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Pain and itch are recognized as antagonistic sensations; pain suppresses itch and inhibition of pain generates itch. There is still a lack of evidence about the neural mechanism of the interaction between pain and itch in the central nervous system. In this study, we focused on the orexin (ORX) neurons in the lateral hypothalamus (LH), which mediate various “defense responses” when animals confront stressors. We found that the scratching behaviors induced by the pruritogen were significantly suppressed in ORX-neuron-ablated (ORX-abl) mice. The exaggerated pain behavior and attenuated itch behavior observed in ORX-abl mice indicated that ORX neurons modulate pain and itch in an opposite way, i.e., pain relief and itch exacerbation. In addition, most of the ORX neurons responded to both pain and itch input. Our results suggest that ORX neurons inversely regulate pain- and itch-related behaviors, which …