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

这项建议是为了开发同时成像的方法和仪器 以及光刺激清醒小鼠大量神经元的活动 大脑在鼠标可以在虚拟环境中自由导航的条件下， 具有在系统神经科学中广泛应用的能力。使能技术是一种 便于在细胞分辨率下进行高分辨率光学成像的设备 最大限度地减少大脑运动。它是基于安装在直立工作台上的双光子显微镜 安装在由一个大的空气支撑球组成的球形跑步机上。老鼠，有 植入的颅窗旨在减少脑部运动，可以在 在成像过程中，当他们的头部保持不动时，球的表面会发生变化。图像 序列显示，与运动相关的大脑运动被限制在~2-5？m 这种运动主要在焦平面内，几乎没有平面外的运动， 为基于离线隐马尔可夫模型的软件方法提供条件 去除残余运动伪影。使用第一代仪器的试点数据 证明来自大型神经元的行为相关钙瞬变和 在清醒的小鼠身上，星形细胞群可以常规地以细胞分辨率成像，甚至 在散步和跑步时。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，以及 通过添加实时运动校正、新的暗室和镜头来扩展其功能 用于更深层次的大脑结构的成像，如海马体，以及 由鼠标的运行行为控制的可视虚拟现实显示系统。 另一个推力将集中在使用脉冲时分复用和多焦平面 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。系统神经科学中的科学问题可以是 使用要开发的工具解决了以下一些最基本的问题 关于大脑如何工作的问题，从确定活动的数量，到 沉默，神经元在特定的行为中，对同步和关联的重要性 在感知、记忆和运动控制方面。能够完整地对活动进行成像 在虚拟环境中导航的小鼠细胞分辨率的神经元群体 将有助于绘制微米级的空间架构和电路连接 把细胞放在海马区，把网格细胞放在大脑皮层。这项研究将开发一种成像方法来测量化学过程 许多单独的神经元同时在大脑中运行时， 清醒状态。这种能力在比较正常和 大脑中的疾病状态。所开发的方法将适用于 小鼠，这是健康领域领先的哺乳动物遗传模型系统 研究。除了测量化学过程外，这些方法还将 提供刺激特定神经元群体的能力，这是 在确定大脑功能的基本机制和在 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.