The Parasubthalamic-Lateral Parabrachial Glutamatergic Circuit Mediates Orofacial Affective Pain

To elucidate how the parasubthalamic nucleus (PSTN) regulates orofacial affective pain via glutamatergic projections to the lateral parabrachial nucleus (LPB). 1.PSTN activation: Immunohistochemistry: c-Fos⁺ neurons in the PSTN of Vglut2::H2B mice were quantified 90 min post 10 μL 4% formalin whisker pad injection. Fiber photometry recording: record calcium signals from PSTN^(Vglut2) neurons during orofacial noxious mechanical stimulation. 2.Circuit tracing: Neural anterograde tracing using AAV2/9-hSyn-DIO-EGFP mapped glutamatergic PSTN→LPB projections. 3.Functional manipulation: Activation: 473 nm light (20 Hz pulses) targeted PSTN^(Vglut2)-LPB during von Frey and conditioned place aversion (CPA) tests. Inhibition: 593 nm light suppressed PSTN^(Vglut2)-LPB activity in mice with orofacial capsaicin-induced pain (0.03%, 10 μL). 1.PSTN nociceptive encoding: Orofacial inflammatory pain (4% PFA) induced c-Fos⁺ expression in PSTN^(Vglut2) neurons (p < 0.05), and orofacial nociception elevated calcium activity in PSTN^(Vglut2) neurons. 2.Projection mapping: Glutamatergic PSTN neurons projected to multiple pain-modulatory regions, including the lateral parabrachial nucleus (LPB). 3.Bidirectional modulation: Optogenetic activation of Vglut2⁺ axon terminals in LPB induced orofacial mechanical hypersensitivity(p < 0.01) and aversion. Optogenetic inhibition of Vglut2⁺ axon terminals in LPB reversed capsaicin-induced orofacial allodynia(p < 0.01) and normalized affective behaviors. The PSTN-LPB glutamatergic circuit drives orofacial affective pain through descending facilitation, with targeted inhibition relieving both sensory and emotional components, offering a therapeutic strategy for refractory orofacial pain.