Fig. 4.

Effect of PI 3-kinase inhibitors on the anti-apoptotic action of NMDA, BDNF, or IGF-1. Cerebellar granule neurons were prepared and grown in medium containing 5 mmKCl, as described in Materials and Methods. On day 4 in vitro, cells were pretreated with vehicle or one of the PI 3-kinase inhibitors, wortmannin (100 nm; A) or LY294002 (10 μm; B), 5 min before treatment with NMDA (100 μm), BDNF (100 ng/ml), or IGF-1 (50 ng/ml). Wortmannin was replenished after 6 hr. Twelve hours after addition of the protective agents, apoptosis was assessed using the ApopTag kit, as described in Materials and Methods. The number of apoptotic cells is expressed as a percent of total cells. Values represent the mean of four to six observations in two separate experiments. A, Kruskal–Wallis ANOVA revealed a significant effect of treatment (H = 39.1; df = 7; p < 0.001). Post hoccomparisons showed that NMDA, BDNF, and IGF-1 significantly inhibited apoptotic cell death (*p < 0.001 compared with the 5 mm KCl group in the absence of wortmannin), wortmannin significantly decreased these effects (**p < 0.001 compared with the appropriate treatment in the absence of wortmannin), and wortmannin alone increased apoptosis (*p < 0.001 compared with the 5 mm KCl group in the absence of wortmannin). B, Kruskal–Wallis ANOVA revealed a significant effect of treatment (H = 34.9; df = 7; p < 0.001). Post hoccomparisons showed that NMDA, BDNF, and IGF-1 significantly inhibited apoptotic cell death (*p < 0.001 compared with the 5 mm KCl group in the absence of LY294002) and that LY294002 significantly reversed these effects (**p< 0.001 compared with the appropriate treatment in the absence of LY294002).