Single-Neuron Connectome of the Central Amygdala in the Mouse Brain

The central amygdala (CeA) orchestrates defensive behaviors, pain processing, and stress responses, yet how its molecularly defined neuronal subtypes are embedded in brain-wide circuits remains unclear. Here, we reconstructed the brain-wide input and output architecture of three neuronal subtypes, CeA Sst , CeA Pkc-δ , and CeA Crh neurons, at single-cell resolution in male mice. Single-cell reconstruction revealed various projection-defined classes within each molecular-defined subtype, ranging from locally restricted neurons to broadly broadcasting neurons that coinnervate hypothalamic, midbrain, and brainstem. CeA outputs exhibit subtype-specific hemispheric asymmetry, providing an anatomical substrate for functional lateralization of CeA circuits. Coprojection analysis showed segregated and convergent CeA output pathways across midbrain and brainstem structures, supporting functional heterogeneity in the selection of appropriate defensive behaviors. Mapping of the input of these neurons further uncovered segregated and spatially organized upstream networks. Cortical projection neurons, particularly from the insular cortex, sent lateralized projections to distinct CeA subregions. Joint analysis of cortical and CeA projection architectures revealed structural signatures of both serial and parallel coordination. Taken together, these findings demonstrate a multilevel, subtype-specific, and lateralized architecture linking molecular identity to brain-wide CeA connectivity.