Figure 2.

A–F, Immunocytochemical staining for the NG2 (C) and O1 antigen (F) were used after confocal calcium imaging (A, B and D, E) to determine the developmental stage at which OLs responded to high K⁺. Four selected OLs in these microscope fields (a–d) responded to high K⁺. Examination of the same field after NG2 (C) and O1 (F) staining indicated that OLs at NG2⁺ stage responded to high K⁺ with large increases in intracellular Ca²⁺, whereas O1⁺ cells displayed small Ca²⁺ uptake. Intracellular Ca²⁺ in these selected cells are plotted with respect to the time of stimulation in G. H, Pure OPCs were cultured for 2 d in vitro (1 and 2 DIV) in defined culture media plus PDGF (10 ng/ml) and bFGF (10 ng/ml). Then the medium was changed and the cells were cultured in a mitogen-free medium (mN2) for another 2 d (3 and 4 DIV). I, Pure OPCs were cultured for 4 DIV in defined culture media plus PDGF (10 ng/ml). The graphs shows the average Ca²⁺ influx amplitude after high K⁺ treatment in each experimental condition (n > 200 cells for each condition). J, Immunocytochemical staining for PDGFrα, NG2, O4, O1, and MBP were used after confocal calcium imaging at 2 DIV. The graphs shows the average Ca²⁺ influx amplitude calculated from 50 responding cells for each OL marker, expressed as percentage of change of the emission intensities. Values are expressed as mean ± SEM of at least four independent experiments. Scale bars: C, 20 μm; F, 40 μm.