MORPHOCHEMISTRY OF CEREBELLAR BRAINSTEM NEURONS AND CIRCUITS

这个项目的总体目标是定义 小脑钙信号的时空特性 神经元。拟议的实验将集中在P型钙通道上， 血浆钙泵与钙浓度管理 分子。需要检验的假设和具体目标分为两个 类别：1)P型钙通道信令和管理 我们提出：i)P型钙通道定位于 浦肯野，颗粒，树突和体细胞上的突触连接， 和小脑核神经元，这种定位与 神经元整合和第二信使信号。二)为了 限制钙浓度水平，钙泵(ATPase)是 分布于浦肯野细胞树突枝上的点状部位 最高浓度出现在树突和棘突的远端 与P通道密切相关。Iii)P型钙通道和 血浆钙泵共同定位，并与移动和 结合的钙缓冲分子，如钙调蛋白和钙调神经磷酸酶，它们 进一步提高这些神经元的[Ca~(2+)]i调节能力。2)用于 我们提出的IP3细胞内钙信号和管理系统 内质网与IP3受体相关 钙通道和顺畅的ER钙泵，形成了一个框架， 建立细胞内钙信号的功能微域并 管理层。这些研究将在大鼠的小脑中进行 在光和电子上使用免疫细胞化学技术 微观层面。超微结构研究将利用最近的 蛋白质大分子高分辨定位研究进展， 我们已经开发了改进的方法来对组织切片进行预处理，并且 最近产生的针对P通道的特异性抗体。一个 对质膜和质膜的形态基础的认识 膜内钙浓度管理系统，以及 细胞内钙调控中各系统间的关系 浓缩，并提供了一种从 神经元是了解神经元功能的关键要素。

The overall goal of this project is to define the morphological basis for the spatial and temporal properties of calcium signaling in cerebellar neurons. The proposed experiments will focus on P-type calcium channels, plasmalemmal calcium pumps, and calcium concentration management molecules. The hypotheses to be tested and specific aims fall into two categories: 1) For the P-type calcium channel signaling and management system we propose that: i) P-type calcium channels are localized at synaptic junctions on the dendritic tree and somata of Purkinje, granule, and cerebellar nuclear neurons and that this localization relates to neuronal integration and second messenger signaling. ii) In order to restrict calcium concentration levels, calcium pumps (ATPases) are distributed at punctate sites over the dendritic arbor of Purkinje cells with the highest concentrations occurring distally on dendrites and spines in close association with P channels. iii) P-type calcium channels and plasmalemmal calcium pumps are co-localized and associated with mobile and bound calcium-buffering molecules, such calmodulin and calcineurin, which further increase the [Ca2+]i regulation abilities of these neurons. 2) For the IP3 intracellular calcium signaling and management system we propose that the endoplasmic reticulum, in association with IP3 receptor-related calcium channels and smooth ER calcium pumps, form a framework which establishes functional microdomains of intracellular calcium signaling and management. The studies will be carried out in the cerebellum of the rat using immunocytochemical techniques at both the light and electron microscopic levels. Ultrastructural studies will take advantage of recent advances in the high resolution localization of protein macromolecules, improved methods we have developed to pre-treat tissue sections, and the recent generation of specific antibodies against the P channel. An understanding of the morphological basis of both the plasmalemmal and the endomembrane calcium concentration management systems, and the relationship between these systems in controlling the cytosolic calcium concentration and in providing a mechanism for extruding calcium from neurons are critical elements in understanding neuronal function.