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

神经回路跨突触标记的分子多物种方法

基本信息

批准号：
10009743
负责人：
Gilad Barnea
金额：
$ 273.18万
依托单位：
DARTMOUTH COLLEGE
依托单位国家：
美国
项目类别：
财政年份：
2020
资助国家：
美国
起止时间：
2020-08-01 至 2024-07-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10009743
关键词：
ARNT gene Address Adopted BRAIN initiative Behavior Biological Assay Brain Brain Diseases Brain imaging Calcium Cell Surface Receptors Cells Chimeric Proteins Communities Complex Cre-LoxP Data Dendrites Drosophila genus Experimental Models Fishes Genetic Genetic Recombination Genetic Techniques Genetic Transcription Genome Glucagon Goals Human Imaging Techniques Injections Invertebrates Knowledge Label Life Ligand Binding Ligands Maps Measurement Mediating Methods Microscope Microscopy Modeling Modification Molecular Molecular Genetics Monitor Names Nervous system structure Neuraxis Neurons Neurophysiology - biologic function Neurosciences Neurosciences Research Noise Optics Organism Pathway interactions Phase Plasmids Population Process Proteins Proteolysis Reagent Receptor Activation Reporter Reporter Genes Reproducibility Research Personnel Rest Signal Pathway Signal Transduction Site Structure Synapses System Technical Expertise Techniques Testing Transgenic Animals Transgenic Organisms Work Zebrafish base design embryo cell experimental study fly in vivo innovation insight interest neural circuit novel novel strategies optogenetics postsynaptic neurons presynaptic neurons receptor relating to nervous system response sensor tool two-photon voltage

项目摘要

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.

项目概要/摘要据估计，人脑包含数量惊人的 1015 个突触，这些突触是正常运作所必需的结构。我们对形成这些关键信号位点的连接的了解，甚至在这些方面。因此，该 BRAIN 计划的既定目标是： “开发和验证新颖的工具，以促进复杂电路的详细分析并提供洞察 “大脑功能背后的细胞相互作用”。这个多 PI 协作项目正是解决了这一目标。它利用了强大的遗传技术 trans-Tango，可将信号传导穿过突触确定突触前神经元及其特定的突触后目标。实验分为三部分：首先，我们将采用 trans-Tango 顺行跨突触信号平台，最初在果蝇模型中建立并成功应用于脊椎动物大脑 - 斑马鱼是首选生物体，因为它能够检测反式 Tango 成分。高效地将质粒构建体注射到 1 细胞胚胎中，并且生成的简便性和快速性其次，我们将独立且独立地利用转基因动物来激活特定神经群体中的反式Tango。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，斑马鱼幼虫非常适合使用光学方法验证体内突触连接。开发新的 trans-Tango 逆行版本，这将允许识别给定的突触前输入 trans-Tango 的模块化允许有效地重新配置和优化突触后神经元。精确电路系统地图 “retro-Tango”版本将首先应用于果蝇。从建立 trans-Tango 中吸取的经验教训，一旦达到最佳效果，就引入斑马鱼神经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的神经系统，我们预计这些技术将被广泛使用神经科学界并应用于其他实验模型。创新策略用于地图神经连接、令人信服的初步数据以及独特且分子遗传学、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为该项目带来了成果。