In vivo translational analysis in neurons

描述(由申请人提供)：一个多世纪以来，神经元一直被认为具有独特的解剖学特征。由于神经元可以有许多树突，每个树突可以有许多突触，很明显，树突是细胞通讯和功能的重要调节器。自从卡哈尔最初观察到这些形态特征以来，这些形态特征如何调节细胞功能就一直是个谜。在显示树枝晶表现出化学区划方面取得了进展。这种区划的例证是特定树突区域内钙水平的刺激变化。这些特征表明树突并不是均匀的，确实不仅表现出形态的异质性，而且还表现出功能的异质性。树突生物学中的一个主要问题是，刺激完整组织中选定的树突区域是如何导致细胞反应的？这被称为树突状整合，其大部分特征是使用电生理和钙输出作为树突状细胞功能的指标。然而，在树突中还发生了其他的具有mRNA靶向和局部翻译的生理过程，这些过程也是树突介导的生理过程的重要调节者，包括突触可塑性。树突的翻译发生在沿着树突长度的位置，被称为“热点”，首次在舒曼和埃伯文实验室中同时展示。最近，来自Eberwin和Kim实验室的体外研究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突出了分析树突翻译的时间和空间动力学以了解突触后反应和树突整合的机制的基本需要。到目前为止，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，正常微环境中的细胞在细胞生物学包括RNA表达方面可以与体外培养的细胞不同。在这一应用中的实验将定义在自然微环境中完整的神经元树突中多个mRNA翻译的基本方面。这些数据将首次通过调节局部树突状蛋白质合成的动力学来量化微环境在调节神经元生理学中的作用。

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.