Rodent nerve-muscle cell culture system for studies of neuromuscular junction development: Refinements and applications

Understanding of vertebrate neuromuscular junction (NMJ) development has be en advanced by experimentation with cultures of dissociated embryonic nerve and skeletal muscle cells, particularly those derived from Xenopus and chi ck embryos. We previously developed a rodent (rat) nerve-muscle coculture s ystem that is characterized by extensive induction of acetylcholine recepto r (AChR) aggregation at sites of axonal contact with myotubes (Dutton et al ., 1995). In this article, we report modifications of this culture system a nd examples of its application to the study of NMJ development: (1) We desc ribe improved methods for the enrichment; of myoblasts to give higher yield s of myotubes with equal or greater purity. (2) We demonstrate lipophilic d ye labeling of axons in cocultures by injection of dye into neuron aggregat es and show the feasibility of studying the growth of living axons on myotu bes during synapse formation. (3) We describe the preparation of a better-d efined coculture system containing myotubes with purified rat motoneurons a nd characterize the system with respect to axon-induced AChR aggregation. ( 4) We demonstrate dependence of the pattern of axon-induced AChR aggregatio n on muscle cell species, by the use of chick-rat chimeric co-cultures. (5) We provide evidence for the role of alternatively-spliced agrin isoforms i n synapse formation by using single cell RT-PCR with neurons collected from co-cultures after observation of axon-induced AChR aggregation, Microsc. R es. Tech. 49:26-37, 2000. Published 2000. Wiley-Liss, Inc.