Function-dependent changes of calcium distribution were studied in the nerve-electroplaque synapses of Torpedo marmorata before and after the transmission of a nerve impulse. For the cytochemical demonstration of calcium at the ultrastructural level the oxalate-pyroantimonate technique was combined with electron spectroscopic imaging. Cholinergic synapses of the electric organ were stimulated in the presence of 4-aminopyridine, a drug which powerfully potentiates transmitter release. A single stimulus evoked a giant electrical discharge, which was followed by a long refractory period. Calcium cytochemistry was performed by fixing the tissue at four well defined functional states: 1. (i) before and 2. (ii) immediately after the giant discharge, and 3. (iii) at 1 min or 4. (iv) at 30 min of subsequent rest, corresponding to partial and complete functional recovery, respectively. In the non-stimulated synapses about 20% of synaptic vesicles contained small electron-dense precipitates. The element specific mapping by electron spectroscopic imaging clearly showed that calcium was present in the vesicular granules. The volume density of synaptic vesicles did not change among the four experimental states, but we detected a significant increase in the proportion of calcium containing vesicles at 1 min after the giant discharge. The vesicular calcium accumulation was transient: it returned to the control value at the end of the recovery period. Our data suggest that the synaptic vesicles play a role in sequestering the excess calcium which enters the nerve terminal during stimulation.
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