Development of neuromuscular transmission in culture with a variety of neurons and in the presence of cholinergic substances and tetrodotoxin

Abstract

Summary The formation of neuromuscular junctions was investigated electrophysiologically in co-cultures of skeletal muscle cells and a variety of neurons obtained from chick embryo and rat. Neurons were plated as dissociated cells or tissue explants. One to 10 days after plating neurons, end-plate potentials (EPPs) were recorded from embryonic chick myotubes co-cultured with embryonic chick spinal cord, medulla oblongata and ciliary ganglion. The EPPs occurred spontaneously with frequency of 0.2–60/sec in 39–83% of myotube. Nerve stimulation also induced EPPs. No selectivity in junction formation on pectoral or thigh muscle was found among cervical, brachial and lumbar regions of the spinal cord. EPPs were observed in cultures of not only chick muscle-chick spinal cord but also rat muscle-rat cord, rat muscle-chick cord and chick muscle-rat cord. On the other hand EPPs were not observed in co-cultures of chick myotubes with chick cerebellum, cerebrum, superior cervical ganglia or dorsal root ganglia. Muscle cells and spinal cord explants of chick embryo were cultured in the presence of high concentrations of acetylcholine (ACh), carbachol, d -tubocurarine, gallamine, atropine, dibenamine or tetrodotoxin. When these substances were removed after 1–5 days of culture, spontaneous EPPs were observed similarly. It is concluded that neither functional ACh receptors nor nerve action potentials were necessary for the development of the neuromuscular junction in culture.