A three dimensional multimodal cellular connectivity atlas of the mouse hypothalamus

项目总结/摘要 哺乳动物下丘脑在生存所必需的行为中起着关键作用。下丘脑外侧 LHA区（LHA）占下丘脑的55%，包含不同的神经元类型，但仍然是下丘脑的一个重要组成部分。 在小鼠图谱中未分化的区域。关于其结构和职能组织仍有许多未知之处 在区域、细胞类型特异性和个体神经元尺度上。此外，下丘脑在结构上和 在功能上性别二态性，但性别差异的大脑图谱是不存在的。我们假设 LHA结构组织的多尺度异质性是其细胞类型的特异性和性质的基础， 具体电路功能组织。我们将使用已建立的和创新的方法来调查这一点， 将分子、细胞和回路级LHA数据关联并合成为多模态、多尺度LHA 参考图集在目标1中，我们将构建男性和女性的多尺度、多模态、3D参考图谱 基于LHA的细胞和化学结构的小鼠下丘脑（目的1a）。这些立体3D 光片数据将被登记到艾伦公司的CCF中，结构描述将使用一种新的 基于云的3D方法。在目标1b中，我们将使用基于连接性的分割策略来细化 基于对>300组预先收集的数据的系统分析，以更高的粒度描绘LHA 通过条件性（Cre依赖性）病毒通路追踪收集的神经通路和其他连接数据 方法.将通过调查LHA亚类的特异性来验证LHA亚类的细化分组。 输入/输出组织。在目标2中，我们将系统地研究LHA分子多样性和网络 组织在中型和微型分辨率。首先，对于每个LHA分区，我们将调查 结合逆行通路研究GABA和GLU靶向投射神经元的分子特性 用RNAscope或HiRNAscope标记（Aim 2a）。然后，对于这些目标特异性GABA和GLU神经元， 类型，我们将系统地检查他们的轴突抵押（目标2b）和神经输入（目标2c）使用2- Vgat-Cre和Vgat 2-Cre基因敲入小鼠的双向病毒标记方法。最后，在Aim 2d中，通过结合一部小说， 遗传稀疏标记方法（MORF）与大脑清除，3D显微成像，和计算 重建算法，我们将重建精细详细的单神经元形态（树突和轴突） >2000 GABA和GLU神经元在男性和女性LHA。所有数据都将注册到新的 在CCF内构建了3D下丘脑图谱（目标1），以构建前所未有的全面 男性和女性下丘脑的细胞图谱。在目标3中，我们将建立一个计算平台， 用于（1）电路、神经元和突触重建、注释和 用有效的地面真实人类评估验证的纠错;以及（2）为了有效的数据传输， 存储和传播。将处理所有CCF注册的神经元形态和连接数据 并通过我们的UCLA BRAIN数据门户传播。

Project Summary/Abstract The mammalian hypothalamus plays a critical role in behaviors essential for survival. The lateral hypothalamic area (LHA) occupies 55% of the hypothalamus and contains diverse neuron types, yet remains an undifferentiated region in mouse atlases. Much remains unknown about its structural and functional organization at the regional, cell-type specific, and individual neuron scales. Further, the hypothalamus is structurally and functionally sexually dimorphic, yet differentiated brain atlases for the sexes is absent. We hypothesize that multiscale heterogeneity of LHA structural organization underlies the specificity and properties of its cell type- specific circuit functional organization. We will investigate this using established and innovative approaches to correlate and synthesize molecular, cellular, and circuit-level LHA data into a multimodal, multiscale LHA reference atlas. In Aim 1, we will construct a multiscale, multimodal, 3D reference atlas of the male and female mouse hypothalamus based on the cyto- and chemoarchitecture of the LHA (Aim 1a). These volumetric 3D lightsheet data will be registered to the Allen CCF and structural delineations will be conducted using a novel cloud-based 3D approach. In Aim 1b, we will use our connectivity-based parcellation strategy to refine the delineation of the LHA at a much higher granularity based on the systematic analysis of >300 sets of pre-collected neural pathways and additional connectivity data collected via conditional (Cre-dependent) viral pathway tracing methods. The refined parcellation of LHA subdivisions will be validated by investigating specificities of their input/output organization. In Aim 2, we will systematically investigate LHA molecular diversity and network organization across meso- and microscale resolutions. First, for each LHA subdivision, we will investigate the molecular identities of target-specific GABA and GLU projection neurons by combining retrograde pathway labeling with RNAscope or HiPlex RNAscope (Aim 2a). Then, for these target-specific GABA & GLU neuron types, we will systematically examine their axon collateralizations (Aim 2b) and neural inputs (Aim 2c) using a 2- way viral labeling method in Vgat-Cre and Vglut2-Cre knock-in mice. Finally, in Aim 2d, by combining a novel genetic sparse labeling method (MORF) with brain clearing, 3D microscopic imaging, and computational reconstruction algorithms, we will reconstruct the fine detailed single neuron morphology (dendrites and axons) of >2000 GABA and GLU neurons in male and female LHA. All of the data will be registered into the newly constructed 3D hypothalamic atlas (Aim 1) within the CCF to construct an unprecedentedly comprehensive cellular atlas of the male and female hypothalamus. In Aim 3, we will establish a computational platform and a specialized computational infrastructure for (1) circuit, neuronal, and synaptic reconstruction, annotation, and error correction validated with efficient ground truth human assessments; and (2) for efficient data transfer, storage, and dissemination. All CCF-registered neuronal morphology and connectivity data will be processed and disseminated through our UCLA BRAIN data portal.