Initial events in the formation of neuromuscular synapse: rapid induction of acetylcholine release from embryonic neuron.

TLDR

We studied electrical events during the initial phase of nerve‑muscle contact in embryonic Xenopus culture. Using a G ω‑seal whole‑cell recording, we continuously monitored muscle membrane currents while bringing a muscle cell into close proximity of a cocultured spinal neuron’s growth cone. After neurite–muscle contact, pulsatile inward currents appeared rapidly, resembling miniature end‑plate currents, were blocked by nicotinic antagonists but not by tetrodotoxin, and increased in frequency over 20 min; these currents, triggered by contact with muscle membrane rather than isolated growth cones, suggest that contact‑dependent acetylcholine release initiates early muscle activity and signals synapse formation.

Abstract

We have studied the electrical events during the initial phase of nerve-muscle contact in embryonic Xenopus culture. Using a G omega-seal, whole-cell recording method, we monitored the membrane current of a muscle cell continuously while it was manipulated into close proximity of the growth cone of a cocultured spinal neuron. We found a rapid appearance of pulsatile inward currents at the muscle cell after the neurite-muscle contact. These currents were abolished by d-tubocurarine and alpha-bungarotoxin but were unaffected by tetrodotoxin. Both the drug sensitivity and the time course of these currents are similar to that of the spontaneous miniature end-plate currents (MEPCs) resulting from spontaneous release of pulses of acetylcholine (AcCho) from the nerve terminal. Unlike the MEPCs at the mature neuromuscular synapse, these early MEPCs varied greatly in their amplitudes, and there was a gradual increase in the frequency of the MEPCs of larger amplitudes during the first 20 min after the contact. Independent measurement of AcCho concentration near the growth cone by an excised patch of AcCho-sensitive muscle membrane showed that very little AcCho is released from the isolated growth cone, and marked release can be triggered by the contact with a muscle cell or with the excised membrane itself. The induction of release is relatively specific: contact with a neuron or the tip of a clean glass pipette was capable of inducing only a transient release, while persistent release was induced by contacts made with muscle membrane. This contact-dependent AcCho release may be responsible for an early induction of muscle activity and serve as a signal for the establishment of synaptic contact.