CALCIUM MEDIATED EXOCYTOSIS OF NEUROTRANSMITTER DURING SYNAPTIC TRANSMISSION

突触传递过程中钙介导的神经递质胞吐作用

• 批准号: 6121827
• 负责人: WILLIAM M ROBERTS
• 金额: $ 2.78万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN DIEGO
• 依托单位国家: 美国
• 财政年份: 1999
• 资助国家: 美国
• 起止时间: 1999-05-15 至 2000-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6121827
• 关键词: biomedical resource; informatics; lipids; microscopy; model design /development; nervous system

The long term goal of these studies is to understand the presynaptic mechanisms that lead to transmitter release at fast chemical synapses. Our specific objective is to reconstruct in three dimensions the ultrastructure of the hair cell afferent synapse in order to test several aspects of our working model of this synapse's function. The hair cell afferent synapse is a ribbon-class synapse, characterized by an electron dense organelle of unknown function, the synaptic body, which appears to hover in the cytoplasm a vesicle's breadth above the active zone. Clear-core vesicles surround the synaptic body, but are also found in the cytoplasm, and associated with the plasma membrane at the active zone. What is the function of the synaptic body, and what is the functional significance of these various pools of vesicles? We will address these issues by determining the precise geometry of the active zone and synaptic body, and map the number and location of synaptic vesicles. By testing the activity-dependence of these measures, we will be able to integrate ultrastructure with ongoing physiological and mathematical modeling studies to build a more detailed understanding of the steps that lead to transmitter exocytosis. We have used electron tomography to reconstruct eleven afferent synapses, and two non-synaptic regions, in frog saccular hair cells. A thick section containing the region of interest was imaged in the IVEM at a series of tilt angles, and the micrographs digitized and back-projected to generate three-dimensional data sets from the two-dimensional projections. By tracing organelles in each plane in which they appeared, we have mapped the locations of nine synaptic bodies, 2573 vesicles, the plasma membranes, presynaptic density, and in some cases, mitochondria and endoplasmic reticulum. Traced organelles were then rendered to reveal their three-dimensional structures and relationships. A shell of vesicles surrounded the synaptic body, and vesicles were distributed uniformly and randomly across the surface, including the space between it and the plasma membrane. At several synapses, the plasma membrane followed the curve of the lower portion of the synaptic body, forming a space one to two vesicles wide between them, and creating a bulge in the cell's surface. In previous, conventional transmission electron micrographs of frog saccular hair cells, synaptic bodies always showed a round profile, arguing that they were spherical. To test this hypothesis, we asked whether spheres fit the synaptic bodies we reconstructed. Our data show that the synaptic body was well fit by a sphere when viewed transected along two perpendicular planes. Since all synaptic bodies were incomplete, it is possible that their unreconstructed portion were not spherical. This is unlikely, however, since each of nine reconstructed synaptic bodies was well fit by spheres, including two which were more than two thirds whole. From these fits, we estimate the average synaptic body diameter to be 470 nm (n = 9). In the cells we study, our capacitance measurements estimated that frog saccular hair cells could maintain exocytosis equivalent to the fusion of 500 vesicles per synapse per second for at least 2 seconds. We therefore counted vesicles at reconstructed synapses to determine the possible ultrastructural basis for this rate. We counted approximately 100 to 200 synaptic body-associated vesicles, which, after correcting for the missing portion of the sphere, gave an average of 376 (n = 9) vesicles per whole synaptic body. Vesicles were packed at 55% of the synaptic body's carrying capacity. Synaptic body-associated vesicles could therefore only account for 752 ms of exocytosis, or 1.37 seconds if all synaptic bodies were loaded at carrying capacity. We also counted outlying vesicles in the reconstructed synapses, and after subtracting the volumes filled by the synaptic body, its associated vesicles, mitochondria, large membranous compartments, and the postsynaptic cytoplasm, we calculated that outlying vesicles occupied 4.2% of available cytoplasm (0.97 um3) in the vicinity of the synapse. To determine whether vesicles were formed at, or actively delivered to synapses, we compared this concentration to the background vesicle density in non-synaptic areas. We used tomography to reconstruct a non-synaptic region of hair cell cytoplasm adjacent to the plasma membrane at an unknown distance from any synapse, but in the basal portion of the cell where synapses are most common. Here, 91 vesicles occupied 0.3% of the available cytoplasm (0.96 um3). Since outlying vesicle concentration was higher in the neighborhood of the synapse, vesicles are either manufactured locally, or translocated toward synapses. Using the background vesicle concentration (0.9% of available volume), and the estimated volumes of three hair cells, we calculated that a hair cell contains approximately 600 000 clear-core vesicles throughout its cytoplasm. If there are 20 afferent synapses per hair cell, then there is a pool of about 30 000 vesicles per synapse, which is two to three times as large as in bipolar terminals. Since some portion of these vesicles will serve non-synaptic functions, and because capacitance measurements estimate that continuous exocytosis may endure for 10 times longer in hair cells than in bipolar, then the larger vesicle pool in hair cells cannot wholly account for the different staminas of exocytosis in these two cells. Miniature postsynaptic potentials (minis), the basis for the quantal hypothesis of transmitter secretion, vary in amplitude at all synapses where they have been observed, and variance in vesicle sizes could account for the mini distribution. Although vesicle diameter distributions from transmission electron micrographs have been reported for many other synapses, including ribbon synapses, data are often pooled between synapses, or are not corrected for sampling biases. Consequently, we have exploited electron tomography to measure many vesicles at single synapses, at high resolution. Dissecting reconstructed volumes in planes one voxel thick allowed us to serial section the synapse with an order of magnitude better resolution than would have been possible with physical sections. We fit the polygon traced at each vesicle's equator to a circle of equal area, and plotted the distribution of equivalent diameters for vesicles at a single synapse, and at all eight synapses. At a single synapse, the synaptic body-associated vesicles (38.8 +/-3.4 nm, n = 139) and outlying vesicles (38.3 +/-3.5 nm, n = 208) were not significantly different, which was also true at

