CELLULAR & MOLECULAR NEUROBIOLOGY: SYNAPSE FORMATION & MODULATION

这个子项目是许多研究子项目中的一个 由NIH/NCRR资助的中心赠款提供的资源。子项目和 研究者（PI）可能从另一个NIH来源获得了主要资金， 因此可以在其他CRISP条目中表示。所列机构为 研究中心，而研究中心不一定是研究者的研究机构。 烟碱型乙酰胆碱受体（nAChR）是一种五聚体膜蛋白，作为阳离子选择性配体门控离子通道发挥功能，广泛分布于脊椎动物神经系统。最丰富的nAChR之一是含有α 7基因产物（α 7-AChR）的物种，其结合蛇毒α-银环蛇毒素并迅速脱敏。这些受体已被证明在突触前位点起作用以调节递质释放1，以及在突触后细胞上介导传递。有趣的是，这些受体对钙具有非常高的相对渗透性。在鸡睫状神经节中，α 7-AChR通过产生大的突触电流发挥突出作用，但受体似乎被排除在细胞上的突触后密度之外。免疫组织化学研究表明，受体在睫状神经节神经元表面形成大簇。我们最近的研究表明，含有α 7的受体集中在体细胞棘簇上，与突触前递质释放的假定位点非常接近。中间电压电子显微镜（IVEM）连同断层重建已允许的三维分析的手指样的预测从细胞体和AchRs的关系的脊柱和突触前萼。这些预测被确定为体细胞棘的基础上，其形态，细胞骨架的内容和接近突触前元件。在原位和神经节分离后，棘在细胞表面聚集并紧密折叠成垫。定量估计表明，这些棘垫有效地使神经元的表面积增加了一倍。含有α 7亚基的受体的免疫金标记显示它们优先集中在体细胞棘上。当与电子断层扫描相结合时，我们能够可视化神经元表面上金颗粒的3D分布。在体脊髓垫连续切片显示，只有不到一半的体细胞脊髓具有突触后密度，但密度通常存在于脊髓附近的索马上。在完整的神经节体细胞棘的高分辨率断层重建表明，突触囊泡填充的预测突触前膜之间的棘相互交错。此外，即使在棘上没有明显的突触后密度时，突触小泡也经常贴在膜上，就像是停靠在一起释放一样。这项工作最近发表在《神经科学杂志》上（Shoop等人，神经科学杂志，19：692-704，1999;其他参考文献在此）。工作正在继续确定脊柱生长和收缩的机制，并确定钙动力学的脊椎垫使用高速多光子显微镜最近开发的国家癌症研究所。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Nicotinic acetylcholine receptors (nAChRs) are pentameric membrane proteins that function as cation selective, ligand-gated ion channels and are widely distributed throughout the vertebrate nervous system. One of the most abundant nAChRs is a species that contains the a7 gene product (a7-AChRs), binds the snake venom a-bungarotoxin, and rapidly desensitizes. These receptors have been shown to function at presynaptic sites to modulate transmitter release1, as well as on postsynaptic cells where they mediate transmission. Interestingly, these receptors have an exceptionally high relative permeability to calcium. In the chick ciliary ganglion, the a7-AChRs play a prominent role, by generating large synaptic currents, but the receptors appear to be excluded from postsynaptic densities on the cell. Immunohistochemical studies have shown that the receptors form large clusters on the surface of the ciliary ganglion neurons. We have recently shown that the a7-containing receptors are concentrated on mats of somatic spines in close proximity to putative sites of presynaptic transmitter release. Intermediate voltage electron microscopy (IVEM) together with tomographic reconstruction has permitted a three-dimensional analysis of the finger-like projections emanating from the cell bodies and the relationship of AchRs to the spines and pre-synaptic calyx. These projections were identified as somatic spines based on their morphology, cytoskeletal content and proximity to presynaptic elements. Both in situ and after ganglionic dissociation the spines were grouped on the cell surface and tightly folded into mats. Quantitative estimations suggested that these mats of spines effectively doubled the surface area of the neuron. Immunogold labeling of receptors containing a7 subunits showed them to be preferentially concentrated on the somatic spines. When combined with electron tomography, we were able to visualize the 3D distribution of gold particles over the surface of the neuron. Serial sections through spine mats in vivo revealed that less than half of the somatic spines have postsynaptic densities, however densities were often present on the soma near spines. High resolution tomographic reconstructions of the somatic spines in intact ganglia showed that the synaptic vesicle-filled projections of the presynaptic membrane were interdigitated among the spines. Moreover, the synaptic vesicles often abutted the membrane as though docked for release even when no obvious postsynaptic densities were juxtaposed on the spine. This work was recently published in the Journal of Neuroscience (Shoop et al., J. Neurosci., 19: 692-704, 1999; other refs here). Work is continuing to determine mechanisms of spine growth and retraction and to determine calcium dynamics in the spine mats using the high-speed multiphoton microscope recently developed at NCMIR.