Release of acetylcholine from embryonic neurons upon contact with muscle cell

When a spherical muscle cell (myoball) was manipulated into contact with either the soma or the neurite of an isolated neuron in 2-day-old Xenopus nerve-muscle cultures, depolarizations similar to miniature endplate potentials (MEPPs) were frequently detected in the muscle cell. These depolarizations occurred within minutes after myoball-soma contact and within seconds after myoball-neurite contact. They had time course and amplitude distribution similar to those of the MEPPs recorded from naturally occurring neuromuscular synapses between neurites and muscle cells found in the same cultures, but they occurred at a lower frequency and had smaller average amplitudes. These depolarizations were induced by acetylcholine (ACh) since they were reversibly blocked by addition of d-tubocurarine into the culture, and they were abolished in muscle cells pretreated with alpha-bungarotoxin before contact with the neuron. Greater than 60% of the neuronal population in these cultures released ACh upon this direct muscle contact. The appearance of MEPP-like potentials in the myoball upon contact with an isolated neuron suggests that the cellular machinery responsible for ACh release is present throughout the neuron and that packages of ACh molecules are available for release prior to nerve-muscle synapse formation. We also found that neurons which had previously made synapse with other muscle cells in the culture all failed to release ACh from the soma and showed reduced release capability at the neurite for the first 30 min to 1 hr of contact with a myoball. This finding suggests that, during synapatogenesis, there is a depletion of ACh molecules and/or substances responsible for the triggering of their release in the extrasynaptic regions of the neuron.