A robotic multi-armed two-photon microscope for imaging neural interactions across multiple brain areas

摘要 大脑倡议最重要的成就之一是对许多新的神经元类型进行基因识别，并创造获得这些细胞类型的遗传工具。然而，揭示这些神经元类型的功能作用以及它们如何跨大脑区域协作产生哺乳动物行为仍然是一个突出的挑战。因此，如果我们要理解全球大脑动力学，发明方法来监控大量遗传识别的神经元如何跨越大脑的多个区域相互作用是至关重要的。今天，电记录方法可以跟踪多个区域的神经活动，但不能轻松地针对特定类型的神经元。Widefield和双光子介观望远镜可以跨毫米尺度的皮质组织区域成像已识别的神经元类型的动态，但无法访问组成大脑感觉、认知或运动回路的主要节点的皮质和皮质下区域的分布集合。为了打破这一僵局，我们发明了“章鱼”，这是一种机器人成像系统，带有多个关节光学手臂，每个手臂都有一个双光子显微镜，可以灵活地放置在大脑周围，同时记录头约束行为的啮齿动物或灵长类动物的多个浅层或深层区域的神经活动。我们设计、制造和测试了一个初始版本的章鱼，它有4个手臂，每个手臂都有5个机械自由度，尖端有一个用于双光子成像的微光学探头。手臂的设计基于外科机器人的想法，并使用远程运动中心运动学来提供全面的机械手臂动作曲目。使用这个系统，视觉神经学家可以同时成像外侧膝状体、视觉皮质、上丘和枕骨的神经活动，运动神经生理学家可以成像运动皮质、基底节、小脑和运动丘脑的活动。在这项计划中，我们会加强每个八达通机械臂的光学和机械设计，并准备透过开源和商业途径广泛推广该系统。每个手臂都将获得最先进的双光子显微镜的光学功能，用于对大规模神经整体活动进行成像。具体地说，每个手臂都将融入光遗传学，并允许在800微米宽的视场内进行双色双光子成像。这些能力将使神经科学家能够监测4个大脑区域中每个区域的两种遗传识别的神经元类型，用光遗传学扰乱这些细胞的动态，并观察这些操作对动物行为和其他3个区域活动的影响。我们亦会简化机械设计，为新用户简化八达通的初步组装，并增加机械臂的灵活性。新的设计也将是机动化的，将为用户提供高度直观的手段来精确操纵机器人手臂。最后，为了反复提高八达通的性能和可用性，并验证其作为一项突破性新技术的传播准备情况，我们将与7个Beta测试器实验室密切合作，在清醒行为的小鼠和绒猴身上实施多区域神经成像研究。

Abstract Among the BRAIN Initiative’s most important achievements are the genetic identification of many new neurons- types and the creation of genetic tools to access these cell types. However, uncovering the functional roles of these neuron types and how they cooperate across brain areas to generate mammalian behavior remains an outstanding challenge. Thus, inventing ways to monitor how large populations of genetically identified neurons interact across multiple regions of the brain is crucial if we are to comprehend global brain dynamics. Today, electrical recording methods can track neural activity across multiple areas but cannot easily target neurons of specific types. Widefield and two-photon mesoscopes can image the dynamics of identified neuron-types across millimeter-scale regions of cortical tissue but cannot access the distributed sets of cortical and subcortical regions that comprise the major nodes of the brain’s sensory, cognitive, or motor circuits. To clear this impasse, we invented the ‘Octopus’, a robotic imaging system with multiple articulated optical arms, each a two-photon microscope, that can be flexibly positioned around the brain to record neural activity concurrently in multiple superficial or deep areas of a head-restrained behaving rodent or primate. We designed, built, and tested an initial version of the Octopus with 4 arms, each of which has 5 mechanical degrees of freedom and a micro-optic probe at its tip for two-photon imaging. The design of the arms is based on ideas from surgical robotics and uses remote center-of-motion kinematics to provide a versatile repertoire of robotic arm movements. Using this system, a visual neuroscientist can concurrently image neural activity in the lateral geniculate nucleus, visual cortex, superior colliculus, and pulvinar, and a motor neurophysiologist can image activity in the motor cortex, basal ganglia, cerebellum, and motor thalamus. In this project, we will enhance the optical and mechanical design of each Octopus arm and prepare the system for wide dissemination through open-source and commercial routes. Each arm will gain the optical functionality of a state-of-the-art, two-photon microscope for imaging large-scale neural ensemble activity. Specifically, each arm will incorporate optogenetics and allow dual-color two-photon imaging over an 800-µm- wide field of view. These capabilities will allow neuroscientists to monitor two genetically identified neuron- types in each of 4 brain areas, to perturb the dynamics of these cells with optogenetics, and to observe the effects of these manipulations on animal behavior and activity in the other 3 areas. We will also streamline the mechanical design to simplify the initial assembly of the Octopus for new users and to endow the robot arms with additional dexterity. The new design will also be motorized and will provide users with highly intuitive means of precisely steering the robot arms. Finally, to iteratively improve the performance and usability of the Octopus and to validate its readiness for dissemination as a groundbreaking new technology, we will work closely with 7 beta-tester labs to implement multi-area neural imaging studies in awake behaving mice and marmosets.