Genetic indicators for dynamic imaging of neuronal signaling in plasticity and de

描述(申请人提供)：在过去的十年中，在成像、分子生物学和生物化学方面取得了惊人的进步，使单一蛋白质在体内的行为得以可视化。在这里，我建议开发和可视化活的神经元内细胞内信号的时间和空间动力学。就像早期的钙成像工作通过定义钙内流的时空特征来重新定义我们对钙内流重要性的理解一样，我相信可视化细胞内信号的空间和时间动力学将对我们理解神经系统有类似的好处。最初，我们已经开发了指示剂，可以可视化许多细胞内信号级联过程中的关键第一步之一：酪氨酸磷酸化。在过去的几年里，我们开发了一种系统，它依赖于对基因编码的磷酸化荧光指示剂的变化进行比率成像。我们现在提出三个具体目标，将这些工具开发成一个系统，用于监测神经元可塑性和发育过程中的信号。我们建议：1)开发一个指标库，以报告特定激酶的活性；2)开发定位于特定细胞间隔的指标；3)开发同时报告多个信号分子活性的指标。使用我们的指示器，工作人员将能够阐明突触可塑性背后的信号动力学。因此，我们的工具将使我们能够对神经元可塑性背后的基本机制有新的见解。公共卫生相关性：项目叙述性神经元可塑性是大脑中许多基本功能的基础，而异常的神经元可塑性与疾病有关。过度可塑性可能是癫痫和成瘾等疾病的基础，而可塑性缺陷可能在癫痫、神经退行性疾病和自闭症谱系障碍中发挥重要作用。我们的研究将通过开发新的工具来可视化具有亚细胞分辨率的动态神经元信号，从而在所有这些水平上产生广泛的影响。

DESCRIPTION (provided by applicant): During the past decade stunning advances have been made in imaging, molecular biology and biochemistry that enable the visualization of the behavior of single proteins in vivo. Here, I propose to develop and visualize the temporal and spatial dynamics of intracellular signaling within living neurons. Much as early work in calcium imaging redefined our understanding of the importance of calcium influx by defining its spatial and temporal characteristics, I believe visualizing the spatial and temporal dynamics of intracellular signaling will have similar benefits to our understanding of the nervous system. Initially we have developed indicators that enable visualization of one of the key first steps in many intracellular signaling cascades: tyrosine phosphorylation. During the past several years, we have developed a system that relies on ratiometric imaging of changes in a genetically encoded fluorescent indicator of phosphorylation. We now propose three specific aims to develop these tools into a system for monitoring signaling during neuronal plasticity and development. We propose to: 1) Develop a library of indicators targeted to report activity of specific kinases; 2) Develop indicators that localize to specific cellular compartments; 3) Develop indicators to report activity of multiple signaling molecules simultaneously. Using our indicators, workers will be able to elucidate the dynamics of signals that underlie synaptic plasticity. Thus, our tools will enable novel insights into essential mechanisms that underlie neuronal plasticity. PUBLIC HEALTH RELEVANCE: Project Narrative Neuronal plasticity underlies many fundamental functions within the brain, while abnormal neuronal plasticity is associated with disease. Excessive plasticity may underlie diseases like epilepsy and addiction, while defects in plasticity could play important roles in epilepsy, neurodegenerative, and autism spectrum disorders. Our research will have broad impacts across all these levels by developing new tools to visualize dynamic neuronal signaling with subcellular resolution.