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

验证跨突触伙伴 (GRASP) 的 GFP 重建解剖方法

• 批准号: 7831834
• 负责人: CHARLES S ZUKER
• 金额: $ 40.12万
• 依托单位: COLUMBIA UNIVERSITY HEALTH SCIENCES
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-09-30 至 2011-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7831834
• 关键词: Address; Area; Automobile Driving; Behavior; Brain; Caenorhabditis elegans; Carrier Proteins; Cell Culture Techniques; Cells; Color; Communities; Drosophila genus; Electron Microscopy; Electrophysiology (science); Engineering; Extracellular Domain; Fiber; Fluorescence; Ganglia; Genetic; Goals; Individual; Insecta; Invertebrates; Label; Maps; Membrane; Membrane Proteins; Methods; Microscopy; Modality; Modeling; Modification; Molecular; Mus; Myxoid cyst; Names; Nervous system structure; Neurons; Neurosciences; Output; Perception; Population; Receptor Cell; Reporter; Savory; Sensitivity and Specificity; Side; Signal Transduction; Site; Specificity; Staining method; Stains; Structure; Synapses; Synaptic Membranes; System; TTA System; Taste Buds; Taste Perception; Technology; Testing; Tetanus Helper Peptide; Time; Toxic effect; Transgenic Mice; Transgenic Organisms; Validation; Variant; Viral; Viral Vector; cell type; driving behavior; extracellular; fly; in vivo; interest; neural circuit; public health relevance; reconstitution; reconstruction; skills; stimulus processing; tool

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.

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，以及具体的挑战主题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图是理解相互连接的神经元组如何产生感知和驱动行为的重要前提。映射神经回路的一个挑战是明确地识别突触伙伴。传统上，突触连接的研究一直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种系统，以便更容易地识别小鼠中选择性标记的神经元之间的突触。最近，在无脊椎动物中开发了一个名为跨突触伙伴的GFP重构系统(GRASH)，用于研究突触连接。它依赖于两个非功能性的、互补的GFP片段的遗传表达，这两个片段暴露在不同细胞群体的胞外侧。GFP重组，因此荧光，发生在这些细胞之间紧密接触的位置(例如突触)。GRAP有许多优点：它可以在基因上针对特定的神经元群体，它是一个很容易用传统显微镜可视化的荧光系统，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。为了验证这项技术在哺乳动物系统中的应用，我们首先将在细胞培养和昆虫模型中测试一组GRAP结构。然后，最有希望的组合将被用来产生通用的转基因株系，这些转基因株系可以与Cre/loxP和tet-TTA系统一起使用，在时间和空间上控制GRAP的表达。此外，我们还将开发GRAP病毒载体，作为替代递送工具和空间限制。我们将使用GRAPH来帮助解决哺乳动物味觉系统中的连通性问题，从而验证其在研究哺乳动物神经回路方面的有效性。最终，我们预计，在这项研究中产生的基因工程GRAPH小鼠品系和病毒载体将提供一个工具箱，将对整个神经科学界具有相当大的价值。 与公共健康相关：绘制神经回路的结构和功能图是理解相互连接的神经元组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种系统，以更容易地识别小鼠选择性标记神经元之间的突触。我们预计，我们的基因工程GRAP小鼠品系和病毒载体将提供一个工具箱，将对整个神经科学界具有相当大的价值。