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

描述（由申请人提供）：本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。映射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先决条件。映射神经回路的一个挑战是明确识别突触伙伴。传统上，突触连接的研究是使用电生理学和电子显微镜-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，以更容易地识别选择性标记的神经元之间的突触在小鼠。最近，在无脊椎动物中开发了一个名为GFP跨突触伙伴重建（GRASP）的系统来研究突触连接。它依赖于两个非功能性互补GFP片段的基因表达，这些片段暴露在不同细胞群的细胞外侧。GFP重建，因此荧光，发生在这些细胞之间的密切接触（如突触）的网站。GRASP有许多优点：它可以在遗传上靶向特定的神经元群体，它是一种荧光系统，可以很容易地使用传统的显微镜观察，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的问题。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构建体。最有希望的组合将用于产生通用的转基因株系，其可以与Cre/LoxP和tet-TTA系统一起使用，以控制GRASP在时间和空间上的表达。此外，我们将开发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，本研究中产生的基因工程GRASP小鼠品系和病毒载体将提供一个工具箱，对整个神经科学界具有相当大的价值。 公共卫生相关性：映射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先决条件。我们的目标是开发和验证一个系统，以更容易地识别选择性标记的神经元之间的突触在小鼠。我们预计，我们的基因工程GRASP小鼠系和病毒载体将提供一个工具箱，将是相当大的价值，为整个神经科学界。

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.