Imaging and stimulation of neural activity at cellular resolution in awake mice

该建议是开发用于同时成像的方法和仪器 以及清醒小鼠中大量神经元的活动的光学刺激 在鼠标可以自由地在虚拟环境中导航的条件下， 在系统神经科学中具有广泛应用的能力。使能技术是一种 本发明提供了一种设备，该设备有助于以细胞分辨率进行高分辨率光学成像， 最小化大脑运动它是基于一个直立的工作台安装双光子显微镜 安装在由一个大的空气支撑球组成的球形跑步机上。小鼠， 植入颅骨窗，旨在减少大脑运动，可以行走和跑步， 在成像过程中球的表面，而他们的头部保持不动。图像 序列表明，与运动相关的大脑运动仅限于~2-5 μ m 并且该运动主要在焦平面内，几乎没有平面外运动， 为基于离线隐马尔可夫模型的软件方法提供条件， 去除残留的运动伪影。使用第一代仪器的试验数据 表明，行为相关的钙瞬变从大的神经元和 星形胶质细胞群体可以在清醒小鼠中以细胞分辨率常规成像， 在行走和跑步时。拟议的研究和开发计划将 进一步验证和优化第一代仪器中使用的方法， 通过增加实时运动校正、新的腔室和镜头来扩展其功能 用于大脑深层结构（如海马体）的成像， 一种由鼠标运行行为控制的可视虚拟现实显示系统。 另一个重点将集中在使用脉冲时间复用和多焦平面 提供同时成像和基于双光子的光刺激的光学设计 使用通道视紫红质。系统神经科学中可以解决的科学问题 使用要开发的仪器解决的是一些最基本的 关于大脑是如何工作的，从确定活跃的数量， 沉默，神经元在特定的行为，同步性和相关性的重要性 感知记忆和运动控制在整个活动中成像的能力 在虚拟环境中导航的小鼠中细胞分辨率的神经元群体 将有助于绘制微尺度的空间结构和电路连接， 将细胞放置在海马体中，将网格细胞放置在皮质中。这项研究将开发一种成像方法来测量化学过程中， 许多单独的神经元同时在大脑中，而它是在运作， 清醒状态。这种能力在比较正常和 大脑中的疾病状态。开发的方法将适用于 小鼠，这是领先的哺乳动物遗传模型系统在健康 research.除了测量化学过程，这些方法还将 提供刺激特定神经元群体的能力， 在确定大脑功能的基本机制和 评估神经假体的新方法

This proposal is to develop methods and instrumentation for the simultaneous imaging and optical stimulation of activity in large populations of neurons in the awake mouse brain under conditions in which the mouse is free to navigate in a virtual environment, a capability with wide application in systems neuroscience. The enabling technology is an apparatus that facilitates high-resolution optical imaging at cellular resolution while minimizing brain motion. It is based on an upright table mounted two-photon microscope mounted over a spherical treadmill consisting of a large air supported ball. Mice, with implanted cranial windows designed to reduce brain motion, can walk and run on the surface of the ball during imaging while their head remains motionless. Image sequences demonstrate that movement-associated brain motion is limited to ~2-5 ¿m and that this motion is predominantly in the focal plane, with little out-of-plane motion, providing the conditions for an offline Hidden Markov Model based software method for removing residual motion artifacts. Pilot data using a first generation instrument demonstrate that behaviorally correlated calcium transients from large neuronal and astrocytic populations can be routinely imaged at cellular resolution in awake mice, even during walking and running. The proposed research and development program will further validate and optimize the methods used in the first generation instrument, and extend its capabilities by adding real-time motion correction, new chambers and lenses for imaging of deeper brain structures such as the hippocampus, and the incorporation of a visual virtual reality display system controlled by the running behavior of the mouse. An additional thrust will focus on using pulse time-multiplexing and a multi-focal plane optical design to provide simultaneous imaging and two-photon based photo-stimulation using channelrhodopsin. The scientific questions in systems neuroscience that can be addressed using the instrumentation to be developed are some of the most fundamental ones about how the brain works, ranging from determining the number of active, versus silent, neurons during a specific behavior, to the importance of synchrony and correlation in perception, memory, and motor control. The ability to image the activity in entire populations of neurons at cellular resolution in mice navigating in a virtual environment will facilitate mapping the micron-scale spatial architecture and circuit connectivity of place cells in the hippocampus and grid cells in the cortex. This study will develop an imaging method to measure the chemical processes in many individual neurons simultaneously in the brain while it is operating in the awake state. This capability would be valuable in comparing normal and diseased states in the brain. The methods developed will be applicable to the mouse, which is the leading mammalian genetic model system in health research. In addition to measuring chemical processes, the methods will also provide the ability to stimulate a specific population of neurons, which is important both in determining basic mechanisms of brain function and in evaluating new methods for neural prostheses.