Membrane targeted rabies virus labeling system: Brain-wide single-neuron mapping of target-specific and intersectionally defined neuronal circuits

Elucidating neural circuit organization at single-neuron resolution is a core goal of modern neuroscience. Brain-wide input connectivity maps underpin the mammalian connectome, but systematic and comprehensive whole-brain input mapping with resolved single-neuron anatomy for defined brain regions remains a formidable technical challenge. Here, we developed a membrane-targeted rabies virus labeling system using mClover3 as the fluorescent reporter, which features high compatibility with fluorescence micro-optical sectioning tomography imaging and enables complete visualization of fine neuronal morphology across the entire brain. Validation in the mouse primary somatosensory cortex barrel field confirms robust full-morphology labeling and faithful reconstruction of brain-wide input neurons, revealing diverse projection patterns with distinct axonal targets. We further generated a dual-recombinase dependent TVA reporter mouse line to enable anatomical interrogation of intersectionally defined neuron populations via EnvA-pseudotyped membrane-targeted rabies virus, allowing specific targeting and single-neuron reconstruction of PV+/vGlut2+ L5b pyramidal neurons in the primary somatosensory cortex barrel field. These neurons exhibited a pathway-conserved population-level projection profile likely representing a subset of pyramidal tract neuron projections, while showing pronounced cellto-cell diversity in axonal target specificity. Overall, this work delivers a robust, versatile toolkit for brain-wide single-neuron mapping of target-specific and intersectionally defined neuronal circuits, with strong potential to advance brain connectomics.