Development of the Postsynaptic Membrane in Xenopus Neuromuscular Cultures Observed by Freeze-fracture and Thin-section Electron Microscopy

The formation of synapses between cultured neurons and muscle cells from Xenopus embryos has been studied with freeze-fracture and thin-section techniques. Clusters of large P-face intramembranous particles (about 11-12 nm) were observed in both innervated and non-innervated muscle cells. They presumably represented clusters of acetylcholine (ACh) receptors because of their close resemblance to the post-junctional particle clusters at the adult neuromuscular (N-M) junctions. In one-day cocultures, particle aggregates could be observed in more than 50% of the N-M contacts. At this stage, these aggregates were diffusely distributed along the contacts. After two days of coculture, extensive and tight clustering of large particles was seen along the length of persisting N-M contacts. Each particle cluster was composed of many particle aggregates and a particle-free groove demarcated each aggregate from its neighbor, thus producing a convoluted appearance of the membrane, which corresponded well with the thin-section image of the membrane profiles at the N-M contacts. In both freeze-fracture and thin-section images, membrane depressions with a diameter of about 0.1 micron were often observed in the vicinity of N-M contacts in newly innervated muscle cells. Within the pits of these depressions a small aggregate of large particles similar to those in the sarcolemma was often encountered. Such particle-rich membrane depressions were also observed in non-innervated muscle cells. They may represent sites for the incorporation of new ACh receptors in light of current theories. Particle aggregates were also closely associated with certain deep membrane invaginations, suggesting that these structures may be involved in the concentration of ACh receptors. Close membrane contacts were observed between nerve endings and muscle cells in young cocultures thin-sectioned. Gap junction-like particle aggregates were also observed in the muscle membrane along identified young N-M contacts. These data suggest that the formation of transient gap junctions may accompany the initial stages of synaptogenesis in Xenopus N-M cultures.