Changes in cytosolic and mitochondrial [Ca2+] produced by brief trains of action potentials were measured in motor nerve terminals using a rapidly scanning confocal microscope. Cytosolic [Ca2+] was measured using ionophoretically injected Oregon Green BAPTA 5N (OG‐5N). Mitochondrial [Ca2+] was measured using rhod‐2, bath loaded as dihydrorhod‐2. In response to 100‐250 stimuli at 25‐100 Hz the average cytosolic [Ca2+] showed an initial rapid increase followed by a much slower rate of increase. Mitochondrial [Ca2+] showed no detectable increase during the first fifteen to twenty stimuli, but after this initial delay also showed an initially rapid rise followed by a slower rate of increase. The onset of the increase in mitochondrial [Ca2+] coincided with the slowing of the rate of rise of cytosolic [Ca2+]. The peak levels of cytosolic and mitochondrial [Ca2+] both increased with increasing frequencies of stimulation. When stimulation terminated, the initial rate of decay of cytosolic [Ca2+] was much more rapid than that of mitochondrial [Ca2+]. After addition of carbonyl cyanide m‐chlorophenyl hydrazone (CCCP, 1‐2 μm) to dissipate the proton electrochemical gradient across the mitochondrial membrane, cytosolic [Ca2+] rose rapidly throughout the stimulus train, reaching levels much higher than normal. CCCP inhibited the increase in mitochondrial [Ca2+]. These results suggest that mitochondrial uptake of Ca2+ contributes importantly to buffering presynaptic cytosolic [Ca2+] during normal neuromuscular transmission.

This study used fluorescent indicator dyes to measure changes in cytosolic and mitochondrial [Ca(2+)] produced by physiological stimulation of lizard motor nerve terminals. During repetitive action potential discharge at 10-50 Hz, the increase in average cytosolic [Ca(2+)] reached plateau at levels that increased with increasing stimulus frequency. This stabilization of cytosolic [Ca(2+)] was caused mainly by mitochondrial Ca(2+) uptake, because drugs that depolarize mitochondria greatly increased the stimulation-induced elevation of cytosolic [Ca(2+)], whereas blockers of other Ca(2+) clearance routes had little effect. Surprisingly, during this sustained Ca(2+) uptake the free [Ca(2+)] in the mitochondrial matrix never exceeded a plateau level of approximately 1 microM, regardless of stimulation frequency or pattern. When stimulation ceased, matrix [Ca(2+)] decreased over a slow ( approximately 10 min) time course consisting of an initial plateau followed by a return to baseline. These measurements demonstrate that sustained mitochondrial Ca(2+) uptake is not invariably accompanied by progressive elevation of matrix free [Ca(2+)]. Both the plateau of matrix free [Ca(2+)] during stimulation and its complex decay after stimulation could be accounted for by a model incorporating reversible formation of an insoluble Ca salt. This mechanism allows mitochondria to sequester large amounts of Ca(2+) while maintaining matrix free [Ca(2+)] at levels sufficient to activate Ca(2+)-dependent mitochondrial dehydrogenases, but below levels that activate the permeability transition pore.

SUMMARY Key components of vesicular neurotransmitter release, such as Ca2+ influx and membrane recycling, are affected by cytosolic pH. We measured the pH-sensitive fluorescence of Yellow Fluorescent Protein transgenically expressed in mouse motor nerve terminals, and report that Ca2+ influx elicited by action potential trains (12.5-100 Hz) evokes a biphasic pH change: a brief acidification (~13 nM average peak increase in [H+]), followed by a prolonged alkalinization (~30 nM peak decrease in [H+]) which outlasts the stimulation train. The alkalinization is selectively eliminated by blocking vesicular exocytosis with botulinum neurotoxins, and is prolonged by the endocytosis-inhibitor dynasore. Blocking H+ pumping by vesicular H+-ATPase (with folimycin or bafilomycin) suppresses stimulation-induced alkalinization and reduces endocytotic uptake of FM1-43. These results suggest that H+-ATPase, known to transfer cytosolic H+ into pre-fused vesicles, continues to extrude cytosolic H+ after being exocytotically incorporated into the plasma membrane. The resulting cytosolic alkalinization may facilitate vesicular endocytosis.

