Non-quantal release of acetylcholine at a developing neuromuscular synapse in culture

Local, pulsed application of d-tubocurarine at neuromuscular synapses in embryonic Xenopus nerve-muscle culture resulted in a transient hyperpolarization of muscle membrane potential. Miniature endplate potentials (MEPPs) were abolished during the hyperpolarization and recovered after the return of resting membrane potential. The magnitude of hyperpolarization was independent of the frequency of MEPPs before curarization, and it had an average peak value of 4.3 mV in medium containing physiological levels of Ca2+. Prolonged application of curare or alpha-bungarotoxin led to sustained hyperpolarizations up to 8 mV in magnitude. Denervation produced by mechanically removing the neurite from the muscle cell also produced similar hyperpolarization, and curarization after denervation was without significant hyperpolarizing effect. Increasing the extracellular Ca2+ concentration to about 8 mM abolished the curare-induced hyperpolarization response, in sharp contrast to its effect in elevating the frequency of MEPPs. Taken together, our results indicate that innervated embryonic muscle cells were maintained at a depolarized state relative to that of uninnervated muscle cells by a steady, spontaneous release of acetylcholine (ACh) from the innervating neurite. The cellular mechanism underlying this mode of ACh release appears to be different from that of the quantal ACh release responsible for MEPPs.