这些研究的长期目标是了解 导致递质快速释放的突触前机制 化学突触。 我们的具体目标是重建三个 毛细胞传入突触的超微结构尺寸 为了测试我们这个突触工作模型的几个方面 功能。 毛细胞传入突触是带状突触， 以功能未知的电子致密细胞器为特征， 突触体，似乎悬浮在囊泡的细胞质中 活动区域上方的宽度。 透明核心囊泡围绕 突触体，但也存在于细胞质中，并且相关 质膜位于活性区。 的作用是什么 突触体，以及这些的功能意义是什么 各种囊泡池？ 我们将通过以下方式解决这些问题 确定活动区和突触体的精确几何形状， 并绘制突触小泡的数量和位置。 经过 测试这些措施的活动依赖性，我们将能够 将超微结构与正在进行的生理和数学相结合 建模研究以更详细地了解步骤 导致递质胞吐作用。 我们使用电子断层扫描 重建十一个传入突触和两个非突触区域， 在青蛙的囊毛细胞中。 包含区域的厚部分 兴趣在 IVEM 中以一系列倾斜角度成像，并且 显微照片数字化和反投影以生成三维 来自二维投影的数据集。 通过追踪细胞器 在它们出现的每个平面中，我们都绘制了它们的位置 九个突触体，2573 个小泡，质膜，突触前 密度，在某些情况下，还包括线粒体和内质网。 然后渲染追踪的细胞器以揭示它们的三维空间 结构和关系。 周围有一层囊泡壳 突触体、小泡均匀随机分布 整个表面，包括表面和等离子体之间的空间 膜。 在几个突触处，质膜遵循曲线 突触体下部，形成一到两个空间 囊泡之间很宽，并在细胞内形成一个凸起 表面。 在以前的传统透射电子显微照片中 青蛙囊毛细胞的突触体总是呈圆形 轮廓，认为它们是球形的。 为了检验这个假设， 我们询问球体是否适合我们重建的突触体。 我们的数据表明，当突触体与球体吻合时 沿两个垂直平面横切观察。 由于所有突触 尸体不完整，可能是未重建的 部分不是球形的。 然而，这是不太可能的，因为每个 九个重建的突触体与球体完美契合，包括 两个，超过了整体的三分之二。 从这些拟合中，我们 估计平均突触体直径为 470 nm (n = 9)。 在 我们研究的细胞，我们的电容测量估计青蛙 囊状毛细胞可以维持相当于融合的胞吐作用 每个突触每秒 500 个囊泡，持续至少 2 秒。 我们 因此，对重建突触处的囊泡进行计数以确定 该速率可能的超微结构基础。 我们数过 大约 100 至 200 个突触体相关囊泡， 校正球体缺失部分后，给出 每个突触体平均有 376 (n = 9) 个囊泡。 囊泡 被包装为突触体承载能力的 55%。 突触 因此，与身体相关的囊泡只能占 752 ms 胞吐作用，或 1.37 秒（如果所有突触体均加载） 承载能力。 我们还计算了外围囊泡 重建突触，并减去填充的体积后 突触体、其相关囊泡、线粒体、大 我们计算了膜室和突触后细胞质 外围囊泡占据了可用细胞质的 4.2% (0.97 um3) 在突触附近。 为了确定囊泡是否 形成于或主动传递至突触，我们比较了这一点 浓度到非突触区域的背景囊泡密度。 我们使用断层扫描重建毛细胞的非突触区域 细胞质与质膜相邻，距离未知 任何突触，但在突触所在的细胞基底部分 最常见。 这里，91 个囊泡占据了可用囊泡的 0.3% 细胞质（0.96 um3）。 由于外围囊泡浓度较高 在突触附近，要么制造囊泡 局部或向突触转移。 使用背景 囊泡浓度（可用体积的 0.9%），以及估计的 三个毛细胞的体积，我们计算出一个毛细胞包含 其细胞质中约有 600 000 个透明核心囊泡。 如果每个毛细胞有 20 个传入突触，那么就有一个池 每个突触大约有 30 000 个囊泡，是普通突触的两到三倍 大如双极端子。 由于这些囊泡的某些部分 将服务非突触功能，并且因为电容 测量估计连续胞吐作用可能持续 10 毛细胞比双极细胞长几倍，然后是较大的囊泡 毛细胞中的池不能完全解释不同的耐力 这两个细胞中的胞吐作用。 微型突触后电位 (minis)，发射机量子假设的基础 分泌，其所在的所有突触的幅度各不相同 观察到，囊泡大小的差异可以解释迷你 分配。 尽管囊泡直径分布 透射电子显微照片已被报道为许多其他 突触，包括带状突触，数据通常汇集在 突触，或者没有纠正采样偏差。 因此，我们 利用电子断层扫描技术一次性测量许多囊泡 突触，高分辨率。 剖析重建的体积 一体素厚的平面使我们能够对突触进行连续切片 分辨率比可能的分辨率高一个数量级 与物理部分。 我们拟合每个囊泡处描绘的多边形 将赤道画成等面积的圆，并绘制其分布 单个突触处囊泡的等效直径，以及 八个突触。 在单个突触处，突触体相关 囊泡（38.8 +/-3.4 nm，n = 139）和外围囊泡（38.3 +/-3.5 nm，n = 208）没有显着差异，这在