Increases in cytosolic [Ca 2ϩ ] evoked by trains of action potentials (20-100 Hz) were recorded from mouse and lizard motor nerve terminals filled with a low-affinity fluorescent indicator, Oregon Green BAPTA 5N. In mouse terminals at nearphysiological temperatures (30-38°C), trains of action potentials at 25-100 Hz elicited increases in cytosolic [Ca 2ϩ ] that stabilized at plateau levels that increased with stimulation frequency. Depolarization of mitochondria with carbonylcyanide m-chlorophenylhydrazone (CCCP) or antimycin A1 caused cytosolic [Ca 2ϩ ] to rise to much higher levels during stimulation. ] measured with fluorescent indicators increases rapidly at first but then stabilizes at a plateau level until stimulation ceases (Steunkel, 1994;Ravin et al., 1997;David et al., 1998). This stabilization of average cytosolic [Ca 2ϩ ] during continued Ca 2ϩ influx is disrupted by agents that inhibit mitochondrial Ca 2ϩ uptake (Steunkel, 1994;David et al., 1998), suggesting that mitochondrial Ca 2ϩ uptake contributes importantly to sequestration of the Ca 2ϩ loads entering stimulated nerve terminals. Ca 2ϩ uptake via the mitochondrial uniporter is driven by the large negative potential (approximately Ϫ150 to Ϫ200 mV) created by proton transport across the inner mitochondrial membrane (for review, see Gunter and Pfeiffer, 1990).For some secretory cells, additional evidence for mitochondrial Ca 2ϩ uptake has been obtained using fluorescent or luminescent indicators localized within the mitochondrial matrix. Increases in matrix [Ca 2ϩ ] evoked by depolarization and/or hormones have been demonstrated in adrenal chromaffin cells (Babcock et al., 1997;Montero et al., 2000) and lizard motor nerve terminals (David et al., 1998). Mitochondrial Ca uptake has also been demonstrated by electron probe microanalysis of total Ca in frog sympathetic ganglion neurons fast-frozen after a 45 sec bath application of 50 mM K ϩ (Pivovarova et al., 1999). Simultaneous imaging of cytosolic and mitochondrial [Ca 2ϩ ] showed that, in lizard motor nerve terminals, mitochondrial Ca 2ϩ uptake begins after as few as 25-50 action potentials delivered at 50 -100 Hz, at approximately the same time that cytosolic [Ca 2ϩ ] reaches a plateau (David et al., 1998). In this preparation, as in crayfish motor nerve terminals, adrenal chromaffin cells, and several types of neuronal somata, mitochondria have been shown to be the dominant means of sequestering moderate to large Ca 2ϩ loads (Friel and Tsien, 1994;Werth and Thayer, 1994;White and Reynolds, 1995;Herrington et al., 1996;Park et al., 1996;Tang and Zucker, 1997;David, 1999;Colegrove et al., 2000).The present study was undertaken to measure cytosolic [Ca 2ϩ ] transients evoked by physiological stimulation in mammalian (mouse) motor nerve terminals and to determine whether mitochondrial Ca 2ϩ sequestration contributes to limiting the magnitude of these transients. We demonstrate that, at temperatures near physiological (33-38°C), the elevation of average cytosolic [Ca 2ϩ ] stab...

We investigated how inhibition of mitochondrial Ca2+ uptake affects transmitter release from mouse motor terminals during brief trains of action potentials (500 at 50 Hz) in physiological bath [Ca2+]. When mitochondrial Ca2+ uptake was inhibited by depolarizing mitochondria with antimycin A1 or carbonyl cyanide m-chlorophenyl-hydrazone, the stimulation-induced increase in cytosolic [Ca2+] was greater (> 10 microM, compared to < or = 1 microM in control solution), the quantal content of the endplate potential (EPP) depressed more rapidly (approximately 84 % depression compared to approximately 8 % in controls), and asynchronous release during the stimulus train reached higher frequencies (peak rates of approximately 6000 s-1 compared to approximately 75 s-1 in controls). These effects of mitochondrial depolarization were not accompanied by a significant change in EPP quantal content or the rate of asynchronous release during 1 Hz stimulation, and were not seen in oligomycin, which blocks mitochondrial ATP synthesis without depolarizing mitochondria. Inhibition of endoplasmic reticular Ca2+ uptake with cyclopiazonic acid also had little effect on stimulation-induced changes in cytosolic [Ca2+] or EPP amplitude. We hypothesize that the high rate of asynchronous release evoked by stimulation during mitochondrial depolarization was produced by the elevation of cytosolic [Ca2+], and contributed to the accelerated depression of phasic release by reducing the availability of releasable vesicles. During mitochondrial depolarization, the post-tetanic potentiation of the EPP observed under control conditions was replaced by a post-tetanic depression with a slow time course of recovery. Thus, mitochondrial Ca2+ uptake is essential for sustaining phasic release, and thus neuromuscular transmission, during and following tetanic stimulation.