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Highlights d Prefrontal CRF GABAergic interneurons are recruited during behavioral challenges d Manipulation of CRF neurons motivates selection of behavioral styles under challenges d Activation of CRF neurons promotes persistent stressresistant behaviors

The speed of high-resolution optical imaging has been a rate-limiting factor for meso-scale mapping of brain structures and functional circuits, which is of fundamental importance for neuroscience research. Here, we describe a new microscopy method of Volumetric Imaging with Synchronized on-the-fly-scan and Readout (VISoR) for high-throughput, high-quality brain mapping. Combining synchronized scanning beam illumination and oblique imaging over cleared tissue sections in smooth motion, the VISoR system effectively eliminates motion blur to obtain undistorted images. By continuously imaging moving samples without stopping, the system achieves high-speed 3D image acquisition of an entire mouse brain within 1.5 hours, at a resolution capable of visualizing synaptic spines. A pipeline is developed for sample preparation, imaging, 3D image reconstruction and quantification. Our approach is compatible with immunofluorescence methods, enabling flexible cell-type specific brain mapping and is readily scalable for large biological samples such as primate brains. Using this system, we examined behaviorally relevant whole-brain neuronal activation in 16 c-Fos-shEGFP mice under resting or forced swimming conditions. Our results indicate the involvement of multiple subcortical areas in stress response. Intriguingly, neuronal activation in these areas exhibits striking individual variability among different animals, suggesting the necessity of sufficient cohort size for such studies.