Since ethanol is teratogenic, and long-term effects may only become evident with time, we investigated whether pre-exposure to ethanol affects the differentiation of NPCs into neurons. Functional, mature neurons expressing MAP2, vGlut1, and synapsin were derived from the NPCs with or without ethanol pre-exposure (Fig. 3). Fewer mature neurons, identified by MAP2 expression, were generated from ethanol pre-exposed NPCs compared to untreated NPCs (Fig. 3a and b). Moreover, synaptic density appears to be reduced in neurons generated from ethanol treated NPCs when compared to neurons derived from control NPCs (Fig. 3a–c). However, in neurons derived from control and ethanol pre-exposed NPCs exhibited both repetitive action potentials as well as synaptic responses (Fig. 3d). Quite remarkably, it appears that inflammasome markers Casp1 and NLRP3 were prominent in neurons derived from ethanol pre-exposed NPCs (Fig. 3e). These findings suggest that neuronal differentiation is compromised (lower efficiency in maturation and less synapse formation) in NPCs pre-exposed to ethanol, and that neurons maintain certain cellular memory of neuroinflammation (i.e. with or without ethanol exposure) from the NPC stage.
