Figure 6.
Transient glutamate excitation results in a hyperpolarization ΔΨm and a late apoptotic injury in cerebellar granule neurons. Cerebellar granule neurons plated on Willco dishes were loaded with 30 nm TMRM and exposed to glutamate/glycine (100 μm/10 μm) for 5 min. TMRM fluorescence was then monitored over time, and images were taken at 5 min intervals. A, Differential interference contrast (DIC) and TMRM fluorescent images were chosen at selected time points (0 min, 2 h, and 14 h after glutamate) from a representative experiment. B, Representative traces for whole-cell TMRM fluorescence in neurons during prolonged glutamate excitation. Two major subgroups are shown: traces that show a secondary collapse of ΔΨm downstream of excitation (i) and traces that show a collapse of ΔΨm within 24 h (ii). C, Whole-cell TMRM fluorescence in control neurons after 120 min and neurons 120 min after glutamate excitation. p < 0.01, difference between TMRM fluorescence for control neurons and apoptotic neurons 120 min after transient glutamate excitation; p < 0.001 difference between TMRM fluorescence for apoptotic neurons 120 min after transient excitation compared with neurons that survive for >24 h after glutamate excitation (control, n = 39; apoptotic, n = 134; live, n = 31). D, Cerebellar granule neurons were loaded with the ΔΨp-sensitive probe DiBAC2(3) (1 μm) and transiently (5 min) exposed to glutamate/glycine (100 μm/10 μm). Fluorescence recovers close to pre-exposure levels (average response dark trace) after the addition of glutamate (traces are representative of those obtained from 3 separate experiments). E, Representative traces for modeled changes in ΔΨp for neurons transiently exposed to glutamate.