A three dimensional multimodal cellular connectivity atlas of the mouse hypothalamus

项目摘要/摘要 哺乳动物的下丘脑在生存所必需的行为中起着关键作用。外侧下丘脑 下丘脑面积(LHA)占下丘脑的55%，含有不同类型的神经元，但仍是 小鼠图谱中的未分化区域。关于其结构和功能组织，仍有许多未知之处 在区域、细胞类型特定和单个神经元的尺度上。此外，下丘脑在结构上和 功能上的性别二态，但性别分化的脑图谱是不存在的。我们假设 LHA结构组织的多尺度异质性是其细胞类型的特殊性和特性的基础。 具体的电路功能组织。我们将使用既定的和创新的方法来调查这一点 将分子、细胞和电路级LHA数据关联并合成为多模式、多尺度LHA 参考图集。在目标1中，我们将构建一个多比例、多模式的男性和女性的3D参考地图集 基于LHA的细胞和化学结构的小鼠下丘脑(目标1a)。这些体积3D LightSheet数据将注册到Allen CCF，并将使用一种新的 基于云的3D方法。在目标1b中，我们将使用我们基于连接的分区策略来细化 基于对&gt；300套预收集数据的系统分析，在更高的粒度上圈定LHA 通过条件性(Cre依赖)病毒路径跟踪收集的神经路径和其他连接性数据 方法：研究方法。LHA细分的精细化划分将通过调查其特殊性来验证 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 跨中、微观分辨率的组织。首先，对于每个LHA分区，我们将调查 结合逆行通路的靶向特异性GABA和GLU投射神经元的分子同一性 使用RNAScope或HiPlex RNAScope进行标记(目标2a)。然后，对于这些靶向特异性的GABA和GLU神经元 类型，我们将系统地检查它们的轴突侧化(目标2b)和神经输入(目标2c)使用2- Vgat-Cre和Vher2-Cre基因敲入小鼠的病毒标记方法。最后，在目标2d中，通过结合一部小说 结合脑透明、3D显微成像和计算的遗传稀疏标记方法(MORF) 重建算法，我们将重建精细详细的单个神经元形态(树突和轴突)。 男性和女性LHA中GABA和GLU神经元的数量。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)，构建了前所未有的全面 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和一个 用于(1)电路、神经元和突触重建、注释和 利用有效的地面实况人类评估来验证纠错；以及(2)为了有效的数据传输， 储存和传播。将处理所有CCF注册的神经元形态和连接数据 并通过我们加州大学洛杉矶分校的大脑数据门户进行传播。

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.