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

描述（由申请人提供）：理解大脑皮层的信息处理需要理解下部和上部皮层中心之间的前馈（FF）和反馈（FB）回路的作用。这些电路的组织原理，决定了它们如何处理感官信息，在很大程度上仍然未知。这是由于区域间回路的复杂性，即它们的解剖和功能特异性，以及缺乏可以揭示特定细胞类型形成的FF和FB回路的精细连通性并将其与皮层的功能结构联系起来的方法。超微结构尺度的电路解剖虽然有助于建立小鼠皮层局部连接的接线图，但不能用于研究由区域间轴突包围的皮质大体积。后者只能在介观尺度上进行研究。我们的目标是开发一种方法来标记和有效地重建，单细胞类型及其区域间轴突。以往的单轴突研究受到轴突标签来源不明确、无法将标签限制在少数神经元上以及人工重建费力的影响。这些研究只提供了不完全重建轴突的小样本，偏向于较少标记的区域，没有确定它们的细胞类型来源。我们的具体目标是：目的：在高分辨率下明确标记单个投射神经元的轴突，并为半自动单轴突重建开发一种新的计算框架。我们将把病毒介导的GFP表达扩展到高分辨率和稀疏的区域间投射神经元轴突的标记。我们将开发一种用于单个轴突快速连续切片重建的新方法，其中包括呈现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型计算算法。目标2。为了应用这些方法来解决文献中关于灵长类动物视觉皮层V1区区间反馈投射功能特异性或缺乏功能特异性的争议。之前的两项关于V2 FB投影在V1方向图上的布局的研究已经证明了定向特异性FB连接和非特异性FB连接。我们的初步数据表明存在两个FB系统，可能与不同的细胞类型有关，它们与皮层表现出独特的关系

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.