The development of clusters of acetylcholine (ACh) receptors at newly formed synapses between embryonic chick spinal cord and muscle cells grown in vitro has been studied by iontophoretic mapping with ACh. A semi-automated technique using on-line computer analysis of ACh responses and a photographic system to record the position of each ACh application permit the rapid construction of extensive and detailed maps of ACh sensitivity. Clusters of receptors, evident as peaks of ACh sensitivity, are present on many uninnervated myotubes. The distribution of ACh sensitivity closely parallels the distribution of 125I-alpha-bungarotoxin binding sites on the same muscle cell. In all cases where individual myotubes were adequately mapped before and after synapse formation, ingrowing axons induced new clusters of receptors rather than seeking out preexisting clusters. Synapses can form at active growth cones within 3 h of nerve-muscle contact. New receptor clusters can appear beneath neurites within a few hours. Many of the uninnervated clusters on innervated myotubes disappear with time. In contrast, receptor clusters on uninnervated myotubes remain in the same location for many hours. Synaptic clusters and clusters on uninervated myotubes are stable even though individual receptors are metabolized rapidly. The morphology of several identified sites of transmitter release was examined. At the scanning EM level, synapses appeared as small, rough-surfaced varicosities with filopodia that radiated outwards over the muscle surface. One synapse was studied by transmission EM. Acetylcholinesterase and a basement lamina were present within the synaptic cleft.

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