Large-scale dual-color two-photon calcium imaging in awake behaving animals

清醒行为动物的大规模双色双光子钙成像

• 批准号: 9788541
• 负责人: MARK J SCHNITZER
• 金额: $ 25.41万
• 依托单位: STANFORD UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-09-15 至 2020-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9788541
• 关键词: Address; Animals; Area; BRAIN initiative; Behavior; Behavioral Assay; Benchmarking; Bioinformatics; Biological; Brain; Brain imaging; Brain region; Calcium; Cells; Color; Communities; Computer Analysis; Computer software; Data; Data Analyses; Databases; Decision Making; Disease Progression; Educational workshop; Feedback; Goals; Grant; Home environment; Human Resources; Image; Image Analysis; Individual; Infrastructure; Institutes; Label; Lasers; Learning; Life Experience; Longitudinal Studies; Methods; Microelectrodes; Microscope; Monitor; Monoclonal Antibody R24; Mus; National Institute of Neurological Disorders and Stroke; Neurons; Neurosciences; Neurosciences Research; Performance; Phase; Population; Publications; Reagent; Reporter; Reporting; Research; Research Personnel; Resolution; Resources; Rodent; Sampling; Scanning; Scientist; Senior Scientist; Services; Speed; System; Techniques; Technology; Time; Training; Transgenic Organisms; United States National Institutes of Health; Universities; Viral Vector; Visual Cortex; Work; awake; brain tissue; cognitive task; commercialization; cost; data sharing; extrastriate visual cortex; flexibility; fluorescence microscope; imaging facilities; imaging study; improved; instrument; instrumentation; neural circuit; relating to nervous system; technology/technique; temporal measurement; two-photon; usability; user-friendly

Today, for lack of appropriate technology there is scant information on how multiple brain areas coordinate their collective neural circuit dynamics. Hence, the recent NIH BRAIN Initiative report calls for new techniques that can provide “multi-area population recording at cellular resolution”. To address this challenge, we built a large-scale two-photon fluorescence microscope with 16 laser beams that together scan a 4 mm2 area of brain tissue in an awake behaving rodent. This new instrument is the world's largest two-photon microscope and enables Ca2+ imaging studies of the simultaneous dynamics of thousands of individual neurons lying across ~3–7 cortical brain areas. The goal of this High Impact Neuroscience Research Resource Grant is to make this groundbreaking new instrumentation a user-friendly and openly accessible resource for neuroscience researchers around the nation. To achieve this, we will first implement key upgrades to the large-scale microscope that will: improve the usability of the software interface; enable multi-color imaging, including simultaneous dual-color imaging for concurrent Ca2+ imaging of two distinct cell populations; and speed the laser-scanning system to 20 Hz imaging frame rates, to meet the needs of users whose research questions about large-scale neural Ca2+ dynamics require this temporal resolution. We will establish core infrastructure, including a data center dedicated to the computational analysis and bioinformatics needs of the Resource users, and a stock of the transgenic reporter mice and viral vectors that neuroscientists most commonly use to express genetically encoded Ca2+ indicators. We will also hire a full-time staff scientist, who will conduct regular tutorial workshops and directly support individual users regarding training and usage of the instrumentation, adaptation of users' animal behavioral assays for compatibility with large-scale two-photon brain imaging, and data analysis. We plan a staged rollout of service, starting with 5 carefully chosen Phase 1 labs to serve as beta- testers, followed by a larger set of 16 Phase 2 labs, soon followed by opening the R24 Resource to all neuroscience research applicants. To oversee and benchmark progress against quantitative milestones, we have established a Steering Committee of senior scientists, from within and outside our home university, who have complementary areas of expertise — including in the management of research imaging facilities and public databases of brain imaging data that are broadly shared across the scientific community. The Steering Committee will monitor Resource usage, evaluate user feedback, provide guidance toward superior usability, performance and data sharing, and track the research progress of each neuroscience lab that is using the R24 Resource toward publication of the biological results. The Resource personnel and Steering Committee will also coordinate with the Stanford Neuroscience Institute to provide broad, equitable, well-advertised, and flexible access to the Resource across the local and national NINDS and neuroscience research communities.

如今，由于缺乏适当的技术，关于多个大脑区域如何协调其集体神经回路动态的信息很少，因此，最近的 NIH BRAIN Initiative 报告呼吁能够提供“以细胞分辨率进行多区域群体记录”的新技术。为了应对这一挑战，我们建造了一台带有 16 束激光的大型双光子荧光显微镜，这些激光束一起扫描清醒行为啮齿动物的 4 mm2 脑组织区域。这种新仪器是世界上最大的双光子显微镜，能够实现这一目标。钙离子对分布在约 3-7 个大脑皮层区域的数千个单个神经元的同时动态进行成像研究，这项高影响力神经科学研究资源拨款的目标是使这种突破性的新仪器成为神经科学研究人员用户友好且可公开访问的资源。为了实现这一目标，我们将首先对大型显微镜进行关键升级，这将： 提高软件界面的可用性，实现多色成像，包括同时对两个不同细胞进行 Ca2+ 成像；人口；并加快激光扫描系统达到 20 Hz 成像帧速率，以满足大规模神经 Ca2+ 动力学研究问题需要这种时间分辨率的用户的需求。我们将建立核心基础设施，包括专门用于计算分析和生物信息学需求的数据中心。资源用户的资源，以及神经科学家最常用来表达基因编码的 Ca2+ 指标的转基因报告小鼠和病毒载体的库存。我们还将聘请一名全职科学家，他将定期举办辅导研讨会并提供直接支持。我们计划分阶段推出服务，从 5 个精心挑选的第一阶段实验室开始。作为 Beta 测试人员，随后是 16 个第二阶段实验室，不久之后向所有神经科学研究申请者开放 R24 资源，为了监督和衡量定量里程碑的进展，我们成立了一个由资深科学家组成的指导委员会，来自我们母校内外的专家，他们具有互补的专业领域——包括在科学界广泛共享的研究成像设施和脑成像数据公共数据库的管理方面。指导委员会将监测资源使用情况，评估用户反馈。 ，为卓越的可用性、性能和数据共享提供指导，并跟踪每个使用 R24 资源的神经科学实验室的研究进展，以发布生物学结果。资源人员和指导委员会还将与斯坦福大学神经科学研究所协调，提供相关信息。广泛、公平、广为宣传、以及灵活地访问本地和国家 NINDS 以及神经科学研究界的资源。