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

描述(申请人提供)：了解大脑皮层的信息处理需要了解较低和较高皮质中心之间的前馈(FF)和反馈(FB)电路的作用。这些回路的组织原则可能决定它们如何处理感觉信息，但在很大程度上仍是未知的。这是由于区域间环路的复杂性，即其解剖学和功能的特殊性，以及缺乏方法来揭示特定细胞类型组成的Ff和Fb环路的精细连接，并将其与皮质的功能结构联系起来。超微结构尺度的电路解剖学虽然有助于建立小鼠皮质局部连接的接线图，但不能用于研究被区域间轴突包围的大皮质体积。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。以前的单一轴突研究受到轴突标记来源不明确、不能将标记限制在少数神经元以及费力的手动重建的影响。这些研究只提供了一小部分未完全重建的轴突样本，偏向于标记较稀疏的区域，没有确定其起源的细胞类型。我们的具体目标是：目标1.在高分辨率下明确地标记单投影神经元的轴突，并开发一种新的半自动单轴突重建的计算框架。我们将把病毒介导的GFP表达扩展到高分辨率标记，并稀疏区域间投射神经元的轴突。我们将开发一种新的方法来快速连续重建单个轴突，其中包括对光学透明的完整组织块进行3D成像，以及用于半自动轴突分割的新的计算算法。目的2.应用这些方法来解决文献中关于区域间反馈投射对灵长类视觉皮质V1区的功能特异性或缺乏的争议。之前两项关于V2 FB投影在V1方位图上的布局的研究已经证明了方向特定的、一种和非特定的FB联系，另一种。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，这两个系统显示出与大脑皮层的独特关系 功能架构，从而提供了一种协调表面上相互矛盾的数据的方法。拟议的研究的贡献是重大的，因为它将为研究提供新的工具 由特定类型的小区构成的区域间电路的精细连接和功能组织。这些联系的一般组织原则将出现，这将为它们的功能的假设驱动的研究提供解剖学基础。这项拟议的研究具有创新性，因为与以前的研究不同：1)新的标记方法可以高分辨率、明确地识别从胞体到轴突的单个区域间神经元；2)从连续切片半自动绘制3D体积图可以快速重建，从而获得更高的重建轴突产量；3)它首次结合了皮质反应的功能成像和单个FB轴突的标记。

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.