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Fig. 4. NMDA modulates the cell cycle characteristics of cultured hippocampal NPCs. (A) Flow-cytometric histograms of DNA content as indicated by fluorescence intensity (FL-2 Area) on the x-axis and number of cells after staining with propidium iodide on the y-axis. Cells were incubated for 4 days with 20 ng/ml bFGF, with or without NMDA (5 µM), thymidine (3 µM), or MK801 (5 µM) for the first 24 hours. The results shown are from a single representative experiment. (B) Cells treated with various stimuli (as described in A) were analyzed for DNA content, and the areas under the peaks were compared. The numbers of cells with DNA contents corresponding to each cell cycle phase are expressed as percentages of the total sorted events. Values are the mean ± s.e.m. of 12 independent experiments. (C) A single-pulse treatment of NMDA decreases the level of Rb and increases that of P-Rb. Quantitative RT-PCR analysis (graph) showing that Rb mRNA levels were decreased by NMDA (5 µM) treatment for 24 hours. The expression of Rb was normalized to the amount of GAPDH to calculate the relative amount of the gene transcript (n=3 per group). Expression levels at each time point are depicted relative to the expression of the control 3 days after the onset of stimulation for comparison. Representative western blots showing that the level of P-Rb^Ser780 was increased by NMDA for up to 6 days, even when NMDA stimulation was restricted to 24 hours (n=3). For comparison, blots were exposed for same duration in each antibody. AP, apoptosis; CTL, control; MK, MK801; Thy, thymidine; ^*P<0.05, ^**P<0.01.

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