Molecular Multi-Species Approach for Trans-Synaptic Labeling of Neural Circuits

项目摘要/摘要 据估计，人脑含有压倒性的1015个突触，这些突触是正常大脑的基本结构。 神经回路的功能。我们对形成这些关键信令点的连接的了解，甚至 最简单的脊椎动物神经系统都严重缺乏。因此，这一大脑计划的一个明确目标是 开发和验证新的工具，以促进复杂电路的详细分析，并提供对 构成大脑功能基础的细胞相互作用“。这个多PI协作项目就是为了实现这一目标。 它利用了一种强大的基因技术--反式探戈，这种技术可以将信号通过突触引导到 识别突触前神经元及其特定的突触后靶点。拟议方案的总体目标 实验有三个方面：第一，我们将适应跨Tango顺行跨突触信号平台， 它最初是在果蝇模型中建立并成功实施的，对脊椎动物的大脑- 斑马鱼。斑马鱼是首选的有机体，因为它有能力检测反式探戈成分 通过将质粒构建物注射到1-细胞胚胎中高效，并容易和快速地产生 转基因动物在确定的神经元种群中激活反式探戈。第二，我们将独立和 严格验证反式探戈揭示的神经连接是功能性突触连接， 利用光遗传学、成像技术和先进的显微技术。由于它的透明度， 斑马鱼幼体非常适合于使用光学方法在体内验证突触连接。第三，我们将 开发一种新的逆行版本的反式探戈，它将允许识别给定的突触前输入 突触后神经元。Trans-Tango的模块化允许高效地重新配置和优化 用于精确电路的系统 地图 平。“复古探戈”版本将首先应用于果蝇，在此基础上 从转基因探戈的建立中吸取的经验教训，一旦优化，就会被引入紧张的斑马鱼 系统。通过组装所提出的用于顺行和逆行跨突触追踪的遗传工具包， 无脊椎动物和脊椎动物神经系统，我们预计这些技术将被广泛应用于 神经科学界，并应用于其他实验模型。这项建议的优点是 创新战略用于 地图 神经连接，令人信服的初步数据，以及独特的 在分子遗传学、电路神经科学和显微镜设计方面的互补专业知识 合作的研究人员为该项目带来了帮助。

PROJECT SUMMARY/ABSTRACT It is estimated that the human brain contains an overwhelming 1015 synapses, structures essential for the normal functioning of neural circuits. Our knowledge of the connections that form these critical signaling sites, in even the simplest vertebrate nervous systems, is sorely lacking. Thus, a stated goal of this BRAIN initiative is to “develop and validate novel tools to facilitate the detailed analysis of complex circuits and provide insights into cellular interactions that underlie brain function”. This multi-PI collaborative project precisely addresses this goal. It takes advantage of a powerful genetic technique, trans-Tango, that directs signaling across synapses to identify both pre-synaptic neurons and their specific post-synaptic targets. The overall objectives of the proposed experiments are three-fold: First, we will adapt the trans-Tango anterograde trans-synaptic signaling platform, which was initially established and successfully implemented in the Drosophila model, to a vertebrate brain - that of the zebrafish. The zebrafish is the organism of choice because of the ability to assay trans-Tango components efficiently from injections of plasmid constructs into 1-cell embryos, and the ease and rapidity of generating transgenic animals to activate trans-Tango in defined neuronal populations. Second, we will independently and rigorously validate the neural connections revealed by trans-Tango as functional synaptic connections, capitalizing on optogenetics, imaging techniques, and advanced microscopy methods. Owing to its transparency, the larval zebrafish is ideally suited to verify synaptic connectivity in vivo using optical approaches. Third, we will develop a new retrograde version of trans-Tango, which will allow identification of the pre-synaptic input of given post-synaptic neurons. The modularity of trans-Tango permits efficient reconfiguration and optimization of the system for accurate circuit map ping. The “retro-Tango” version will first be applied to Drosophila, building upon lessons learned from the establishment of trans-Tango and, once optimal, introduced to the zebrafish nervous system. By assembling the proposed genetic toolkit for anterograde and retrograde trans-synaptic tracing in both invertebrate and vertebrate nervous systems, we expect these techniques to become widely used by the neuroscience community and applied to additional experimental models. The strengths of this proposal are the innovative strategies used to map neural connectivity, the compelling preliminary data, and the unique and complementary expertise in molecular genetics, circuit neuroscience and microscopy design that the collaborating researchers bring to the project.