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

项目概要/摘要 据估计，人类大脑包含压倒性的1015个突触，这些突触是正常大脑活动所必需的结构。 神经回路的功能。我们对形成这些关键信号位点的连接的了解，即使是在 最简单的脊椎动物神经系统，是非常缺乏的。因此，BRAIN倡议的一个既定目标是 “开发和验证新的工具，以促进复杂电路的详细分析，并提供以下方面的见解： 细胞间的相互作用是大脑功能的基础。这个多PI合作项目正是为了实现这一目标。 它利用了一种强大的遗传技术trans-Tango，这种技术可以引导信号穿过突触， 识别突触前神经元及其特定的突触后靶点。建议的总体目标 实验有三个方面：首先，我们将调整trans-Tango顺行跨突触信号平台， 最初在果蝇模型中建立并成功实施，到脊椎动物的大脑- 斑马鱼。斑马鱼是生物体的选择，因为它有能力分析反式探戈成分 有效地从质粒构建体注射到1-细胞胚胎中，并且产生的容易和快速 转基因动物激活trans-Tango在确定的神经元群体。第二，我们将独立和 严格验证trans-Tango揭示的神经连接作为功能性突触连接， 利用光遗传学、成像技术和先进的显微镜方法。由于其透明度， 幼鱼非常适合使用光学方法验证体内突触连接性。三是 开发一种新的逆行版本的trans-Tango，这将允许识别给定的突触前输入。 突触后神经元trans-Tango的模块化允许有效的重新配置和优化 用于精确电路系统 地图 平.“复古探戈”版本将首先应用于果蝇， 从建立trans-Tango的经验教训，一旦优化，引入斑马鱼神经 系统通过组装所提出的遗传工具包，在两个组织中进行顺行和逆行跨突触追踪， 无脊椎动物和脊椎动物的神经系统，我们希望这些技术成为广泛使用的 神经科学界和应用到其他实验模型。这项建议的优点是， 创新战略， 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， 合作研究人员带来的项目。

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.