Proapoptotic BH3-only protein Bim is essential for NMDA-mediated excitotoxic apoptosis. (A) Neocortical neurons were exposed to 100 µM NMDA/10 µM Gly or sham conditions for 5 min and allowed to recover for 24 h. Nuclear morphology was assessed by Hoechst staining. (B) Neocortical neurons were treated as described in A and allowed to recover for the indicated times. The expression of the BH3-only proteins Bim, Bid, and PUMA was analyzed by Western blotting. Probing for β-actin served as loading control. (C–E) Mouse neocortical neurons from bim^−/− (C), puma^−/− (D), or bid^−/− (E) mice and WT controls were treated with 100 µM NMDA or sham conditions for 5 min and scored for pyknotic nuclei 24 h after excitation. Three subfields containing ∼300 neurons each were captured and quantified per well. Data represent means ± SEM from n = 4 cultures. This experiment was repeated three times with similar results. *, P < 0.05 compared with NMDA-treated WT controls. (F) Representative images of OHSCs treated with 50 µM NMDA or exposed to sham conditions for 30 min and allowed to recover for 24 h. Cell death was assessed by PI staining. (G) Quantification of cell death. OHSCs were treated as described in F, and cell death was assessed by PI staining. Experiments were performed in triplicate in three separate cultures for each strain of mice. Mean ± SEM is shown. *, P < 0.05 compared with NMDA-treated WT controls (ANOVA post-hoc Tukey). Bars: (A) 10 µm; (F) 500 µm.