Depolarization-Induced Ca2+ Entry Preferentially Evokes Release of Large Quanta in the Developing Xenopus Neuromuscular Junction

Abstract

The amplitude histogram of spontaneously occurring miniature synaptic currents (mSCs) is skewed positively at developing Xenopus neuromuscular synapses formed in culture. To test whether the quantal size of nerve-evoked quanta (eSCs) distributes similarly, we compared the amplitude histogram of single quantum eSCs in low external Ca2+ with that of mSCs and found that nerve stimulation preferentially released large quanta. Depolarization of presynaptic terminals by elevating [K+] in the external solution or by direct injection of current through a patch pipette increased the mSC frequency and preferentially, but not exclusively, evoked the release of large quanta, resulting in a second broad peak in the amplitude histogram. Formation of the second peak under these conditions was blocked by the N-type Ca2+ channel blocker, ω-conotoxin GVIA. In contrast, when the mSC frequency was elevated by thapsigargin- or caffeine-induced mobilization of internal Ca2+, formation of the second peak did not occur. We conclude that the second peak in the amplitude histogram is generated by Ca2+ influx through N-type Ca2+ channels, causing a local elevation of internal Ca2+. The mSC amplitude in the positively skewed portion of the histogram varied over a wide range. A competitive blocker of acetylcholine (ACh) receptors, d-tubocurarine, reduced the amplitude of smaller mSCs in this range relatively more than that of larger mSCs, suggesting that this variation in the mSC amplitude is due to variable amounts of ACh released from synaptic vesicles. We suggest that Ca2+ influx through N-type Ca2+ channels preferentially induces release of vesicles with large ACh content.