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

描述（由申请人提供）：GRASP（GFP Reconstitution Across Synaptic Partners）最初在无脊椎动物中开发，是一种遗传可控的基于荧光的系统，用于识别两种细胞（或细胞群）之间的接触位点。它依赖于膜栓系分裂（即非功能性）GFP模块的表达，这些模块必须聚集在一起以重建GFP荧光（spGFP 1 -10和spGFP 11）;由于这种功能互补需要膜之间的紧密并置（<100 nm），GRASP是一种强大的系统来识别两个细胞之间的突触。本申请的目的是开发和验证GRASP方法用于哺乳动物和果蝇的神经回路。我们将生成一组GRASP工具，包括用于靶向表达spGFPs的病毒载体，在Cre或四环素诱导系统的控制下产生表达spGFPs的转基因（和敲入）系，并在正交表达控制系统下工程化含有GRASP组分的果蝇染色体。总之，这些试剂将提供GRASP表达的时间和空间选择性，并为电路映射提供多功能平台。我们建议在两种不同的模型中测试这些组件：苍蝇中的热敏电路屏幕和小鼠中的Cre报告屏幕。我们还将设计一种新的“突触指纹”技术。我们希望这些研究的结果，以显着提高武器库的工具可用于电路映射，并有助于正常和患病的神经系统的特性。 公共卫生相关性：映射神经回路的连接性是理解神经元集合如何处理感觉刺激和驱动行为的重要前提。为了实现这一目标，我们将开发和验证新的策略，以更容易地识别小鼠中定义的神经元群体之间的突触。我们预计，诱导型和多色GRASP，以及新的Grainbow转基因系和病毒载体将提供一个工具箱，这将是相当大的价值，为整个神经科学界。

DESCRIPTION (provided by applicant): GRASP (GFP Reconstitution Across Synaptic Partners), originally developed in invertebrates, is a genetically controllable fluorescence-based system for identifying sites of contact between two cells (or cell populations). It relies on the expression of membrane-tethered split (i.e. non-functional) GFP modules that must come together to reconstitute GFP fluorescence (spGFP1-10 & spGFP11); since this functional complementation requires close apposition between membranes (<100 nm), GRASP is a powerful system to identify synapses between two cells. The objective of this application is to develop and validate GRASP methods for use in mammalian and Drosophila neural circuits. We will generate a battery of GRASP tools, including viral vectors for targeted expression of spGFPs, produce transgenic (and knock-in) lines expressing spGFPs under the control of the Cre or tetracycline inducible systems, and engineer Drosophila chromosomes harboring GRASP components under orthogonal expression control systems. Together, these reagents will afford temporal and spatial selectivity of GRASP expression, and provide a versatile platform for circuit mapping. We propose to test these components in two different models: a thermosensory circuit screen in flies and a Cre reporter screen in mice. We will also engineer a multicolor GRASP as a novel "synaptic fingerprinting" technique. We expect the results of these studies to significantly enhance the arsenal of tools available for circuit mapping, and be helpful in the characterization of normal and diseased nervous systems. PUBLIC HEALTH RELEVANCE: Mapping the connectivity of neural circuits is an important prerequisite to understand how neuronal ensembles process sensory stimuli and drive behavior. Towards this goal, we will develop and validate new strategies to more easily identify synapses between defined neuronal populations in the mouse. We anticipate that inducible and multi-color GRASP, as well as the novel Grainbow transgenic lines and viral vectors will provide a toolbox that will be of considerable value for the entire neuroscience community.