Increased expression of Pax6 and glutamatergic neuronal differentiation by ethanol treatment. NPCs were treated with ethanol and Western blot and immunocytochemistry were performed to determine the expression of Pax6 and downstream transcription factors (A) as well as glutamatergic and GABAergic neuronal subtype markers (B). (C) Immunocytochemical staining of GABAergic marker GAD67 and a regulator of excitatory neuronal differentiation, NeuroD, in NPCs treated with ethanol. (D) Triple immunocytochemical staining of neuronal marker Tuj1 (red) and vGluT1 (blue), a marker for glutamatergic neuron along with BrdU (green) staining, a marker for proliferated cells. Most of the vGluT1-positive cells were co-localized with BrdU staining.