Project Summary This proposal's objective is to develop synchrotron-based X-ray imaging technologies to enable high-resolution imaging of brain-wide neuronal circuits. Comprehensively mapping brain-wide circuits is not currently feasible, even in small mammalian model systems, because light microscopy (LM) lacks sufficient resolution and electron microscopy (EM) cannot be applied over large volumes. Leveraging the unprecedented qualities of the new 4th generation synchrotron source at the European Synchrotron, we will develop X-ray nano-holography (XNH) imaging techniques for large-scale imaging of brain circuits. Taking advantage of improvements in source coherence and brightness, we will improve imaging resolution to allow direct visualization of synaptic connections between neurons, and develop imaging protocols that allow imaging of centimeter-scale circuit volumes within a typical beamline experiment. We will combine non- destructive XNH with EM and LM imaging techniques to rigorously and quantitatively validate the accuracy of XNH- based circuit reconstruction. We will then use this correlative workflow to study the relationships between long-range sensory inputs, local synaptic micro-circuitry, and single-neuron activity, investigating how circuits in the posterior parietal cortex (PPC) support perceptual decision-making. Lastly, we will apply XNH circuit-mapping over an entire cortical hemisphere, and utilize deep-learning based machine vision algorithms to obtain a comprehensive atlas of cortical connectivity. This atlas will in principle resolve all long-range connections between cortical areas at single-axon resolution, lending insight into how distinct cortical areas achieve specialized function, and how distributed cortical networks support cognition and are affected by psychiatric disorders.