As shown in Figure 1, undifferentiated NSCs in neurospheres were characteristically round and small with a distinct nucleus in the center of the small cytoplasm, as reported previously (Singh et al., 2009a). After a two-day culture on substrate-coated chamber slides, the neurospheres were flattened in appearance and cells in the outer layer of the neurosphere increased in size (Fig 1A). On the fourth day in culture (DIC4), noticeable zoning for a differential differentiating state was evident, as reported previously (Singh et al., 2009a). In brief, in the core of the neurosphere, the NSCs remained round and small (Fig 1A). However, in the periphery of the neurosphere, much like the undifferentiated groups, many NSCs remained round, but a subpopulation of the cells formed angular cytoplasmic expansions, some with fiber extensions. In the migrated zone (out of neurosphere), more differentiated cells grew processes, MAP2-immunostained (im), and acquired neuronal morphology with varicosities (Figure 1A); on the other hand, the OCT4-im was reduced in the peripheral area. The alcohol-treated NSC neurospheres, on the fourth day, displayed decreased migration, i.e. migrated cells traveled in smaller
