In vivo translational analysis in neurons

描述（由申请人提供）：世纪以来，已知神经元具有不同的解剖学规格。由于神经元可以具有许多树突，并且每个树突可以具有许多突触，因此很明显树突是细胞通信和功能的重要调节剂。这些形态特征如何调节细胞功能，自卡哈尔最初观察以来一直是个谜。在显示树突表现出化学区室化方面已经取得了进展。这种区室化的例子是在特定的树突状区域的Ca++水平的刺激变化。这些特征表明，树突是不均匀的，实际上不仅表现出形态异质性，而且功能异质性。树突生物学中的一个主要问题是，刺激完整组织中树突的选定区域如何导致细胞反应？这被称为树突整合，其大部分表征使用电生理和Ca++输出作为树突功能的指标。然而，存在在树突中发生的具有mRNA靶向和局部翻译的其他生理过程，其也是树突介导的生理学（包括突触可塑性）的重要调节剂。树突翻译发生在沿着树突长度的位点，称为“热点”，首先在舒曼和埃伯温实验室同时证明。最近，Eberwine和Kim实验室的体外研究已经证明了一个高度复杂的树突状翻译过程。这些数据，以及其他人，突出的根本需要分析的时间和空间动态的翻译树突了解突触后反应和树突整合的机制。迄今为止，大部分翻译工作都利用了培养中分散的神经元，虽然适用于许多实验问题，但越来越清楚的是，在正常微环境中的细胞在其细胞生物学（包括RNA表达）中可以在功能上不同于其体外对应物。本申请中的实验将定义在其天然微环境中的完整神经元树突中多mRNA翻译的基本方面。这些数据将是第一个量化的作用，微环境在调节神经元生理学通过调制本地化的树突蛋白合成的动力学。

DESCRIPTION (provided by applicant): Neurons have been known to have distinct anatomical specifications for over a century. As neurons can have many dendrites and each dendrite can have many synapses it is clear that dendrites are an important modulator of cellular communication and function. How these morphological features modulate cellular function has been a mystery since the time of Cajal's initial observations. Progress has been made in showing that dendrites exhibit chemical compartmentalization. This compartmentalization is exemplified by stimulated changes in Ca++ levels in specific dendritic areas. These features show that dendrites are not homogeneous and indeed not only exhibit morphological heterogeneity but also functional heterogeneity. One of the dominant questions in dendrite biology is how does stimulation of selected regions of dendrites in the intact tissue result in a cellular response? This has been termed dendritic integration with much of its characterization using electrophysiological and Ca++ outputs as indicators of dendritic function. There are however other physiological processes that occur in dendrites with mRNA targeting and local translation that are also important modulators of dendrite-mediated physiologies including synaptic plasticity. Dendritic translation occurs at sites along the length of the dendrites called "hotspots" first demonstrated simultaneously in the Schuman and Eberwine labs. Recent, in vitro studies from the Eberwine and Kim labs have demonstrated a highly complex dendritic translational process. These data, and those of others, highlight the fundamental need to analyze the temporal and spatial dynamics of translation in dendrites to understand the mechanism of post-synaptic responsiveness and dendritic integration. Much of the translation work to date has utilized dispersed neurons in culture and while appropriate for many experimental questions, it is increasingly clear that cells in their normal microenvironment can be functionally distinct from their in vitro counterparts in their cell biology including RNA expression. Experiments in this application will define the fundamental aspects of multi-mRNA translation in intact dendrites of neurons that are in their natural microenvironment. These data will be the first to quantify the role of the microenvironment in modulating neuronal physiology through modulation of the dynamics of localized dendritic protein synthesis.