Bim is required for DCD, mitochondrial depolarization, and AIF translocation during excitotoxic apoptosis. Cortical neurons cultured on Wilco dishes were loaded with Fluo-4 AM and TMRM for 30 min at 37°C before being mounted on a thermostatic chamber of a confocal microscope. The neurons were exposed to 100 µM NMDA/10 µM Gly or sham conditions for 5 min, after which neuronal injury was monitored over a 24-h period. (A and B) Individual traces of initial Ca²⁺ responses in WT (A) and bim^−/− (B) neurons after NMDA receptor activation. (C) Quantification of peak initial Fluo-4 fluorescence in WT (n = 91) and bim^−/− (n = 70) neurons revealed no significant differences between genotypes. (D) Analysis of the frequency of WT (n = 90) and bim^−/− (n = 70) neurons undergoing DCD. *, P < 0.05 compared with WT-treated neurons. (E) Individual traces of TMRM fluorescence in WT and bim^−/− neurons after NMDA treatment. (F) Analysis of the frequency of WT (n = 91) and bim^−/− (n = 70) neurons undergoing loss of TMRM fluorescence. *, P < 0.05 compared with WT-treated neurons. (C, D, and F) Mean ± SEM is shown. (G) Representative images of AIF immunofluorescence in NMDA- or sham-treated cultures. (H) Quantification of the number of neurons with nuclear-localized AIF as analyzed by immunofluorescence. Mouse cortical neurons from WT and bim^−/− mice were treated with 100 µM NMDA/10 µM Gly or sham conditions for 5 min and allowed to recover for 16 h. Six subfields containing ∼50 neurons each were captured and quantified for AIF release per culture. Data represent mean ± SEM from n = 4 cultures. The experiment was repeated twice with similar results. *, P < 0.05 compared with NMDA-treated WT neurons (ANOVA post-hoc Tukey). Bar, 10 µm.