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

 描述(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，活体钙质成像实验已经开始揭示在头部固定的小鼠行为过程中，新皮质不同区域发生的时空活动模式。然而，通常情况下，成像细胞活动的视场相当小，只有几百微米左右。这一限制限制了可以研究的神经元网络的大小，从而留下了局部神经元网络如何与遥远的突触连接区域通信的问题。因此，需要一种新的成像设备，能够同时从两个区域进行高分辨率功能成像，在最好的情况下识别相互投射的神经元。我们开发了一种新型的多区域双光子显微镜(MA2PM)，通过在一个大的全局扫描场(直径1.8毫米，相当于并排排列100,000个神经元细胞体，假设每个神经元20微米)内同时成像两个子区域来满足这一需求。这两个区域可以独立而灵活地定位，例如，当老鼠用胡须执行触觉纹理识别任务时，可以同时测量小鼠新皮质初级和次级躯体感觉区域的神经元(相距1毫米)。使用MA2 PM原型，我们在清醒的、行为正常的小鼠的躯体感觉皮层中进行了第一次原理验证实验，使用了一种遗传编码的钙指示器。我们应用病毒逆行标记策略来识别引起区域间联系的神经元亚群。我们在拟议项目中的目标是在几个方面进一步优化和推广这一创新的、转化显微技术。目标1：我们将致力于最终完成用于两区域成像的全显微镜设计，并展示其在皮质处理研究中的实用性。我们完全致力于构建一个模块化的、精心设计的仪器，执行量化的系统表征，并将这一新仪器推广到更广泛的神经科学界，特别是向越来越多的应用双光子成像分析皮质处理的研究小组。目标2：我们将进一步将系统扩展到更大的视野，并将其扩展到四个分区的同时成像。为此，我们将应用最新的激光技术，并使用最先进的红移遗传编码钙指示器进行深度成像。我们特别致力于在两个相连的皮质区域中实例化两层成像(例如，在层L2/3和L5中)。总体而言，我们预计新的多区域成像技术将是一项使能技术，将局部微电路水平连接到沟通大脑区域的‘大电路’水平，因此将立即引起神经科学界的广泛兴趣和最高意义。

 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.