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

今天，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力学的信息很少。因此，最近的NIH大脑倡议报告呼吁使用新技术，以提供“以细胞分辨率进行的多区域人口记录”。为了应对这一挑战，我们建造了一个大型双光子荧光显微镜，它有16束激光，在一只清醒的行为啮齿动物身上共同扫描4mm2的脑组织。这一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单个神经元的同时动力学进行钙成像研究。这项高影响力神经科学研究资源基金的目标是使这一突破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；启用多色成像，包括同时进行两个不同细胞群的钙成像的双色成像；并将激光扫描系统加速到20赫兹成像帧速率，以满足用户对大规模神经钙动力学的研究问题需要这种时间分辨率的需求。我们将建立核心基础设施，包括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来表达遗传编码的钙指示剂的转基因报告小鼠和病毒载体。我们还将聘请一名全职科学家，他将定期举办教程研讨会，直接支持个人用户培训和使用仪器，调整用户的动物行为分析以与大规模双光子脑成像兼容，以及数据分析。我们计划分阶段推出服务，首先是精心挑选的5个第一阶段实验室作为测试者，然后是更大规模的16个第二阶段实验室，然后很快向所有神经科学研究申请者开放R24资源。为了监督和参照量化里程碑的进展，我们成立了一个由来自我们所在大学内外的资深科学家组成的指导委员会，他们拥有互补的专业领域-包括在研究成像设施的管理和脑成像数据的公共数据库方面，这些数据在科学界广泛共享。指导委员会将监控资源使用情况，评估用户反馈，为实现卓越的可用性、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在发表生物学结果方面的研究进展。资源人员和指导委员会还将与斯坦福神经科学研究所协调，为地方和国家NINDS和神经科学研究社区提供广泛、公平、宣传良好和灵活的资源访问机会。

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.