Validating GFP Reconstitution Across Synaptic Partners (GRASP) Methods to Dissect

描述（由申请人提供）：该申请涉及广泛的挑战领域（06）使能技术和特定的挑战主题，06- ns -106：验证研究大脑连接的新方法。绘制神经回路的结构和功能是理解相互连接的神经元群如何产生感知和驱动行为的重要前提。绘制神经回路图的一个挑战是明确地识别突触伙伴。传统上，突触连通性是通过电生理学和电子显微镜来研究的，这些方法提供了关键的细节，但不可能大规模应用。我们的目标是开发和验证一个系统，以更容易地识别小鼠选择性标记神经元之间的突触。最近，一种名为跨突触伙伴GFP重构（GRASP）的系统在无脊椎动物中被开发出来，用于研究突触连接。它依赖于两个非功能性互补的GFP片段的遗传表达，这些片段暴露在不同细胞群的细胞外侧面。绿色荧光蛋白重组，因此荧光，发生在这些细胞之间的密切接触的位置（如突触）。GRASP有许多优点：它可以针对特定的神经元群体进行遗传靶向，它是一个荧光系统，可以很容易地使用传统显微镜进行可视化，并且可以很容易地适应于回答关于神经元突触连接的各种不同问题。为了验证该技术在哺乳动物系统中的应用，我们将首先在细胞培养和昆虫模型中测试一组GRASP构建物。最有希望的组合将用于产生通用的转基因系，可以与Cre/LoxP和tet-TTA系统协同使用，以控制时间和空间上的GRASP表达。此外，我们将开发用于GRASP的病毒载体，作为递送和空间限制的替代手段。我们将使用GRASP来帮助解决哺乳动物味觉系统中的连接问题，从而验证其在研究哺乳动物神经回路中的实用性。最终，我们预计在本研究中产生的基因工程小鼠系和病毒载体将为整个神经科学界提供一个具有相当价值的工具箱。

DESCRIPTION (provided by applicant): This application addresses broad Challenge Area (06) Enabling Technologies, and specific Challenge Topic, 06-NS-106: Validating new methods to study brain connectivity. Mapping the structure and function of neural circuits is an important prerequisite to understand how groups of interconnected neurons produce perceptions and drive behavior. One challenge in mapping neural circuits is to unambiguously identify synaptic partners. Traditionally, synaptic connectivity has been studied using electrophysiology and electron microscopy - methods that provide critical detail but are impossible to apply in large scale. We aim to develop and validate a system to more easily identify synapses between selectively tagged neurons in the mouse. Recently, a system named GFP Reconstitution Across Synaptic Partners (GRASP), has been developed in invertebrates to study synaptic connectivity. It relies on genetic expression of two non-functional, complementary GFP fragments that are exposed on the extracellular sides of different cell populations. GFP reconstitution, and therefore fluorescence, occurs at the sites of close contact (e.g. synapses) between these cells. GRASP has many advantages: it can be genetically targeted to specific neuronal populations, it is a fluorescent system that can be readily visualized using traditional microscopy, and can be easily adapted to answer a wide variety of different questions about synaptic connectivity of neurons. To validate this technology for use in mammalian systems, we will initially test a battery of GRASP constructs in cell culture and an insect model. The most promising combinations will then be used to generate general-use transgenic lines that can be employed in concert with the Cre/LoxP and the tet-TTA systems to control expression of GRASP in time and space. In addition, we will develop viral carriers for GRASP as an alternate means of delivery and spatial restriction. We will use GRASP to help address questions of connectivity in the mammalian taste system, thereby providing validation of its utility to study mammalian neural circuits. Ultimately we anticipate that the genetically engineered GRASP mouse lines and viral vectors generated in this study will provide a toolbox that will be of considerable value for the entire neuroscience community. PUBLIC HEALTH RELEVANCE: Mapping the structure and function of neural circuits is an important prerequisite to understand how groups of interconnected neurons produce perceptions and drive behavior. We aim to develop and validate a system to more easily identify synapses between selectively labeled neurons in the mouse. We anticipate that our genetically engineered GRASP mouse lines and viral vectors will provide a toolbox that will be of considerable value for the entire neuroscience community.