Ultrastructural Observations on Rapid Formation of Neuro-muscular Junctions in Vitro

The development of neuro-muscular junctions (mouse, rat) from the time of first contact between neurons and myotubes in culture and the changes which lead to the formation of functional synaptic contacts have been investigated using light microscopy and ultrastructural techniques. An extensive basal lamina was present when the neuronal cell population was added to the developing myotubes in culture. The nerve cells were initially strongly attracted to each other and nerve cell aggregates formed rapidly. It was only when nerve fibers began to grow out of these aggregates to contact developing myotubes that changes within the cytoplasm of the two adjacent cells were observed. These developments included accumulations of filaments, membrane densities, mitochondria and large clear vesicles within both cells in the region of contact. In addition, collections of glycogen granules and an extensive membrane reticular complex were found within myotubes, and an extensive granular material filled many of the nerve processes. The basal lamina within the intercellular space appeared more electron-dense that elsewhere and was traversed by strands linking the two cell membranes. These features all appeared to be stages of the initial formation of neuro-muscular junctions. It was only after these events had occurred that presynaptic vesicles gradually appeared within the future nerve terminal. The results of this paper therefore support the view that synaptic transmissions at developing mammalian neuro-muscular junctions is not necessarily dependent on the presence of presynaptic vesicles.