Ouabain (0.1 and 0.05 mM) was applied to frog cutaneous pectoris nerve-muscle preparations bathed in modified Ringer's solution containing either 1.8 mM Ca2+ (and 4 mM Mg2+) or no added Ca2+ (4 mM Mg2+ and 1 mM EGTA). During the intense quantal release of acetylcholine (ACh) induced by ouabain, the parameters of the miniature endplate potentials (mepps) were deduced from the variance, skew, and power spectra of the endplate recordings by applying a recently described modification of classical fluctuation analysis. Often the high frequency of mepps is not stationary; therefore, the signal was high-pass filtered (time constant of the resistance-capacitance filter of 2 ms) to remove the errors introduced by nonstationarity. When ouabain was applied in the presence of Ca2+, mepp frequency started to rise exponentially after a lag of 1.5-2 h, reached an average peak frequency of 1,300/s in approximately 30 min, and then suddenly subsided to low level (10/s). In Ca2+-free solution, after a shorter lag (1-1.5 h), mepp frequency rose to peak rate of 700/s in approximately 20 min and then gradually subsided. In spite of the different time course of secretion in the two experimental conditions, the cumulative quantal release was not significantly different (7.4 +/- 1.3 X 10(5) in Ca2+-containing and 8.8 +/- 2.7 X 10(5) in Ca2+-free solutions). 60 min after the peak secretion, the muscles were fixed for observation in the electron microscope. Morphometric analysis on micrographs of neuromuscular junctions revealed in both cases a profound depletion of synaptic vesicles and deep infoldings of presynaptic membrane. This rapid depletion and the lack of uptake of horseradish peroxidase suggest that ouabain impairs the recycling process that tends to conserve the vesicle population during intense secretion of neurotransmitter. The good correlation observed between the reduction in the store of synaptic vesicles and the total number of quanta of ACh secreted in the absence of a vigorous membrane recycling strongly supports the view that the secretion of a quantum of ACh requires the fusion of a synaptic vesicle with the axolemma.

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