The postulated role of the acetylcholine receptor in the formation of neuromuscular synapses during the course of embryonic development was investigated in the superior oblique muscle of white Peking duck embryos. The possibility that the number of receptors could be experimentally lowered by chronic injections of the anticholinesterase agent, neostigmine methylsulfate, was determined using 125I-alpha-bungarotoxin. The total number of acetylcholine receptors on incubation day 12, 2 d subsequent to the onset of treatment, was reducted 45% as compared to saline-treated controls. A similar reduction in total receptor content (49%) was also observed on day 19. Radioautographic preparations showed that clusters of acetylcholine receptors were rare and that the grain density of extrajunctional receptors was also reduced. Hence, chronic treatment with neostigimine during development was observed to exert an effect on both the number and distribution of receptors in the developing superior oblique muscle. These changes occurred in the absence of any apparent effect on muscle differentiation in general. Myoblasts and myotubes were present on day 14 and further differentiated into myofibers by day 18 in both neostigmine and saline-treated muscles. The cytology of the develop;ing muscle cells also appeared normal. This is in contradistinction to the striking morphological changes that take place in adult mammalian and avian muscle after anticholinesterase treatment. More significantly, the decreased total receptor content and sparsity of clusters had no apparent effect on the formation of developing neuromuscular junctions at the electron microscopic level. The frequency of neuromuscular junctions in neostigmine-treated muscles was similar to that of the controls. It is concluded that acetylcholine receptor clusters are not required for the events leading to the morphological formation of neuromuscular junctions during in vivo development.

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