Figure 5. bok-deficient neurons fail to recover their mitochondrial membrane potential in response to NMDA. WT and bok−/− cortical neurons, preloaded with TMRM (20 nm) and Fluo-4AM (3 μm), were exposed to NMDA (100 μm/5 min NMDA) and monitored by confocal microscopy (LSM 510). A, B, Representative TMRM traces measuring alterations in mitochondrial membrane potential (Δψm) of NMDA-treated WT and bok−/− cortical neurons. WT neurons showed a Δψm repolarization after the initial NMDA excitation (A). bok−/− neurons showed an incomplete Δψm repolarization to basal levels even at later times after excitation (B). C, TMRM fluorescence in WT and bok−/− cortical neurons plated on Willco dishes was monitored over time (before, during, and after NMDA excitation). DIC and TMRM fluorescent images were chosen at selected time points (baseline, Δψm depolarization, 1 h, and 3.5 h) from a representative experiment. D, E, Analysis of baseline levels of Δψm (D) and average of TMRM fluorescence in WT (n = 178) and bok−/− (n = 246) neurons during and after NMDA excitation are represented. A significant increase in the whole-cell TMRM fluorescence of the WT neurons was identified within a 1–2 h period after NMDA excitation. In contrast, bok gene deletion did not produce a considerable repolarization of the Δψm. Means ± SEMs are shown from at least three independent experiments for each genotype. *p ≤ 0.05. p values are the following: p = 0.015 (60 min), p = 0.007 (90 min), p = 0.00008 (120 min) versus Δψm WT cultures (ANOVA, post hoc Tukey's test). #p ≤ 0.05. p values are the following: p = 0.0055 (60 min), p = 0.00002 (90 min), p = 0.00004 (120 min) versus WT cultures (ANOVA, post hoc Tukey's test). F, Whole-cell TMRM fluorescence in NMDA-treated WT (n = 178) and bok−/− (n = 246) neurons at the 120 min time point dividing neurons undergoing cell death from neurons that survive for >24 h after NMDA excitation. *p ≤ 0.05. p values are the following: p = 3.11E-13 (dead, WT), p = 0.0000001 (alive, bok−/−), p = 2.59E-13 (dead, bok−/−) versus alive neuron WT cultures (ANOVA, post hoc Tukey's test). #p ≤ 0.05. p = 1.16E-07 (dead, bok−/−) versus alive neuron bok−/− cultures (ANOVA, post hoc Tukey's test).