glial cells (Fig. 8, A and B), which is consistent with the microglia proportion in the human brain (61). We first analyzed synapse formation after a 7-day intermittent ethanol exposure with 75 mM ethanol. After IHC staining with the presynaptic marker synapsin and the neuronal marker MAP2, we used Intellicount (62) to quantify the synaptic density per cell. As a control, we also included iN cultures (on mouse glia) without microglia. We found that synaptic density was increased in human iN cultures after intermittent ethanol exposure (Fig. 8, C and D). While adding low-PRS microglial cells had no substantial impact on the synaptic density, high-PRS microglial cells reversed the increase of synapse number after intermittent ethanol exposure (Fig. 8, C and D). To further validate the impact of high-PRS microglial cells on synaptic formation/function, we conducted patch-clamp electrophysiology on iNs with or without microglial cells. Consistent with the findings of the synaptic density analyses, after intermittent ethanol exposure, we found increased frequencies of miniature excitatory postsynaptic currents in iNs without human microglia or with low-PRS microglial cells but not in high–PRS-iN cocultures (Fig. 8, E and F).
