Neuromuscular transmission to identified primary and secondary myotubes: A reevaluation of polyneuronal innervation patterns in rat embryos☆

Abstract The early morphogenesis of rat skeletal muscle is a biphasic process involving two sequentially generated populations of myotubes. A small population of primary myotubes appears early and is followed by a much larger population of secondary myotubes which appear progressively over a number of days. All previously published electrophysiological studies of developing muscle have failed to appreciate the relevance of biphasic myotube production. Here we reevaluate the status of early motor innervation, taking into account the wide range of sizes and levels of maturity within the two myotube populations. Evoked end-plate potentials (EPPs) were recorded from fibers of E17-20 rat sternocostalis muscles. Impaled fibers were then marked by ejection of HRP from the recording pipet, enabling ultrastructural identification of fibers from which recordings had been made. The average number of synaptic inputs per fiber increased to a peak at E19, and the rate of rise of the EPPs increased with age. The majority of impaled fibers (76%) were subsequently found to be primary myotubes, even at ages when secondary myotubes formed the majority of fibers in the muscle. Electrophysiological studies during early stages of secondary myotube development therefore sample largely from the more mature primary fibers and probably give the wrong impression of the extent and degree of polyneuronal innervation and of synaptic rearrangement within the muscle. In addition, the results show that very young secondary myotubes are distinguished by EPPs of longer latency, slower rate of rise, and smaller size than those of other types of myotubes. These results suggest that young secondary myotubes are predominantly activated by EPPs which originate in adjoining primary myotubes and propagate electrotonically to the secondary myotube. We propose a new model of early synaptic rearrangement which accommodates the biphasic nature of muscle development. We also suggest that secondary myotubes do not require direct neural input for the initiation of their development.