Xie, G.; Wang, M.; Liu, Y.; Zhang, Y.

Chronic pain is a leading cause of human suffering, affecting about 20% of the population. The insular cortex (IC) is a central hub for the multidimensional experience of pain, yet how the diverse neurons in IC are organized and assembled into functional circuits remains unclear. Here, combining multiplexed error-robust fluorescence in situ hybridization (MERFISH), neural tracing and in vivo functional analyses, we resolve the molecular, cellular and circuit architecture of the IC. We find that neuronal projections are specified by both transcriptomic identity and spatial topography. We further show that three molecularly defined neuron types in the posterior IC form discrete brain-wide networks that differentially regulate pain. Layer 5 pyramidal tract (PT) neurons regulate mechanical, thermal and affective pain. In contrast, layer 5 intratelencephalic (IT) neurons selectively modulate thermal nociception, whereas layer 6 corticothalamic (CT) neurons surprisingly relieve negative pain affects. Together, these findings reveal the spatially resolved cellular and circuit organization of the IC governing different dimensions of pain and uncover targets for precision pain therapy.