We have observed structural remodelling of neuromuscular junctions (NMJ's)by in vivo monitoring of identified junctions in growing, mature, and aged mice. The goal of this proposal is to test a new hypothesis as to mechanisms which account for the observed neuromuscular stability and remodelling. The working hypothesis is that the extent of neuromuscular remodelling and its location within the synapse is the resultant of two opposing forces: those which stabilize existing synaptic components -- specifically focal identifiable nerve terminal adhesive complexes with synaptic matrix and with Schwann cell -- and those which favor outgrowth: ongoing lamellipodial outgrowth, forming new synaptic extensions when the perisynaptic substrate favors focal adhesion. Several key predictions of this hypothesis will be tested with techniques mostly already demonstrated. Pre- and post-synaptic components of identified NMJ's will be visualized in living animals by specific fluorescent ligands. An adhesion assay based on hypertonic shrinkage will be correlated with ultrastructural quantitation of detachment of nerve terminal adhesion sites, and the extent of co-localization of the adhesions with active zones will be analyzed. Also, a paradigm of following nerve terminal synaptic regeneration in vivo (3 days after specific focal destruction by spider venom) over 2 hr intervals during synapse re-establishment, combined with correlated E.M. will be used to test the relation between number and type of adhesion sites and in vivo synaptic adhesion. The functional role of molecular components of the synaptic adhesive complex will be defined by perturbing regeneration with specific antibodies, analogs or enzymes. Also, the contribution of the Schwann cell adhesion to synaptic stability and regeneration will be tested by selective laser ablation of identified junctional Schwann cell nuclei. Other studies, combining E.M. and fluorescence microscopy will determine the 'life cycle' of synaptic adhesions (including active zones) and duration of its phases. It is predicted that the observed structural instability of aging synapses reflects altered synaptic adhesion and perisynaptic environment; therefore, aging NMJ's will be analyzed as above. It is anticipated that the results will clarify both age changes in synaptic remodelling and the general mechanisms of neuromuscular synaptic stabilization and plasticity.
