Repetitive nerve stimulation decreases the acetylcholine content of quanta at the frog neuromuscular junction

We investigated how elevated quantal release produced by motor nerve stimulation affects the size of the quanta. The motor nerve was stimulated at 10 Hz in preparations in which excitation-contraction coupling was disrupted. Two hundred stimuli reduced the size of the time integrals of the miniature endplate currents ([integral]MEPCs), measured at the same junction immediately after stimulation, by 16 %. Three thousand stimuli reduced size by 23 %. When the solution contained 10 microM neostigmine (NEO) 3000 stimuli reduced [integral]MEPCs by 60 %, because with acetylcholinesterase (AChE) inhibited, [integral]MEPC size is more sensitive to changes in acetylcholine (ACh) content. Similar decreases in miniature endplate potential size ([integral]MEPP) followed repetitive stimulation of contracting preparations. The depolarization produced by iontophoretic pulses of ACh was scarcely changed by 3000 nerve stimuli at 10 Hz, suggesting that the decreases in miniature sizes are largely due to less ACh released per quantum. Following 3000 stimuli at 10 Hz the sizes of the [integral]MEPCs increased back to pre-stimulus values with a half-time of 8-10 min. Recovery was blocked by (-)-vesamicol (VES), by hemicholinium-3 (HC3) and by nicotinic cholinergic agonists - all of which inhibit ACh loading into synaptic vesicles. The number of quanta in the total store was estimated by releasing them with carbonyl cyanide m-chlorophenylhydrazone (CCCP). CCCP releases fewer quanta after stimulation than from unstimulated controls. After resting for hours following stimulation, the releasable number increased, even when ACh loading inhibitors were present. We conclude that the inhibitors do not block a significant fraction of the ACh loading into reformed reserve vesicles and propose that ACh can be loaded in a series of steps.