Live imaging of neuron circuit assembly in Drosophila olfactory system

项目总结/摘要 大脑的正常功能依赖于特定的神经元连接及其组装成功能电路。 然而，由于电路组装的动态过程的困难，许多详细的机制尚不清楚， 在活体大脑中进行可视化。采用基于活体成像的外植体系统，在这里，我建议调查 细胞生物学基础的神经电路组装使用果蝇嗅觉系统。 在果蝇的嗅觉回路中，50类嗅觉受体神经元（ORN）将它们的轴突发送到 触角叶（AL）中的特定肾小球，在那里它们与相应的细胞进行一对一的连接。 二阶嗅觉投射神经元（PNs）。这提供了一个研究神经元如何形成的优秀系统 在电路组装过程中的特定连接。最近，我开发了一个移植系统， 成像以揭示嗅觉回路组装的动态过程。在这里，我建议使用这个外植体：1。 成像单个ORN轴突如何靶向其肾小球; 2.探索ORN-ORN或ORN-PN相互作用 通过手术切断一条触角神经来调节靶向; 3.描述错误的表型 使用活体成像的果蝇亲latrophilin突变体（Aim 1和Aim 3的一部分，K99训练阶段）。 K99训练阶段的研究主要集中在使用双光子来定义粗目标 基于显微镜的长期成像。为了进一步探索有趣行为背后的详细机制， ORN轴突，先进的成像方法，允许更高的时间和空间分辨率是必需的。 因此，我建议使用自适应光学晶格光片来执行高速双色成像 显微镜结合用于细胞骨架标记和操作的遗传工具， ORN轴突靶向关键步骤的生物学机制。这项研究将与埃里克博士合作 在Janelia Research Campus的Betzig。我们已经从试点实验中获得了有希望的结果。我也会 使用活体成像研究潜在的Latrophilin-Ten-a介导的轴突-轴突或轴突-树突排斥（目的2 和目标3，R 00训练阶段的一部分）。一般来说，R 00培训阶段研究将涉及更多内容 在成像策略和生物学背景下的复杂情况。 我的导师团队由世界著名神经科学家罗立群博士（导师）和康博士组成 Shen（共同导师），经验丰富的细胞生物学家和发育神经生物学家和Eric Betzig （合作者），诺贝尔奖得主和世界著名的显微镜学家，以及博士迈克尔林（顾问），一个 基因成像工具开发专家。在他们的指导下，我相信我将获得广泛的培训， 成为独立私家侦探的过渡期

Project Summary/Abstract Proper functions of the brain rely on specific neuron connections and their assembly into functional circuit. However, many detailed mechanisms of the dynamic process of circuit assembly are unclear due to difficulty to visualize it in live brains. Employing an explant system based live imaging, here I propose to investigate the cell biology underpinnings of neural circuit assembly using the Drosophila olfactory system. In the Drosophila olfactory circuit, 50 classes of olfactory receptor neurons (ORNs) send their axons to specific glomeruli in the antennal lobe (AL), where they make one-to-one connections with corresponding second-order olfactory projection neurons (PNs). This provides an excellent system to study how neurons form specific connections during circuit assembly. Recently I developed an explant system that allows me to use live imaging to reveal the dynamic process of olfactory circuit assembly. Here I propose to use this explant to: 1. image how single ORN axon targets its glomerulus; 2. explore how ORN-ORN or ORN-PN interaction regulates targeting through surgical cutting one antennal nerve; 3. characterize the mistargeting phenotype in Drosophila latrophilin mutant using live imaging (Aim 1 and part of Aim 3, K99 training phase). The K99 training phase studies mostly focus on defining the coarse targeting using two-photon microscope based long term imaging. To further pursue detailed mechanisms underlying interesting behaviors of the ORN axons, advanced imaging method allowing higher temporal and spatial resolution is required. Therefore I propose to perform high speed two color imaging using adaptive optical lattice lightsheet microscope. Combined with genetic tool for cytoskeleton labeling and manipulation, I aim to understand cell biology mechanism of crucial steps in ORN axon targeting. This study will be in collaboration with Dr. Eric Betzig at Janelia Research Campus. We have already got promising results from pilot experiments. I will also pursue potential Latrophilin-Ten-a mediated axon-axon or axon-dendrite repulsion using live imaging (Aim 2 and part of Aim 3, R00 training phase). In general the R00 training phase studies will deal with more complex situation in both imaging strategy and biological context. My mentoring team consists of Dr. Liqun Luo (mentor), a world-renowned neuroscientist, and Drs. Kang Shen (co-mentor), an experienced cell biologist and developmental neurobiologist and Eric Betzig (collaborator), Nobel laureate and world-renowned microscopist, as well as Dr. Michael Lin (consultant), an expert on developing genetic imaging tools. With their guidance, I believe that I will gain extensive training in my transition to be an independent PI.