposure, demonstrated by higher fluorescence intensity and the proportion of microglia containing synaptic proteins (fig. S8, D and E). Notably, this enhanced phagocytic activity was reversible upon treatment with phagocytosis inhibitors such as minocycline and cytochalasin D (fig. S8, C to E).
