Although MTT assay can measure cytotoxicity (loss of viable cells) of cultured cells by assessing cell metabolic activity, it may also potentially suggest cytostatic activity (shift from proliferation to quiescence) of cells induced by EtOH exposure. To gain more insight into EtOH-mediated cytotoxicity in different lineages of neuronal cells, hNSPs, hNPCs, immature neurons, and mature neurons were also plated in chamber slides, treated with EtOH (50 mM) for 24 h, fixed and processed by immunocytochemistry for cleaved caspase-3, an apoptosis marker. As shown in Fig. 3a, e, EtOH exposure had a significant impact on the morphology of hNPCs with a robust cleaved caspase-3 induction. Similarly, neural progenitors (hNPCs) derived from hNSPs (Fig. 3b, f) and immature neurons (Fig. 3c, g) were also very sensitive to EtOH treatment with an extensive cleaved caspase-3 activation. Interestingly, mature neuronal cultures had no visible sign of cellular toxicity and cleaved caspase-3 activation (Fig. 3d, h). These results suggest that while neural progenitors and immature neurons are highly sensitive, mature neurons show resistance to the neurotoxic effects of EtOH.
