We do basic research on the cellular and molecular mechanisms involved in development of the mammalian neuromuscular junction, utilising a novel nerve-muscle culture system. Postsynaptic acetylcholine receptor aggregation is a critical early event in junction formation. We previously showed that the capacity to induce receptor aggregation resides primarily in the axons of ventral spinal cord neurons. This suggests that one or more of the signals for the induction of postsynaptic differentiation has a polarized distribution in these neurons. Agrin is a proteoglycan that is required for normal postsynaptic differentiation in muscle. We are using immunofluorescence to determine if the expression of neuronal agrin is polarized. We are also using single-cell PCR to examine the expression of alternatively-spliced isoforms of agrin mRNA in cultured ventral spinal cord neurons and the effect of myotube contact or synaptogenesis on that expression. Our results suggest that one of three """"""""neuronal"""""""" agrin isoforms predominates in neurons that have formed synapses with muscle. We are exploring the possibility that adhesive interactions with muscle are specific to axons as opposed to dendrites, and that they may be important for the initiation of postsynaptic differentiation. We have used scanning and transmission electron microscopy to show that the closest interactions between nerve and muscle cells occur at sites of contact between myotubes and axons that induce acetylcholine receptor aggregation. Moreover, the myotube surface adjacent to these contacts exhibits ruffles and microvilli, indicating a motile response to axonal contact. This response appears to occur very early after contact and, together with adhesion, may facilitate cell-surface interactions between the axon and myotube. We are now exploring the possible relationship between this response and the signal-transduction processes involved in acetylcholine receptor accumulation. Specifically, we are attempting to determine if agrin or ARIA (a growth-factor-like protein that regulates acetylcholine receptor expression by muscle) play a role in the induction of this response.
