Abstract In this 5 year R01 grant entitled “Unraveling the superficial white matter of the primate brain: Tracer-based histology and diffusion MRI tractography validation,” we will map superficial white matter (SWM) in the primate cerebrum using experimental tract tracing methods. We will use this ground truth information to validate high- resolution in vivo and ultra-high resolution ex vivo diffusion MRI (dMRI) based tractography in the same rhesus macaque monkeys. The SWM is a continuous layer located between the cerebral cortex of the forebrain and the underlying white matter association pathways. It comprises axons that interconnect cerebral cortical areas, including U-shaped fibers (U-fibers) under the cerebral sulci. This axonal layer plays a role in a broad range of neurological conditions such as autism spectrum disorder, multiple sclerosis, and Alzheimer’s disease, and knowledge of the SWM is essential for accurately interpreting dMRI-based tractography. Critically, the fundamental connectional neuroanatomy of the SWM is largely unknown due to our inability to visualize the specific origins, terminations, and trajectories of axons in the human brain. Our knowledge of human SWM connectional neuroanatomy is thus derived almost exclusively from experimental tract tracing results in the non- human primate (NHP) model, but comprehensive studies of the NHP SWM, from origin through trajectory to termination, have not been performed. Therefore, knowledge of human SWM connectivity and organization can be improved by invasive tract tracing studies in the NHP. The goal of the proposed research is to carry out the first detailed neuroanatomical study of the SWM in the NHP brain, with dMRI validation in the NHP and translation to human brains. To achieve this goal, we will use a range of histological techniques in conjunction with dMRI scans obtained with the Massachusetts General Hospital (MGH) Connectom scanner to produce superior quality dMRI data. We will first datamine the research literature to produce a compendium of existing knowledge of SWM connectivity in the rhesus monkey. We will also examine the Pandya-Rosene Archive, a vast collection of neuroanatomy cases that has formed the foundation of data on white matter connections in the NHP. We will utilize this archive to chart the organization of the SWM in frontal brain areas. We will then use modern histological methods, including CLARITY-based tissue clearing and neuroanatomical tract tracing, to interrogate the structure, topography, and connectivity of the SWM, and thereby produce ground truth data. We will perform in vivo and ex vivo dMRI in the same animals in which we perform neuroanatomical tract tracing experiments, allowing for direct comparisons between histological and neuroimaging-based connectivity. Moreover, we will disseminate the dMRI data to the neuroimaging community and host a competition to determine the optimal tractography method for SWM. Finally, we will translate kno...