ANATOMY RESEARCH SEGMENT SUMMARY The overarching goal of neuroanatomy is to establish a structural framework to integrate multiscale and multi-modal information and to provide a road map that guides the exploration of neural dynamics and brain function. “Anatomic” neuron types can be described by their location, morphology, and connectivity. As morphology is one of the most intuitive depictions of cell types that reflects their input-output connectivity, the visualization, characterization and quantification of their complete and detailed shapes are key to the identification and classification of anatomic neuron types. However, these have remained enduring challenges for over a century, as most vertebrate neurons are simultaneously small and large, spanning vast spatial scales from submicron to centimeter (e.g. corticospinal neurons connecting cortex to spinal cord). The overall goals of the Anatomy Segment are to: 1) establish a Forebrain Projection Cell Atlas that integrates in-depth multi-modality anatomic and molecular information across cerebral cortex, basal ganglia, thalamus, and hypothalamus. A forebrain projection neuron atlas will provide a core skeleton that anchors nearly all other brain structures and the transcriptome cell atlas constructed in the Molecular Segment;; 2) advance current and emerging imaging technologies and pipelines with improved resolution, throughput and lower cost. We will use several state-of-the-art imaging technologies and pipelines to obtain multi-modal, high- resolution whole brain datasets on cell location, morphology, projection, and connectivity of vast sets of forebrain neurons, all registered in the mouse brain Common Coordinate Framework (CCF). To achieve high-resolution brain-wide imaging, we will use 1) Serial Two-Photon tomography (STP) to image marker- defined neuronal body distribution, 2) STP to image axon projection patterns of genetic and virally-labeled neuron subpopulations, 3) fluorescence Micro-Optical Sectioning Tomography (fMOST) to image and reconstruct the complete morphology of single neurons – the ultimate resolution for anatomic cell typing. Importantly, this anatomical forebrain atlas will integrate single-cell molecular information collected and positionally mapped for the same cell types in the Molecular Segment, towards a close-to-completion Molecular and Anatomical atlas of the mouse forebrain.