Abstract
Changes in cytosolic and mitochondrial [Ca
2+
] produced by brief trains of action potentials were measured in motor nerve terminals using a rapidly scanning confocal microscope. Cytosolic [Ca
2+
] was measured using ionophoretically injected Oregon Green BAPTA 5N (OG-5N). Mitochondrial [Ca
2+
] was measured using rhod-2, bath loaded as dihydrorhod-2.
In response to 100-250 stimuli at 25-100 Hz the average cytosolic [Ca
2+
] showed an initial rapid increase followed by a much slower rate of increase. Mitochondrial [Ca
2+
] 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 [Ca
2+
] coincided with the slowing of the rate of rise of cytosolic [Ca
2+
]. The peak levels of cytosolic and mitochondrial [Ca
2+
] both increased with increasing frequencies of stimulation.
When stimulation terminated, the initial rate of decay of cytosolic [Ca
2+
] was much more rapid than that of mitochondrial [Ca
2+
].
After addition of carbonyl cyanide m-chlorophenyl hydrazone (CCCP, 1-2 μ
m
) to dissipate the proton electrochemical gradient across the mitochondrial membrane, cytosolic [Ca
2+
] rose rapidly throughout the stimulus train, reaching levels much higher than normal. CCCP inhibited the increase in mitochondrial [Ca
2+
].
These results suggest that mitochondrial uptake of Ca
2+
contributes importantly to buffering presynaptic cytosolic [Ca
2+
] during normal neuromuscular transmission.
