EVIDENCE THAT AGRIN DIRECTLY INFLUENCES PRESYNAPTIC DIFFERENTIATION AT NEUROMUSCULAR-JUNCTIONS IN-VITRO

The synaptic protein agrin is required for aspects of both pre-and pos tsynaptic differentiation at neuromuscular junctions. Although a direc t effect of agrin on postsynaptic differentiation, presumably through the MuSK receptor, is established, it is not clear whether agrin direc tly affects the presynaptic nerve. To provide evidence on this point, we used anti-agrin IgG to disrupt agrin function in chick ciliary gang lion (CG) neuron/myotube cocultures. In cocultures grown in the presen ce of 200 mu g/ml anti-agrin IgG, clustering of acetylcholine receptor s (AChRs), extracellular matrix proteins, and the synaptic vesicle pro tein synaptotagmin (syt) at nerve-muscle contacts was inhibited. Syt c lustering was still inhibited in the presence of 100 mu g/ml blocking antibody, while the postsynaptic clustering of AChRs, heparan sulphate proteoglycan, and s-laminin was retained. Additionally, in CG neurons cultured with COS cells expressing agrin A(0)B(0), which lacks the ab ility to signal postsynaptic differentiation, syt clustering was induc ed and this clustering was also blocked by anti-agrin IgG. Our results demonstrate that agrin function is acutely required for pre-and posts ynaptic differentiation in vitro, and strongly suggest that agrin is d irectly involved in the induction of presynaptic differentiation.