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Rodent nerve‐muscle cell culture system for studies of neuromuscular junction development: Refinements and applications
Author(s) -
Daniels Mathew P.,
Lowe Brian T.,
Shah Sanjiv,
Ma Jianxin,
Samuelsson Steven J.,
Lugo Brian,
Parakh Tehnaz,
Uhm ChangSub
Publication year - 2000
Publication title -
microscopy research and technique
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.536
H-Index - 118
eISSN - 1097-0029
pISSN - 1059-910X
DOI - 10.1002/(sici)1097-0029(20000401)49:1<26::aid-jemt4>3.0.co;2-8
Subject(s) - agrin , myogenesis , neuromuscular junction , myocyte , acetylcholine receptor , microbiology and biotechnology , synapse , axon , biology , axon guidance , xenopus , motor neuron , neuroscience , receptor , biochemistry , spinal cord , gene
Understanding of vertebrate neuromuscular junction (NMJ) development has been advanced by experimentation with cultures of dissociated embryonic nerve and skeletal muscle cells, particularly those derived from Xenopus and chick embryos. We previously developed a rodent (rat) nerve‐muscle coculture system that is characterized by extensive induction of acetylcholine receptor (AChR) aggregation at sites of axonal contact with myotubes (Dutton et al., 1995). In this article, we report modifications of this culture system and examples of its application to the study of NMJ development: (1) We describe improved methods for the enrichment of myoblasts to give higher yields of myotubes with equal or greater purity. (2) We demonstrate lipophilic dye labeling of axons in cocultures by injection of dye into neuron aggregates and show the feasibility of studying the growth of living axons on myotubes during synapse formation. (3) We describe the preparation of a better‐defined coculture system containing myotubes with purified rat motoneurons and characterize the system with respect to axon‐induced AChR aggregation. (4) We demonstrate dependence of the pattern of axon‐induced AChR aggregation 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 in synapse formation by using single cell RT‐PCR with neurons collected from co‐cultures after observation of axon‐induced AChR aggregation. Microsc. Res. Tech. 49:26–37, 2000. Published 2000 Wiley‐Liss, Inc.