Project summary: This project will develop and validate a comprehensive toolset of novel technologies for imaging axonal projections across scales, and will deploy this toolset to map a complex system of cortico- subcortical projections in the macaque and human brain. We will combine the complementary strengths of three innovative microscopy techniques. First, polarization-sensitive optical coherence tomography (PS-OCT) will provide label-free, undistorted imaging of axonal orientations at the scale of microscopic fascicles, allowing us to follow fascicles across the brain without the need for axon segmentation. Second, whole-mount light- sheet microscopy (LSM) of cleared and immunolabeled sections will allow us to image fascicles at the sub- micron scale, resolving individual axons. Third, hierarchical phase-contrast tomography (HiP-CT) will allow us to image both the axons and their micro-environment, at a range of scales from a few microns down to sub- micron. We will scale these three microscopy techniques up to image a large sub-volume of the brain (up to two thirds of a hemisphere) that contains subcortical projections of the motor, premotor, and prefrontal cortex. In the macaque brains, fluorescent tracer injections will allow direct validation of our novel microscopy techniques. In combination with an extensive collection of prior tracer injections, the macaque data will also provide the topographic organizational rules of fibers in cortico-subcortical bundles, which we will then use to validate our novel microscopy techniques in human brains. In both macaque and human specimens, we will also collect extensive, cutting-edge, whole-brain diffusion MRI data, which will provide the link to non-invasive neuroimaging. The unprecedented datasets generated by our project will enable research discovery in two use cases. In the first use case, we will annotate projections of the motor, premotor, and prefrontal cortex to the subthalamic nucleus (STN). We will use them to advance our understanding of circuits associated with clinical improvements in four diseases that are treated with deep-brain stimulation in neighboring subzones of the STN: dystonia, Tourette’s syndrome, Parkinson’s disease, and obsessive-compulsive disorder. In the second use case, we will investigate the mapping from the axonal orientations and microstructural features obtained from the microscopy data to dMRI signals acquired in the same brains. In addition to the unprecedented datasets and the two use cases described above, this project will generate state-of-the-art pipelines for pre- processing, co-registration, axon segmentation, tractography, and quantification, across the scales spanned by the acquired data. We will develop a novel platform for sharing the microscopy, tracer, and MRI data with the research community. This will go well beyond a static data repository, allowing the user to interact with the data remotely and providing a “validation engine” for testing neuroimaging software tools against the gold standard post mortem data collected by this project. If successful, this project will generate a scalable and validated toolset for imaging connectional anatomy, with a direct link it to its applications in the study of human disease.

Project narrative: This project will generate connectional diagrams of the monkey and human brain at unprecedented resolutions. These diagrams will be linked both to the neuroanatomic literature and to in vivo neuroimaging techniques, bridging between the rigor of the former and the clinical relevance of the latter. The data to be generated by this project will advance our understanding of brain circuits that are implicated in motor and psychiatric disorders, and that are targeted by deep-brain stimulation to treat these disorders.