Organization of retrosplenial cortex projections at the single-neuron level

The retrosplenial cortex (RSP) supports spatial cognition, memory, and brain-wide information integration, yet its output architecture remains poorly resolved at single-neuron resolution. Using fluorescence micro-optical sectioning tomography (fMOST), we reconstruct sparsely labeled neurons sampled predominantly from the anterior RSP across the whole brain. We find three major projection routes-cortical-restricted (CR), thalamic-restricted (TR), and thalamic-brainstem collateral (TBC)-and classify neurons into ten projection subtypes with distinct target-selective profiles. Somatic location and volumetric features are associated with axonal projection patterns. TR-pattern neurons show subregion-dependent thalamic targeting, whereas TBC-pattern neurons display greater axonal complexity and subtype-specific collateralization toward thalamic, midbrain, and hindbrain targets. Bilaterally projecting neurons across distinct projection patterns show cross-network projection modes with an overall ipsilateral preference and laminar-specific commissural architectures. Together, these findings provide a single-neuron anatomical map of RSP cortical, thalamic, and cortico-subcortical output patterns and a framework for examining distributed information processing across brain-wide networks.