Temporal single-cell profiling of the parabrachial Calca neurons reveals molecular dynamics driving nociplastic pain

Parabrachial Calca neurons are necessary for chronic pain and sufficient to drive nociplastic pain in mice, but how they sustain a pain state that outlasts its trigger is unknown. We performed temporal single-cell mRNA sequencing of the parabrachial nucleus (PBN) across the onset, chronic, and recovery phases of Calca neuron-driven tactile allodynia, using fixed-tissue profiling and reference-atlas registration to track molecularly defined populations over time. Activation broadly induced immediate-early genes, after which Calca neurons displayed changes in expression of genes that affect signaling and synaptic plasticity, with bidirectional changes that mirrored the onset and resolution of allodynia. The gene encoding brain-derived neurotrophic factor (Bdnf) remained persistently elevated in the chronic phase. BDNF infusion in the PBN prolonged allodynia, whereas blockade of its receptor (TrkB) attenuated it, and inactivating the Bdnf gene in Calca neurons abolished their hyperexcitability, attenuated allodynia, and relieved pain in a migraine model. These observations pinpoint BDNF as a driver of persistent nociplastic pain.