Multi-color optical voltage imaging of neural activity in behaving animals

摘要 美国国立卫生研究院大脑计划的开创性工作揭示了许多新型神经元和它们的 基因签名。这项研究的红利将包括允许选择性遗传的尖端工具 获取这些类型的细胞，例如用于成像、光遗传学或示踪研究。为了补充这些强大的 基因工具，同样重要的是拥有新的成像技术，可以揭示多个神经元是如何- 不同类型的人在活的大脑中一起工作，以支持信息处理并构建不同的大脑状态。 为了应对这一挑战，斯坦福大学和耶鲁大学约翰·B·皮尔斯实验室 将创造光学技术来成像多达4种不同神经元类型的并发电压动态 表现得像动物一样。首先，我们将结合机器学习方法和自动化的高通量蛋白质 用于设计4种不同类别的基因编码荧光光学指示剂的筛选平台 神经元跨膜电压。然后，我们将创新几种类型的光学仪器，以便在 与新的电压指示器配合使用。这些仪器将使对电压的前所未有的研究成为可能 大鼠脑浅层和深层2-4种神经元类型的节律和放电动力学 醒来后表现得像动物一样。一台仪器将允许我们跟踪并发的总体电压振荡 2自由行为啮齿动物的神经元类型。另一种仪器，光学介镜，将使成像成为可能 对表现良好的小鼠整个新皮质表面的电压波动和振荡的研究。第三种设备 将是一种高速微型显微镜，用于在单细胞、单峰电位分辨率下跟踪神经动力学 行为自由的老鼠。最后，我们将开发以毫秒级精度成像的能力 大脑皮层或脑深部4种靶向神经元的同步放电动力学。五个外部 测试者实验室将在活的老鼠和苍蝇身上评估所有这些创新，并提供关键的用户反馈。 如果我们的工作取得成功，它将为大脑动力学研究带来至关重要的知识，从而改变游戏规则 关于不同的神经元类型如何协同它们的动力学来塑造动物的行为和大脑的全局 在健康和疾病方面。为了促进这一成果，我们计划了一项5倍的资源共享战略：(I)所有 电压指示器构建、病毒载体、转基因苍蝇、软件和筛查数据将存放在 公开分发的公共储存库；(2)所有仪器设计将广泛详细地发布到 促进复制；(Iii)我们的新型成像设备将集成到现有的NIH支持的、公开的 可使用的啮齿动物脑成像设施；(Iv)在项目第2-4年，我们将举办4个培训工作坊 每年40名访问科学家(总共120名)第一手学习新技术。这些访客还将 提供广泛的用户反馈；(V)我们将许可我们的成像仪器进行商业分销。 总体而言，我们预计我们的项目将导致脑科学的重大概念进步和多个新的 将重塑哺乳动物大脑成像实践的技术。

Abstract Groundbreaking work within the NIH BRAIN Initiative has revealed many new types of neurons and their genetic signatures. The dividends from this research will include sophisticated tools allowing selective genetic access to these cell-types, such as for imaging, optogenetic or tracing studies. To complement these powerful genetic tools, it will be equally important to have new imaging techniques that can reveal how multiple neuron- types work together in the live brain to support information-processing and construct different brain states. To address this challenge, Stanford University and The John B. Pierce Laboratory at Yale University will create optical techniques for imaging the concurrent voltage dynamics of up to 4 separate neuron-types in behaving animals. First, we will combine machine learning methods and an automated, high-throughput protein screening platform to engineer 4 different categories of genetically encoded fluorescent optical indicators of neuronal transmembrane voltage. We will then innovate several types of optical instruments tailored to work in conjunction with the new voltage indicators. These instruments will enable unprecedented studies of voltage rhythms and spiking dynamics in 2–4 genetically identified neuron-types in superficial and deep brain areas of awake behaving animals. One instrument will allow us to track the concurrent, population voltage oscillations of 2 neuron-types in freely behaving rodents. Another instrument, an optical mesoscope, will enable imaging studies of voltage waves and oscillations across the entire neocortical surface of behaving mice. A third device will be a high-speed miniature microscope for tracking neural dynamics at single cell-, single spike-resolution in freely behaving mice. Lastly, we will develop the capability to image with millisecond-scale precision the simultaneous spiking dynamics of 4 targeted neuron-types in either cortical or deep brain areas. Five external beta-tester labs will evaluate all these innovations in live mice and flies and provide critical user-feedback. If our work succeeds, it will be a ‘game-changer’ for studies of brain dynamics, yielding vital knowledge about how different neuron-types synergize their dynamics to shape animal behavior and the brain’s global states in health and disease. To facilitate this outcome, we plan a 5-fold strategy for resource sharing: (i) All voltage-indicator constructs, viral vectors, transgenic flies, software and screening data will be deposited at public repositories for open distribution; (ii) All instrument designs will be published in extensive detail to facilitate replication; (iii) Our novel imaging devices will be integrated into an existing NIH-supported, publicly accessible facility for brain-imaging in rodents; (iv) In project years 2–4, we will conduct 4 training workshops for 40 visiting scientists per year (120 in total) to learn the new technologies firsthand. These visitors will also provide extensive user-feedback; (v) We will license our imaging instruments for commercial distribution. Overall, we expect our project will lead to major conceptual advances in brain science and multiple new technologies that will reshape the practice of mammalian brain imaging.