Brain-wide atlas of outputs and inputs of mouse auditory cortex revealed by modified viral labeling strategies

Understanding brain-wide connectivity is necessary to understand the principles of neural functions. While traditional neuron labeling methods have contributed to structural mapping, they often fall short in accurately identifying neuronal outputs because of limitations in solely using axon tracing. In this study, we improved labeling specificity by incorporating nuclear-localized fluorescent markers into retrograde and monosynaptic anterograde viral tracing strategies. Combined with high-resolution whole-brain imaging, this method enabled precise mapping of input-output relationships in the mouse auditory cortex (ACx). Soma-based labeling allowed for the clear identification of upstream and downstream neurons across the entire brain. Our analysis revealed that ACx projections to the primary somatosensory (SSp) and primary visual (VISp) cortices were largely restricted to layer 5, contrasting with previous reports suggesting stronger innervation in layers 1–3. We also identified downstream neurons within the corpus callosum (CC), a region not previously known to receive direct input from the ACx. These findings highlight the advantages of soma-targeted approaches in connectivity research and offer new insights into the organization of auditory networks. This approach lays the groundwork for future studies of circuit morphology and function across the brain.