Studies of nerve-muscle interactions in Xenopus cell culture: fine structure of early functional contacts

We have studied the fine structure of nerve-muscle contacts during the first few hours of synaptogenesis in embryonic Xenopus cell cultures. The precise timing of contact was achieved by manipulating isolated spherical myocytes (myoballs) into contact with growth cones or neurites of co-cultured spinal neurons. The contacts were shown to be functional by whole-cell voltage-clamp recording of nerve-evoked synaptic currents in the muscle cell. The ultrastructure of these functional contacts was examined by thin-section electron microscopy. In total, 20 nerve-muscle pairs were studied with contact periods ranging from 20 min to 12 hr, during which time a substantial increase in the amplitude of synaptic currents occurred. The structure of noncontacting cells and of nerve-muscle contacts formed between the cells by natural encounters in 1-d-old cultures were also examined in order to identify the features and the time course of morphological differentiation of early functional contacts. Prominent features of the contact area during the first few hours included: close apposition of the nerve and muscle membranes, greater frequency of coated pits and vesicles, and thickening of postsynaptic muscle membrane. Occasionally, clusters of clear vesicles occurred near presynaptic membrane, but no further sign of active zone differentiation was observed. In comparison, definitive active zone structure, well-formed extracellular basal lamina, and widened cleft were seen in natural contacts less than 24 hr old. This study of the identified functional contacts may help us to understand the structural basis for early nerve-muscle interaction and the functional significance of various synaptic specializations.