Evidence that agrin directly influences presynaptic differentiation at neuromuscular junctions in vitro

The synaptic protein agrin is required for aspects of both pre- and postsynaptic differentiation at neuromuscular junctions. Although a direct effect of agrin on postsynaptic differentiation, presumably through the MuSK receptor, is established, it is not clear whether agrin directly affects the presynaptic nerve. To provide evidence on this point, we used anti-agrin IgG to disrupt agrin function in chick ciliary ganglion (CG) neuron/myotube cocultures. In cocultures grown in the presence of 200 microg/ml anti-agrin IgG, clustering of acetylcholine receptors (AChRs), extracellular matrix proteins, and the synaptic vesicle protein synaptotagmin (syt) at nerve-muscle contacts was inhibited. Syt clustering was still inhibited in the presence of 100 microg/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 A0B0, which lacks the ability to signal postsynaptic differentiation, syt clustering was induced and this clustering was also blocked by anti-agrin IgG. Our results demonstrate that agrin function is acutely required for pre- and postsynaptic differentiation in vitro, and strongly suggest that agrin is directly involved in the induction of presynaptic differentiation.