Live imaging of neuron circuit assembly in Drosophila olfactory system

果蝇嗅觉系统神经元回路组装的实时成像

• 批准号: 10038736
• 负责人: Tongchao Li
• 金额: $ 12.77万
• 依托单位: STANFORD UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-08-01 至 2022-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10038736
• 关键词: Actins; Adhesions; Axon; Behavior; Biological; Brain; Cells; Cellular biology; Collaborations; Color; Complex; Contralateral; Cues; Cytoskeleton; Data; Dendrites; Development; Drosophila genus; Esthesia; Exhibits; F-Actin; G-Protein-Coupled Receptors; Genes; Genetic; Goals; Guanosine Triphosphate Phosphohydrolases; Image; Imaging Device; In Vitro; Integral Membrane Protein; Ipsilateral; Knowledge; Label; Light; Link; Lobe; Mammals; Mediating; Membrane; Mentors; Microscope; Microscopy; Mus; Nerve; Neurons; Odors; Olfactory Pathways; Olfactory Receptor Neurons; Operative Surgical Procedures; Organ; Outcome; Pattern; Phase; Phenotype; Presynaptic Terminals; Process; Regulation; Research; Resolution; Scanning; Sensory; Side; Specificity; Speed; Stereotyping; Structure; Synapses; System; Time; Training; Visualization; alpha-latrotoxin receptor; base; experience; experimental study; fly; genetic manipulation; imaging genetics; imaging modality; imaging study; in vivo; insight; mutant; neural circuit; neuron development; optical lattices; relating to nervous system; synaptogenesis; tool; two photon microscopy; two-photon

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.

项目概要/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，由于难以理解，电路组装动态过程的许多详细机制尚不清楚。 在活人大脑中想象它。采用基于实时成像的外植体系统，在这里我建议研究 使用果蝇嗅觉系统进行神经回路组装的细胞生物学基础。 在果蝇嗅觉回路中，50 类嗅觉受体神经元 (ORN) 将其轴突发送至 触角叶 (AL) 中的特定肾小球，在那里它们与相应的肾小球进行一对一的连接 二阶嗅觉投射神经元（PN）。这为研究神经元如何形成提供了一个优秀的系统 电路组装期间的特定连接。最近我开发了一个外植体系统，可以让我使用活体 成像揭示嗅觉回路组装的动态过程。在这里，我建议使用该外植体来： 1. 图像单个 ORN 轴突如何靶向其肾小球； 2.探索ORN-ORN或ORN-PN如何相互作用 通过手术切割一根触角神经来调节目标； 3. 描述误定位表型 使用实时成像的果蝇 latrophilin 突变体（目标 1 和目标 3 的一部分，K99 训练阶段）。 K99 训练阶段研究主要集中于使用双光子定义粗目标 基于显微镜的长期成像。进一步探究有趣行为背后的详细机制 ORN 轴突的研究需要允许更高时间和空间分辨率的先进成像方法。 因此我建议使用自适应光学晶格光片进行高速双色成像 显微镜。结合细胞骨架标记和操作的遗传工具，我的目标是了解细胞 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 贝齐格在珍妮莉亚研究园区。我们已经从试点实验中获得了有希望的结果。我也会 使用实时成像追踪潜在的 Latrophilin-Ten-a 介导的轴突-轴突或轴突-树突排斥（目标 2 以及目标 3、R00 训练阶段的一部分）。一般来说，R00 训练阶段的研究将涉及更多 成像策略和生物背景中的复杂情况。 我的导师团队由世界著名神经科学家罗立群博士（导师）和炕 Shen（共同导师），一位经验丰富的细胞生物学家和发育神经生物学家和 Eric Betzig 诺贝尔奖得主、世界著名显微镜学家 Michael Lin 博士（顾问） 开发遗传成像工具的专家。在他们的指导下，我相信我将获得广泛的培训 我向独立 PI 的转变。