Mapping Connectivity of the Human Brainstem in a Nuclear Coordinate System

The ~1 billion neurons that form the human brainstem are organized at multiple scales, ranging from their cell type-specific patterns of dendritic arborization, to local circuits embedded within large-scale projection systems spanning the brainstem, and a complex nuclear architecture. In this project, we will image across this vast range of scales to build technologies to create a multiscale atlas akin to Google Earth for the human brainstem to visualize brainstem-wide networks and zoom in to the level of individual, labeled cells and their connectivity at micrometer resolution within the context of individual nuclei. This dramatic advance will be made possible through the use of an array of imaging technologies, including light-sheet fluorescence microscopy (LSFM), tissue clearing, immunohisto-chemistry (IHC), 2-photon expansion microscopy (2PEM), magnetic resonance imaging (MRI) and newly developed techniques in polarization-sensitive optical coherence tomography (PS- OCT). PS-OCT in particular is a potentially transformative technology as it provides micrometer resolution over large volumes of tissue, images all of the tissue (as opposed to fluorescence), does not require mounting and staining, can be automated, is essentially distortion free as it images the tissue prior to cutting, and with innovations we propose in our project, allows direct measures of 3D axonal orientation. LSM-based IHC will provide molecular, morphological and spatial properties of cells and their projections that will enable us to nuclear boundaries to place the connections in a nuclear context, 2PEM will provide direct validation of the 3D-PSOCT, and the OCT will also enable us to remove the distortions induced by cutting and clearing, and transfer information to intact brainstem and whole-hemisphere MRI for quantitative atlasing and in vivo inference.