The long-term objectives are to elucidate cellular and molecular mechanisms of development, maintenance, and plasticity of synapses. Recent studies have shown that peanut agglutinin (PNA) recognizes synapse-specific molecules in the extracellular matrix, and its fluorescent conjugates stains living frog neuromuscular junctions. This proposal aims to characterize further the PNA-binding and to investigate the possible roles of PNA-binding molecules (PNA-BM) in frog endplates. (1) Postembedding staining with gold-conjugates PNA will be used to examine the ultrastructural localization of PNA-BM in neuromuscular junctions. (2) The effects of sialic acid removal by neuraminidase on PNA binding will be examined in normal and developing junctions. (3) Changes in the distribution of PNA-BM in relation to innervation will be examined with electron microscopy and epi-fluorescence light microscopy of whole mounts and cryosections of denervated, reinnervated, and developing neuromuscular junctions. (4) PNA will be applied to adult muscles during reinnervation of PNA-BM and acetylcholine receptors in culture also will be examined. (5) Nerve terminals and synaptic matrix in normal junctions stained with 4-di-2-Asp and rhodamine- PNA will be examined in situ repeatedly with video-enhanced microscopy. Dynamics changes in synaptic matrix during synapse formation and elimination will also be examined in situ. (6) Synaptic extracellular matrix of normal, degenerating, and developing endplates will be examined by gel electrophoresis and probed with peroxidase-conjugated PNA for biochemical characterization of PNA-BM. This research will characterize a new molecular probe for living neuromuscular junctions and provide further insights into cellular and molecular mechanisms of development, maintenance and plasticity of synapses. Elucidation of these basic mechanisms will enhance fundamental understanding of certain developmental and neuromuscular disorders.
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