Fig. 1.

Concentration and time dependence of the anti-apoptotic effect of NMDA. A, Cerebellar granule neurons were maintained in medium containing 5 mm KCl for 4 d in vitro and were then treated with the indicated concentrations of NMDA for 24 hr, as described in Materials and Methods. Apoptosis was assessed with the ApopTag kit on day 5in vitro. Results are expressed as the percent decrease in apoptosis produced by NMDA. In the absence of NMDA, apoptosis was detected in 41% of the cells. Values represent the mean ± SEM of 11–33 observations in four separate experiments. Kruskal–Wallis ANOVA was performed on the raw data (percent apoptotic cell death) and revealed a significant effect of NMDA (H = 46.9; df = 3; p < 0.001). Post hoccomparisons showed significant effects with 10, 30, and 100 μm NMDA compared with that with no NMDA (control).B, NMDA (100 μm) was added to the culture medium of the cerebellar granule neurons on day 4 in vitro, and at the indicated times after addition, cells were washed to remove NMDA. The cells were maintained until day 5 in vitro in conditioned medium containing 5 mm KCl. Cells were then fixed for determination of apoptosis using the ApopTag kit. Results are expressed as the number (percent) of apoptotic (fluorescein-positive) cells per total cell number (propidium iodide-labeled cells). Values represent the mean ± SEM of 12–33 observations in four separate experiments. Kruskal–Wallis ANOVA revealed a significant effect of time of exposure to NMDA (H = 74.9; df = 4; p < 0.001); *p < 0.001 compared with all other groups (post hoc comparisons).