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: 9346634
• 负责人: MARK J SCHNITZER
• 金额: $ 27.13万
• 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/9346634
• 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; 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 Infrastructure; 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 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，关于多个大脑如何协调其集体神经元电路动态的信息很少。因此，最近的NIH脑倡议报告呼吁采用新技术，这些技术可以提供“蜂窝分辨率下的多区域人口记录”。为了应对这一挑战，我们用16个激光梁建造了一个大尺度的两光子荧光显微镜，共同扫描了一个4 mm2的脑组织，在醒着的啮齿动物中。这种新仪器是世界上最大的两光子显微镜，可以对〜3-7个皮质大脑区域的数千个单个神经元的简单动力学进行CA2+成像研究。这种高影响神经科学研究资源赠款的目标是使这种开创性的新仪器成为全国神经科学研究人员的用户友好且公开访问的资源。为了实现这一目标，我们将首先实施将关键升级到将：提高软件接口的可用性；启用多色成像，包括对两个不同细胞种群的并发CA2+成像的简单颜色成像；并将激光扫描系统加速至20 Hz成像框架速率，以满足用户的需求，这些用户的需求对大规模神经CA2+动力学的研究需要此临时分辨率。我们将建立核心基础架构，包括一个专门用于资源用户的计算分析和生物信息学需求的数据中心，以及转基因记者小鼠的库存和病毒载体的库存，这些库存最常用于表达一般代表的CA2+指标的神经科学家。我们还将聘请一名全职员工科学家，他们将进行定期的教程研讨会，并直接支持各个用户有关培训和使用仪器的使用，适应用户的动物行为分析，以兼容与大规模的两光脑大脑成像以及数据分析。我们计划进行分阶段的服务推出，从5个精心选择的1阶段实验室开始，以作为β测试仪，然后是一组较大的16阶段2实验室，然后很快向所有神经科学研究申请人开放R24资源。为了监督和基准反对定量里程碑的进步，我们已经建立了一个由我们家庭大学内外的高级科学家指导委员会，这些委员会具有互补的专业知识领域，包括研究成像设施的管理和脑成像数据的公共数据库，这些数据在科学社区中广泛共享。指导委员会将监视资源使用情况，评估用户反馈，为优越的可用性，性能和数据共享提供指导，并跟踪每个神经科学实验室的研究进度，该研究的进度使用R24资源来发布生物学结果。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进行协调，以提供广泛，公平，良好的，良好的和灵活的访问到本地和国家NINDS和神经科学研究社区的资源。