Development of neuromuscular junctions in the mouse esophagus: morphology suggests a role for enteric coinnervation during maturation of vagal myoneural contacts

The time course of establishment of motor endplates and the subsequent developmental changes in their enteric and vagal innervation were examined in esophageal striated muscle of perinatal and adult C57/Bl6 mice by using immunocytochemistry and confocal laser scanning microscopy. Nicotinic acetylcholine receptors were visualized with alpha-bungarotoxin; vagal motor nerve terminals with antisera against vesicular acetylcholine transporter; and enteric nerve fibers with antisera against neuronal nitric oxide synthase, vasoactive intestinal peptide, and galanin. Because the various stages of esophageal striated myogenesis advance caudocranially, i.e., more mature stages are found cranial to immature stages, longitudinal cryosections through the esophagus were investigated. Synaptogenesis was divided into several distinct stages. 1) Mononucleated cells express acetylcholine receptors over their entire surface. 2) They start to cluster receptors without nerve fiber contacts. 3) The first nerve contact on a growing receptor cluster is made by a vagal nerve terminal, followed by an enteric terminal. 4) Vagal terminals grow until they match the size of endplate areas, and one to three enteric terminals intertwine with them on every receptor cluster. 5) After vagal terminals have covered the whole endplate area, enteric terminals are withdrawn from the majority of motor endplates. In a minority of endplates, enteric coinnervation persists through adulthood. The enteric innervation of all developing motor endplates, shortly after vagal terminals have contacted them, and the removal of enteric nerve fibers from the majority of mature motor endplates suggest a major role of enteric nerve fibers during maturation of esophageal neuromuscular junctions.