Slow time course of presynaptic mitochondrial [Ca2+] transients. A, DIC image of the presynaptic terminal. Scale bar, 15 μm. B,Projected stack of fura-FF images focused through the entire terminal.C, Projected stack of rhod-2 images showing that fluorescence was concentrated in the terminal but was absent from the pipette and axon regions. D,[Ca2+]mito rose rapidly on stimulation (as in Fig. 1C, black trace) but decayed slowly. Four groups of four stimuli (the same protocol as in Fig. 1Crepeated 4 times 150 msec apart, gray trace) had a similar time course and amplitude, indicating saturation of the dye. Mitochondrial depolarization (perfusion of 25 μm rotenone and 1 μm FCCP in the presence of 5 μg/ml oligomycin) completely blocked calcium accumulation by the mitochondria after one group of four stimuli (bottom black trace).E, Rhod-FF fluorescence signals were not saturated by four groups of four stimuli (gray trace) compared with one group of stimuli (black trace). Thetraces in D and E are shown normalized to the change in fluorescence after one group of stimuli. F, Summary data show that mitochondrial calcium uptake was inhibited by mitochondrial depolarization and 10 μm TPP+ but not 1 μmthapsigargin. Mitochondrial calcium sequestration saturated rhod-2 (Kd, 570 nm) but not rhod-FF (Kd, 19 μm).Asterisks indicate statistical significance (p < 0.05).
