EAGER: Development of the Novel Recombinase System for the Intersectional Dissection of Neural Circuitry

对脑细胞多样性和细胞间通讯（通过称为突触的细胞间专门连接的连接）的全面理解对于理解脑功能的解剖学基础和细胞机制至关重要。阐明特定神经回路的详细突触组织是实现这一目标的必要组成部分。神经科学这一领域的进展受到了工具的限制，这些工具可以同时揭示同一大脑区域内具有不同分子和生理特性的多种细胞类型，并详细强调这些细胞如何相互连接。该项目利用一种新的分子生物学策略来定义小鼠大脑中的多种细胞类型及其相互联系，使研究人员能够标记表达工程遗传元件组合的单个细胞。这项技术与现有的方法相结合，该方法将荧光分子的两个不完整部分插入到不同神经细胞的表面。如果这些细胞通过相互形成连接（突触）进行通信，荧光分子的片段就会紧密接触，重新发挥功能，突触就可以被识别为彩色的荧光点。研究生和本科生都将参与这些工具的开发和优化，遗传构建体将通过非营利机构分发;所有协议和序列信息将通过PI的网站广泛提供。这些新工具将大大提高神经科学家研究大脑回路详细组织以及特定神经元突触连接的速度和精度。该项目介绍了新的分子和病毒策略，用于定义多种细胞类型及其突触组织。它是基于一种新的病毒策略，以实现在表达Cre，翻转酶（FLP）和其他重组酶的转基因小鼠系中特定神经元群体之间的转基因交叉表达。一种新的位点特异性重组酶系统（phiC 31和phiC 31依赖性单反向开放阅读框[pSIO]）用于在体外和体内实现病毒介导的特异性转基因表达，而不与其他重组酶发生交叉反应。然后将该phiC 31/pSIO重组酶系统与“增强型绿色荧光蛋白跨突触伴侣重建”（eGRASP）组合以标记由特定输入产生的突触接触。通过提供一个新的实验框架，用于识别局部神经回路的细胞成分，并检查特定神经元上的多个突触前输入的突触组织，并通过优化这些工具以供普遍使用，并使其免费提供给科学界，该项目有望加速对神经回路的组织和功能特性的发现。该奖项反映了美国国家科学基金会的法定使命并通过使用基金会的知识价值和更广泛的影响审查标准进行评估，被认为值得支持。

A comprehensive understanding of brain cell diversity and cell-to-cell communication (connectivity via specialized junctions between cells called synapses) is essential for understanding the anatomical substrates and cellular mechanisms that underlie brain function. Elucidation of the detailed synaptic organization of specific neural circuits is a necessary component for achieving this goal. Progress in this area of neuroscience has been limited by a lack of tools that can simultaneously reveal multiple cell types with distinct molecular and physiological properties within the same brain areas, as well as highlighting in detail how these cells are connected to each other. This project utilizes a novel molecular-biological strategy for defining multiple cell types and their interconnections in mouse brains, enabling investigators to mark individual cells that express combinations of engineered genetic elements. This technique is combined with an existing method that inserts two incomplete parts of a fluorescent molecule onto the surfaces of different nerve cells. If those cells communicate by forming a connection (synapse) with each other, the fragments of the fluorescent molecule come into close enough contact to become functional again, and synapses are identifiable as colored dots of fluorescent light. Both graduate and undergraduate students will be involved in the development and optimization of these tools, and the genetic constructs will be distributed via a non-profit agency; all protocols and sequence information will be made widely available through the PI's website. These new tools will greatly enhance the speed and precision with which neuroscientists can study the detailed organization of brain circuitry, and the synaptic connections made onto specific neurons. This project introduces novel molecular and viral strategies for defining multiple cell types and their synaptic organization. It is based on a novel viral strategy to achieve the intersectional expression of transgenes among specific neuronal populations in transgenic mouse lines that express Cre, flippase (FLP), and other recombinases. A novel site-specific recombinase system (phiC31 and a phiC31-dependent single inverted open reading frame [pSIO]) is used to acheive virus-mediated, specific-transgene expression without cross-reactivity to other recombinases, both in vitro and in vivo. This phiC31/pSIO recombinase system is then combined with "enhanced Green fluorescent protein Reconstitution Across Synaptic Partners" (eGRASP) to label synaptic contacts made by specific inputs. By providing a novel experimental framework for identifying the cellular constituents of local neural circuits and examining the synaptic organization of multiple presynaptic inputs onto specific neurons, and by optimizing these tools for general use and making them freely available to the scientific community, this project is expected to accelerate discovery about the organizational and functional properties of neural circuitry.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.