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 ...