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

描述（由申请人提供）：本提案旨在开发方法和仪器，用于在小鼠在虚拟环境中自由导航的条件下，对清醒小鼠大脑中大量神经元的活动进行同步成像和光学刺激，这是一种在系统神经科学中广泛应用的能力。使能技术是一种设备，可以促进细胞分辨率的高分辨率光学成像，同时最大限度地减少大脑运动。它是基于一个直立的桌子安装双光子显微镜安装在一个球形跑步机组成的一个大的空气支撑球。植入了旨在减少大脑运动的颅骨窗口的老鼠，在成像过程中可以在球的表面上行走和奔跑，而它们的头部保持不动。图像序列表明，与运动相关的大脑运动被限制在~2-5 μ m，并且这种运动主要在焦平面内，很少有平面外运动，这为基于隐马尔可夫模型的离线软件方法去除残余运动伪影提供了条件。使用第一代仪器的试验数据表明，大神经元和星形细胞群体的行为相关钙瞬态可以在清醒小鼠中以细胞分辨率常规成像，甚至在行走和跑步时也是如此。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，并通过增加实时运动校正，用于海马等更深脑结构成像的新腔室和透镜以及由鼠标运行行为控制的视觉虚拟现实显示系统来扩展其功能。一个额外的推力将集中在使用脉冲时间复用和多焦平面光学设计，以提供同时成像和使用通道视紫红质的双光子光刺激。系统神经科学中的科学问题可以使用即将开发的仪器来解决，这些问题是关于大脑如何工作的一些最基本的问题，从确定特定行为中活跃与沉默神经元的数量，到感知、记忆和运动控制中同步和相关性的重要性。在虚拟环境中以细胞分辨率成像小鼠整个神经元群活动的能力，将有助于绘制海马体中位置细胞和皮层中网格细胞的微米尺度空间结构和电路连接。这项研究将开发一种成像方法来测量大脑在清醒状态下同时运行的许多单个神经元的化学过程。这种能力在比较大脑的正常和患病状态方面是有价值的。开发的方法将适用于小鼠，这是健康研究中领先的哺乳动物遗传模型系统。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的能力，这对于确定脑功能的基本机制和评估神经假体的新方法都很重要。

DESCRIPTION (provided by applicant): 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 um 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.