ELEMENTAL AND STRUCTURAL ORGANIZATION OF NEURONS AND GLIA

这两部分的项目研究的组织和功能， 神经元和神经胶质中的特殊膜。 第一部分旨在 表征突触活动期间的钙调节 小脑皮质的纤维/浦肯野细胞突触。 新冻结 切片技术，结合扫描透射电子 显微镜（STEM），允许研究协调变化， 胞浆总钙伴随着游离钙的调节 内质网（ER）介导的细胞内钙。 结果表明 （先前描述的）特征钙浓度 树突棘中ER的状态与特定的 细胞质钙水平。 这导致了三种不同的状态， 钙动员，这可能反映了 从细胞内储存释放，然后通过 膜通道 在有刺的树突中，有一类 富含钙的ER，在棘中不存在，表明更复杂 树突中钙调节的模式。 一种新的方法，基于 暗场质量映射的STEM，已被开发用于确定 神经过程中细胞器的原位分子量。 这 这种能力对于揭示蛋白质结合的变化很有价值 和反映突触活动的水含量。 结构分析 直接冷冻制备各种器官型培养物， 海马体继续定义树突和棘的组织 在这个组织的锥体细胞中。 在第二部分中， 在髓磷脂组装的背景下研究专门的膜。 共聚焦显微镜显示雪旺细胞依赖于 微管为基础的细胞内运输和组装髓鞘特异性 proteins. 此外，在神经细胞中合成的各种髓鞘蛋白质， 在trans-Golgi网络中，核周区被分为不同的 运输囊泡，这一过程似乎也是由 微管 有强大的和重要的协会之间 微管和细胞质内的其他细胞骨架成分 节间的通道;施旺细胞ER的组织 似乎取决于这些相互作用。

This two-part project studies the organization and function of specialized membranes in neurons and glia. The first part aims to characterize calcium regulation during synaptic activity in parallel fiber/Purkinje cell synapses of the cerebellar cortex. New frozen sectioning techniques, in combination with scanning transmission electron microscopy (STEM), have permitted studies of coordinated changes in cytoplasmic total calcium which accompany regulation of free intracellular calcium by endoplasmic reticulum (ER). The results show that the (previously described) characteristic calcium concentration states of the ER in dendritic spines are associated with specific cytoplasmic calcium levels. This leads to three distinct states of calcium mobilization, which likely reflect the sequential processes of release from intracellular stores, followed by calcium uptake through membrane channels. In spine-bearing dendrites, there is a class of calcium-rich ER which is not present in spines, suggesting more complex patterns of calcium regulation in dendrites. A new method, based on darkfield mass mapping in the STEM, has been developed for determining the in situ molecular mass of organelles within neuronal processes. This capability has proven valuable for uncovering changes in protein binding and water content that reflect synaptic activity. Structural analysis of directly frozen preparation of various organotypic cultures of hippocampus continues to define the organization of dendrites and spines in the pyramidal cells of this tissue. In Part Two, formation of specialized membranes is studied in the context of myelin assembly. Confocal light microscopy has shown that Schwann cells depend on microtubule based intracellular transport and assembly of myelin-specific proteins. Moreover,the various myelin proteins synthesized in the perinuclear region are sorted in the trans-Golgi network into distinct transport vesicles, and this process also appears to be directed by microtubules. There are strong and important associations between microtubules and other cytoskeletal components within cytoplasmic channels of the internode; the organization of the Schwann cell ER appears to depend on these interactions.