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它们的集体神经回路动力学的信息很少。因此，最近的美国国立卫生研究院BRAIN倡议报告呼吁开发新技术，以提供“细胞分辨率的多区域人口记录”。为了应对这一挑战，我们建造了一个大型双光子荧光显微镜，用16束激光扫描一只清醒行为的啮齿动物的脑组织，面积为4平方毫米。这台新仪器是世界上最大的双光子显微镜，能够对位于~ 3-7个大脑皮质区域的数千个单个神经元的同时动态进行Ca2+成像研究。这项高影响力神经科学研究资源资助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；支持多色成像，包括同时双色成像，用于两个不同细胞群的同时Ca2+成像；并将激光扫描系统的成像帧率提高到20 Hz，以满足需要这种时间分辨率的大规模神经Ca2+动力学研究问题的用户的需求。我们将建立核心基础设施，包括一个数据中心，专门用于资源用户的计算分析和生物信息学需求，以及神经科学家最常用来表达基因编码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，并直接支持个人用户对仪器的培训和使用，适应用户的动物行为分析，以兼容大规模双光子脑成像，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beta测试者，然后是16个第二阶段实验室，不久之后将向所有神经科学研究申请者开放R24资源。为了监督和衡量量化里程碑的进展，我们成立了一个由来自我们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——包括研究成像设备的管理和在科学界广泛共享的脑成像数据的公共数据库。指导委员会将监控资源的使用情况，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神经科学研究所协调，为当地和全国的NINDS和神经科学研究界提供广泛、公平、充分宣传和灵活的资源访问。

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.