CELLULAR & MOLECULAR NEUROBIOLOGY: SYNAPSE FORMATION & MODULATION

这个子项目是许多研究子项目中利用 资源由NIH/NCRR资助的中心拨款提供。子项目和 调查员(PI)可能从NIH的另一个来源获得了主要资金， 并因此可以在其他清晰的条目中表示。列出的机构是 该中心不一定是调查人员的机构。 烟碱型乙酰胆碱受体(NAChRs)是一种作为阳离子选择性、配体门控离子通道的五聚体膜蛋白，广泛分布于脊椎动物神经系统。最丰富的nAChRs之一是一种含有A7基因产物(A7-AChRs)的物种，它与蛇毒a-银环蛇毒素结合，并迅速脱敏。这些受体已被证明在突触前部位起作用，调节递质释放，以及在突触后细胞上调节传递。有趣的是，这些受体对钙的相对渗透率非常高。在雏鸡睫状神经节中，A7-AChRs通过产生大的突触电流发挥重要作用，但受体似乎被排除在细胞突触后密度之外。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。我们最近发现，含有A7的受体集中在躯体棘垫上，与突触前递质释放的假定位置非常接近。中电压电子显微镜(IVEM)与断层重建相结合，可以对细胞体发出的指状投射以及AchRs与脊椎和突触前花瓣的关系进行三维分析。根据它们的形态、细胞骨架含量和与突触前元件的接近程度，这些投射被鉴定为体细胞棘突。无论是在原位还是在神经节分离后，棘突都聚集在细胞表面，并紧密地折叠成垫子。定量估计表明，这些棘突有效地使神经元的表面积增加了一倍。含有A7亚单位的受体的免疫金标记法显示，它们优先集中在体刺上。当结合电子断层扫描时，我们能够可视化金粒子在神经元表面的3D分布。活体脊柱垫的连续切片显示，只有不到一半的体棘具有突触后密度，但体棘附近的体棘上常有密度。完整神经节体棘的高分辨率断层重建显示，突触前膜充满突触小泡的投射在棘间交错排列。此外，即使没有明显的突触后密度并列在脊椎上，突触小泡也经常与膜毗邻，就像停靠在一起准备释放一样。这项研究最近发表在神经科学杂志上(Shoop等人，J.Neurosci，19：692-704,1999；其他参考文献在这里)。NCMIR最近开发的高速多光子显微镜正在继续确定脊柱生长和回缩的机制，并确定脊柱垫中的钙动力学。

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.