Multi-area two-photon microscopy for revealing long-distance communication between multiple local brain circuits

多区域双光子显微镜揭示多个局部脑回路之间的长距离通信

• 批准号: 8826876
• 负责人: Fritjof Helmchen
• 金额: $ 36.51万
• 依托单位: UNIVERSITY OF ZURICH
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-09-30 至 2017-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8826876
• 关键词: Address; Animals; Area; Behavior; Behavioral; Biological; Brain; Brain region; Calcium; Caliber; Cells; Communication; Communities; Complex; Computer software; Custom; Dendrites; Detection; Discrimination; Distant; Ensure; Fiber; Fluorescence; Functional Imaging; Generations; Goals; Head; Image; Image Analysis; Imaging technology; Individual; Label; Lasers; Life; Measurement; Measures; Methods; Microscope; Microscopy; Monitor; Mus; Neocortex; Neurons; Neurosciences; Operating System; Optics; Pattern; Performance; Population; Population Dynamics; Positioning Attribute; Process; Property; Research; Resolution; Rodent; Scanning; Sensory; Side; Signal Transduction; Somatosensory Cortex; Speed; Support System; Synapses; System; Tactile; Technology; Testing; Texture; Tissues; Vibrissae; Viral; Work; awake; base; calcium indicator; cell type; cellular imaging; design; experience; fluorescence microscope; imaging modality; improved; in vivo; in vivo imaging; information processing; innovation; instrument; interest; neocortical; neural circuit; neuronal cell body; novel; optical imaging; prototype; public health relevance; relating to nervous system; research study; somatosensory; spatiotemporal; two-photon

 DESCRIPTION (provided by applicant): Two-photon microscopy is a widely used, key method for functional imaging of cellular activity in living animals. Most recently, in vivo calciu imaging experiments have started to reveal the spatiotemporal activity patterns that occur in various areas of the neocortex during head-fixed mouse behavior. Typically, however, the field-of- view for imaging cellular activity is fairly small, on the order of a few hundred micrometers. This restriction limits the size of neuronal networks that can be studied and thus leaves open the question how local neuronal networks communicate with distant, synaptically connected regions. What is therefore needed is a new imaging instrument that allows high-resolution functional imaging from two regions simultaneously, in the best case identifying the mutual projection neurons. We have developed a novel multi-area 2-photon microscope (MA2PM) that fulfills this need by enabling simultaneous imaging of two sub-areas within a large global scan- field (1.8 mm diameter, a distance equivalent to lining 100,000 neuronal cell bodies side-by-side assuming 20 um per neuron). Such two areas can be independently and flexibly positioned, making it for example possible to simultaneously measure neurons in the primary and secondary somatosensory areas of mouse neocortex (> 1 mm apart) while the mouse is performing a tactile texture discrimination task with its whiskers. The Using the MA2PM prototype we have conducted first proof-of-principle experiments in somatosensory cortex of awake, behaving mice using a genetically-encoded calcium indicator. We apply viral retrograde labeling strategies to identify the subsets of neurons that give rise to the inter-areal connection. Our goal in the proposed project is to further optimize and extend this innovative, transforming microscopy technology in several ways. Aim 1: We will work on finalizing a full microscope design for 2-area imaging and demonstrate its usefulness for research on corticocortical processing. We are fully dedicated to build a modular, carefully designed instrument, perform a quantitative system characterization, and disseminate this new instrument to the broader neuroscience community, especially to the growing number of research groups applying two-photon imaging for the analysis of cortical processing. Aim 2: We will furthermore expand the system to an even larger field-of-view and extend it for simultaneous imaging of four sub-areas. To this end we will apply newest laser technology and employ state-of-the-art red-shifted genetically encoded calcium indicator for deep imaging. We particularly aim at instantiating two-layer imaging (e.g. in layers L2/3 and L5) in two connected cortical areas. Overall, we expect that the new multi-area imaging technology will be an enabling technology to bridge the level of local microcircuits to the 'macrocircuit' level of communicating brain areas and thus will be of immediate and broad interest and highest significance to the neuroscience community.

 描述（由申请人提供）：双光子显微镜是活体动物细胞活动功能成像的一种广泛使用的关键方法。最近，体内钙成像实验已经开始揭示在头部固定的小鼠行为期间在新皮层的各个区域中发生的时空活动模式。然而，通常，用于成像细胞活动的视场相当小，大约几百微米。这种限制限制了可以研究的神经元网络的大小，因此留下了一个问题，即局部神经元网络如何与遥远的突触连接区域进行通信。因此，需要一种新的成像仪器，允许同时从两个区域进行高分辨率的功能成像，在最好的情况下识别相互投射神经元。我们已经开发了一种新颖的多区域双光子显微镜（MA 2PM），其通过使得能够在大的全局扫描场（1.8mm直径，假设每个神经元20 μ m，该距离相当于并排排列100，000个神经元细胞体）内同时成像来满足该需求。这两个区域可以独立且灵活地定位，使得例如可以在小鼠用其胡须执行触觉纹理辨别任务时同时测量小鼠新皮层的初级和次级躯体感觉区域（相隔> 1 mm）中的神经元。使用MA 2PM原型，我们使用遗传编码的钙指标在清醒的行为小鼠的体感皮层中进行了第一次原理验证实验。我们应用病毒逆行标记策略来识别引起区域间连接的神经元子集。我们在拟议项目中的目标是以多种方式进一步优化和扩展这种创新的、变革性的显微镜技术。目标1：我们将致力于完成一个完整的显微镜设计的2区域成像，并证明其有用的研究皮质处理。我们完全致力于建立一个模块化的，精心设计的仪器，进行定量系统表征，并传播这种新的仪器到更广泛的神经科学界，特别是越来越多的研究小组应用双光子成像的皮层处理的分析。目标2：我们将进一步扩展系统到更大的视场，并将其扩展为四个子区域的同时成像。为此，我们将采用最新的激光技术，并采用最先进的红移遗传编码钙指示剂进行深度成像。我们的目标特别是在两个连接的皮层区域中实例化两层成像（例如，在层L2/3和L5中）。总的来说，我们预计新的多区域成像技术将是一种使能技术，将局部微电路水平与沟通大脑区域的“宏电路”水平连接起来，从而对神经科学界具有直接和广泛的兴趣和最高的意义。