A novel approach for mapping single-cell long-range connections in the cerebral c

描述（由申请人提供）：理解大脑皮层中的信息处理需要理解较低和较高皮层中心之间的前馈（FF）和反馈（FB）回路的作用。这些回路的组织原则，可以决定它们如何处理感官信息，仍然在很大程度上未知。这是由于区域间回路的复杂性，即它们的解剖学和功能特异性，以及缺乏可以揭示由特定细胞类型形成的FF和FB回路的精细尺度连接性并将其与皮质的功能结构相关联的方法学。超微结构规模的电路解剖，而有用的构建布线图的局部连接在小鼠皮层，不能用于研究大的皮层体积所涵盖的区域间轴突。后者只能在介观尺度上进行研究。我们的目标是开发一种标记和有效重建单细胞类型及其区域间轴突的方法。以前的单轴突研究受到轴突标签起源不明确，无法将标签限制在少数神经元和费力的手动重建的影响。这些研究只提供了一小部分不完全重建的轴突样本，偏向于标记稀疏的区域，没有确定它们的起源细胞类型。我们的具体目标是：目标1。以高分辨率明确标记单投射神经元的轴突，并开发一种新的半自动单轴突重建计算框架。我们将扩大病毒介导的表达GFP标记在高分辨率，稀疏的区域间投射神经元的轴突。我们将开发一种新的方法，用于单轴突的快速连续切片重建，其中包括光学透明的完整组织块的3D成像，以及用于半自动轴突分割的新计算算法。目标2.应用这些方法来解决文献中关于灵长类动物视觉皮层V1区的区域间反馈投射功能特异性或缺乏功能特异性的争议。两个以前的研究V2 FB投影到V1方向图的布局已经证明了方向特异性，一个，和非特异性FB连接，另一个。我们的初步数据表明存在两种FB系统，可能与不同的细胞类型有关，它们与皮质细胞有着独特的关系。 功能架构，从而提供了一种方法来协调明显矛盾的数据。这项研究的贡献是重大的，因为它将为研究提供新的工具。 由特定细胞类型形成的区域间电路的精细尺度连通性和功能组织。这些连接的一般组织原则将出现，这将提供一个解剖学基础的假设驱动的研究他们的功能。这项研究是创新的，因为与以前的研究不同：1）新的标记方法允许高分辨率，明确识别单个区域间神经元，从索马到轴突; 2）从连续切片的3D体积的半自动映射允许快速重建，因此重建轴突的产量更高; 3）首次将皮层反应的功能成像与单个FB轴突的标记相结合。

DESCRIPTION (provided by applicant): Understanding information processing in the cerebral cortex requires understanding the role of feedforward (FF) and feedback (FB) circuits between lower and higher cortical centers. Organizing principles for these circuits, that could determine how they process sensory information, remain largely unknown. This is due to the complexity of inter-areal circuits, i.e. their anatomical and functional specificity, and the lack of methodologis that can reveal the fine-scale connectivity of FF and FB circuits made by specific cell types, and relate it to the functional architecture of the cortex. Ultrastructural-scale circuit anatomy, whil useful for building wiring diagrams of local connections in mouse cortex, cannot be used to study the large cortical volumes encompassed by inter-areal axons. The latter can be studied only at mesoscopic scale. Our goal is to develop a methodology for labeling and efficiently reconstructing, single cell types and their inter-areal axons. Previous single axon studies were affected by ambiguity in the origin of the axonal label, inability to restrict label to few neurons and laborious manual reconstructions. These studies have provided only a small sample of incompletely reconstructed axons, biased towards regions of sparser labeling, with no identification of their cell types of origin. Our Specific Aims are: Aim 1. To label unambiguously at high resolution the axon of single projection neurons, and to develop a novel computational framework for semi-automated single axon reconstruction. We will extend viral-mediated expression of GFP to labeling at high resolution, and sparsely the axons of inter-areal projection neurons. We will develop a novel approach for fast serial section reconstruction of single axons, which includes 3D imaging of intact tissue blocks rendered optically-transparent, and novel computational algorithms for semi-automated axon segmentation. Aim 2. To apply these methods to resolve controversies in the literature on the functional specificity, or lack thereof, f inter-areal feedback projections to primate visual cortical area V1. Two previous studies of the layout of V2 FB projections onto the V1 orientation map have demonstrated orientation-specific, one, and unspecific FB connections, the other. Our preliminary data suggest existence of two FB systems, likely related to different cell types, which show unique relationships to the cortical functional architecture, thus providing a way to reconcile apparently contradictory data. The contribution of the proposed research is significant because it will provide new tools for studying the fine-scale connectivity and functional organization of inter-areal circuits made by specific cel types. General organizing principles for these connections will emerge that will provide an anatomical foundation for hypothesis-driven studies of their function. The proposed research is innovative because unlike previous studies: 1) the novel labeling method permits high-resolution, unambiguous identification of single inter-areal neurons, from soma to axon; 2) semi-automated mapping of 3D volumes from serial sections allows for fast reconstruction and, thus, higher yield of reconstructed axons; 3) it combines for the first time functional imaging of corticl responses with labeling of single FB axons.