ACETYLCHOLINE-RECEPTOR AGGREGATION AT NERVE-MUSCLE CONTACTS IN MAMMALIAN CULTURES - INDUCTION BY VENTRAL SPINAL-CORD NEURONS IS SPECIFIC TOAXONS

We used a novel mammalian coculture system to study ACh receptor (AChR ) redistribution and synaptic structure at nerve-muscle contacts, Vent ral spinal cord (VSC) neurons were plated on cultures containing exten sive myotubes but few fibroblasts, Neurite-induced redistribution of A ChRs occurred within 6 hr after plating neurons and was maximal betwee n 36-48 hr, This AChR redistribution appeared in two patterns: (1) ACh R density at sites directly apposed to the neurite where neurites cros sed preexisting AChR patches was sharply reduced, (2) Newly aggregated AChRs formed swaths lateral to the neurite path. VSC neurons induced more AChR aggregation than hippocampal, superior cervical ganglion and dorsal root ganglion neurons. The 43 and 58 kDa postsynaptic proteins were colocalized with AChR-enriched domains in all VSC neurite-induce d aggregates whereas the colocalization of laminin was variable. Elect ron microscopy of regions with neurite-induced AChR aggregation showed postsynaptic membrane specializations characteristic of developing sy napses and, in older cultures, features of more mature synaptic struct ure, Thus, the coculture system is useful for studying early stages of neuromuscular junction (NMJ) formation. Neurites in these cocultures were identified as axons or dendrites by morphological criteria and by their immunoreactivity for synaptophysin and phosphorylated heavy neu rofilament subunits or for microtubule associated protein 2 (MAP2), re spectively, Axons showed a 10-fold higher induction of AChR aggregatio n than did dendrites, Thus, at least one essential signaling molecule necessary for the induction-of AChR aggregation at sites of interactio n with muscle appears to be expressed in a polarized fashion in develo ping VSC neurons.