Synaptic localization and axonal targeting of agrin secreted by ventral spinal cord neurone in culture

Agrin secreted by motor neurons is a critical signal for postsynaptic diffe rentiation at the developing neuromuscular junction. We used cultures of ch ick ventral spinal cord neurons with rat myotubes and immunofluorescence wi th species-specific antibodies to determine the distribution of agrin secre ted by neurons and compare it to the distribution of agrin secreted by myot ubes. Ln addition, we determined the distribution of agrin secreted by isol ated chick ventral spinal cord neurons and rat motor neurons grown on a sub strate that binds agrin. In cocultures, neuronal agrin was concentrated alo ng axons at sites of axon-induced acetylcholine receptor (AChR) aggregation and was found at every such synaptic site, consistent with its role in syn aptogenesis. Smaller amounts of agrin were found on dendrites and cell bodi es and rarely were associated with AChR aggregation. Muscle agrin, recogniz ed by an antibody against rat agrin, was found at nonsynaptic sites of AChR aggregation but was not detected at synaptic sites, in contrast to neurona l agrin, in cultures of isolated chick neurons or rat motor neurons, agrin was deposited relatively uniformly around axons and dendrites during the fi rst 2-3 days in culture. In older cultures, agrin immunoreactivity was mark edly more intense around axons than dendrites, indicating that motor neuron s possess an intrinsic, developmentally regulated program to target agrin s ecretion to axons. (C) 2000 John Wiley & Sons, Inc.*.