In vivo translational analysis in neurons

描述（由申请人提供）：一个多世纪以来，人们已经知道神经元具有不同的解剖规格。由于神经元可以有许多树突，每个树突可以有许多突触，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。在表明树突具有化学区隔性方面已经取得了进展。这种区隔化可以通过特定树突区域钙离子水平的刺激变化来证明。这些特征表明树突不是均匀的，不仅表现出形态的异质性，而且表现出功能的异质性。树突生物学的一个主要问题是如何刺激完整组织中树突的选定区域导致细胞反应？这被称为树突整合，它的许多表征使用电生理和Ca++输出作为树突功能的指标。然而，在树突中发生的mRNA靶向和局部翻译的其他生理过程也是树突介导的生理（包括突触可塑性）的重要调节剂。树突翻译发生在树突长度上被称为“热点”的位点上，舒曼和埃伯温实验室首次同时证明了这一点。最近，来自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翻译过程。这些数据，以及其他数据，强调了分析树突翻译的时空动态的基本需要，以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分散的培养神经元，虽然适合许多实验问题，但越来越清楚的是，正常微环境中的细胞在细胞生物学（包括RNA表达）方面与体外细胞的功能不同。本应用中的实验将定义在其自然微环境中完整神经元树突中多mrna翻译的基本方面。这些数据将首次量化微环境通过调节局部树突蛋白合成的动态来调节神经元生理的作用。

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.