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.

今天，由于缺乏适当的技术，关于多个大脑区域如何协调集体神经回路动力学的信息很少。因此，最近的NIH BRAIN Initiative报告呼吁新技术可以提供“细胞分辨率的多区域人口记录”。为了应对这一挑战，我们建立了一个大规模的双光子荧光显微镜，有16个激光束，一起扫描一个清醒的行为啮齿动物的脑组织的4平方毫米的区域。这台新仪器是世界上最大的双光子显微镜，能够对分布在3-7个大脑皮层区域的数千个神经元的同步动力学进行Ca 2+成像研究。 这项高影响力神经科学研究资源补助金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和开放访问的资源。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，包括同时双色成像，用于两个不同细胞群的同时Ca 2+成像;并将激光扫描系统加速到20 Hz成像帧速率，以满足用户的需求，其关于大规模神经Ca 2+动力学的研究问题需要这种时间分辨率。我们将建立核心基础设施，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，以及神经科学家最常用于表达遗传编码的Ca 2+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，并直接支持个人用户进行仪器的培训和使用，调整用户的动物行为测定，以适应大规模双光子脑成像和数据分析。 我们计划分阶段推出服务，首先是5个精心挑选的第一阶段实验室作为beta测试人员，然后是16个第二阶段实验室，很快就会向所有神经科学研究申请人开放R24资源。为了监督和基准进度对量化的里程碑，我们已经建立了一个指导委员会的资深科学家，从内部和外部我们的家大学，谁拥有互补的专业知识领域-包括在研究成像设施的管理和公共数据库的大脑成像数据是广泛共享整个科学界。指导委员会将监测资源使用情况，评估用户反馈，提供关于上级可用性、性能和数据共享的指导，并跟踪使用R24资源的每个神经科学实验室的研究进展，以发表生物学结果。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，在当地和国家NINDS和神经科学研究社区提供广泛、公平、广而告之和灵活的资源访问。