Cultures were prepared of embryonic cells from Drosophila melanogaster. Neurons and myocytes differentiated in vitro from their respective stem cells. Electron microscopy showed that neuromuscular junctions formed where axons contacted myocytes. Electrical stimuli were applied to axons and these caused contractions of innervated myocytes. This is the first report of insect or other invertebrate neuromuscular junctions differentiating in vitro. In addition, this is the first system reported in which the neurons, myocytes, and junctions are completely differentiated in vitro from neuroblasts and myoblasts.For the study of neuromuscular junctions in cultured material, ideally the culture should contain fully-differentiated, functional neurons, myocytes, and junctions, and these elements should be easily accessible to observation and handling. Cultures in which mature elements arise from incompletely differentiated precursor cells offer opportunities for investigation of the properties of immature elements and for the study of the differentiation processes.Several culture systems using vertebrate cells have already been described (1-6). Typically, portions of spinal cord and muscle are taken from 4-to 14-day-old chick embryos or 10-to 19-day-old rat or mouse fetuses and placed side by side in culture (1-5). Axons grow from the cord to the muscle explants and establish junctions with the muscle cells. In one system, cord and muscle tissues were dispersed by trypsinization, the cells were mixed in culture, and junctions were later detected (6). In these studies, neuromuscular junctions were detected by various techniques, but little has been done so far to describe their characteristics. In each culture system, the neuromuscular junctions differentiated in vitro, but the progress of the nerve and muscle cells, themselves, was not followed closely. The We are reporting a system in which neurons, myocytes, and neuromuscular junctions differentiate in vitro from an inoculum of neuroblasts and myoblasts. Electron microscopy and electrophysiologic techniques show the junctions to be morphologically complete and functional. The system is amenable to neurologic investigations and should also provide a description of the neuromuscular junction at all stages in its differentiation. Eventually, pertinent mutants should become available, and these will broaden the possibilities for investigation.
MATERIALS AND METHODSDrosophila melanogaster strain Oregon-R was the source of all cells. We prepared cultures by disaggregating whole embryos and plating the cells in plastic dishes, as described (8), with the following modifications. Cells from single embryos were introduced to 2 ml of medium, and oil overlay was omitted. NaHCO3 was omitted from the medium, and the cultures were incubated in air.Cells were taken from gastrulating embryos, none older than 40-min after the onset of gastrulation (8). Cultures were kept at 26 i 0.50C. Cells were prepared for electron microscopy as described (8).We tested neuromuscular junctions ...
