FORMATION AND ELIMINATION OF SYNAPSES

长期目标是阐明细胞和分子 突触的发育、维持和可塑性的机制。 最近的研究表明，花生凝集素(PNA)可以识别 细胞外基质中的突触特异性分子及其 荧光偶联物染色活的青蛙神经肌肉接头。 这项提案旨在进一步说明具有PNA约束力的特点，并 研究PNA结合分子(PNA-BM)的可能作用 在青蛙端板上。 (1)包埋后用金标记物PNA染色 PNA-BM的超微结构定位观察 神经肌肉接头。(2)去除唾液酸的效果 神经氨酸酶对PNA结合的检测将在正常和 发展中的连接点。(3)PNA-BM分布的变化 与神经支配有关的部分将用电子手段检查 整体马匹的显微镜和表观荧光显微镜 以及去神经、再神经和发育中的冷冻切片 神经肌肉接头。(4)PNA将应用于成人肌肉 在PNA-BM和乙酰胆碱受体的再神经支配过程中 文化也将被审查。(5)神经末梢和突触 4-二-2-天冬氨酸和罗丹明染色的正常连接的基质 PNA将通过视频增强反复进行现场检查 显微镜。突触过程中突触基质的动态变化 形成和消除也将在现场进行检查。(6) 突触细胞外基质的正常、退变和 正在形成的终板将通过凝胶电泳法和 过氧化物酶标记的PNA用于生化探针的研究 PNA-BM的特性研究。 这项研究将表征一种新的生命分子探针 神经肌肉连接，并提供对细胞 以及发育、维持和可塑性的分子机制 突触的特征。对这些基本机制的阐明将加强 对某些发展和发展的基本认识 神经肌肉疾病。

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.