PROJECT SUMMARY A structural connectome can provide critical information for animal brain studies to study white matter bundles and correlate brain circuits with experimental findings. As more animal models for neurological disorders are widely used to understand the disease mechanism and test possible treatments, tractography atlases will help those studies to correlate white matter pathways with brain function or dysfunction. Here, we aim to construct tractography atlases at unprecedented details as structural connectome for mouse, rat, and rhesus macaque and developed associated toolboxes for novel tractography analytics. To achieve this goal, we have formed a multidisciplinary team between Duke Center for In Vivo Microscopy and the "DSI Studio" team at the University of Pittsburgh. At Duke CIVM, we will acquire the ultra-high resolution "beyond-tensor" diffusion MRI on the mouse, rat, and rhesus macaque brains to resolve complicated axonal structures. The image acquisition will be accelerated using compressed sensing and further reconstructed using a super-resolution approach. Besides MRI, the rat and mouse brain tissue will be processed by tissue clearing and scanned by light-sheet fluorescence microscopy to capture cell-level histology details of the entire brain. The diffusion MRI data will then be processed at the University of Pittsburgh to construct tractography atlases and develop novel automated tractography, shape analysis, and bundle analytics toolboxes. We will track whole-brain pathways and cluster them into bundles. Our neuroanatomist team will validate tractography against publicly available tracer data and aggregate the results in tractography atlases, which will be used to build their associated atlas- based automated tractography and associated analysis. The completion of this study will provide a mesoscale structural connectome with integrated histologic images at unprecedented quality as well as their associated toolboxes. The data and tools combined will serve as invaluable resources for animal studies to investigate brain disorders.