Fig. 6. Excitation increases acquisition of the neuronal phenotype and neuronal maturation of NPCs. Cells were induced to differentiate by withdrawal of bFGF in the presence of NMDA or MK801 for 3-5 days, followed by either dual immunocytochemistry or western blotting. (A) Diagram illustrating the experimental procedure. (B,C) Immunolabeling for Tuj1 (red). (B) Tuj1⁺ cells were counted and numbers are expressed as the change in percentage compared with DAPI-positive cells in four experiments. (D) Dual-label immunocytochemistry for MAP2⁺ (green) and GFAP⁺ (red) cells 5 days after inducing differentiation. (E) Western blots showing that NMDA increased the expression of Tuj1 and MAP2 in differentiating NPCs and had little effect on the expression of the astroglial marker GFAP. (F) Immunocytochemical analysis showing that NMDA increased the number of neurons with axonal arborizations. Asterisks indicate soma; arrows indicate branch points. (G) The number of axons exiting the soma (1' axon) and axonal branches arborizing from 1' axons (2' axons) were counted in neurons immunostained for Tuj1 (n=100 cells per treatment). (H) Synaptic varicosities (arrows) were increased by NMDA treatment. Inset, enlargement of the field indicated by the box. (I) Representative images from Ca²⁺ imaging. Differentiated cells (8 DIV) were treated as described for Fig. 2A. The mean values of all loaded cells are shown (n=10 for KCl; n=16 for NMDA). Images show a field of cells at time points corresponding to 1 and 2. MK, MK801. ^*P<0.05, ^***P<0.001. Bars, 50 µm (B,C), 20 µm (F), 5 µm(H).