The absence of calcium blocks impulse-evoked release of acetylcholine but not de novo formation of functional neuromuscular synaptic contacts in culture.

We have examined the role of calcium-dependent, impulse-evoked release of acetylcholine (ACh) in the formation of functional neuromuscular contacts. Dissociated cell cultures prepared from Xenopus laevis embryos were maintained either in standard medium containing calcium or in calcium-free medium. Both the number of neuron-myocyte contacts and the number of terminations of neurites on myocytes were reduced in Ca-free medium. Intracellular recordings from neuron-myocyte pairs during perfusion with a standard saline revealed that functional synaptic contacts were formed in Ca-free medium, although with a reduced frequency (approximately 30% of controls). Postsynaptic potentials elicited by neuronal action potentials were smaller than those observed after growth in standard medium. The frequency of small, spontaneously occurring potentials was reduced by a factor of 10. Ca-free saline prevented the impulse-evoked release of transmitter from neurons in Ca-free and control cultures. Labeling with alpha-bungarotoxin and iontophoretic application of ACh revealed no ACh receptor clusters in the membrane of myocytes grown in Ca-free medium. Our results suggest that evoked, vesicular release is not required for the initial formation of neuromuscular contacts, although it may be involved in further maturation of synapses. Roles for spontaneous quantal or non-quantal release have not been excluded.