in vivo experimental animal models45–48. In our neuronal culture model, alcohol toxicity assessments by cell viability and apoptosis analysis showed a robust toxicity in neural progenitor cells with only modest effect in neurosphere cultures. This could be due to the nature of these cultures been growing in suspension as spheres rather than monolayers on the surface of the plates as progenitors do. As one would expect, EtOH may not diffuse efficiently and reach the inner layers of neurospheres due to the growth characteristics of these cells. In fact, immunocytochemical analysis of caspase-3 activation suggest that while the outer layer of neurospheres showed higher cleaved caspase-3 activation, inner layers had fewer response to apoptosis activation. Another challenge in this type of studies is the maintenance of same number of cells from different cell types in culture during the duration of assays. While immature and mature neurons are post mitotic, neurospheres and progenitors grow and replicate. We believe that some of the differences in the levels of Mcl-1S alternative splicing and sensitivity to the EtOH across cell types could be explained by differences in cell characteristics.
