Formalin-induced pain preferentially activates orexin neurons in male mice

Sex differences are well-documented across many functions of the orexin system; however, whether such differences extend to pain processing has been examined far less thoroughly. Therefore, we investigated whether males and females differ in orexin system engagement during nociception, and whether any differences reflect structural organization or functional recruitment. Adult C57BL/6N mice of both sexes were used, with females tested during proestrus and metestrus. Formalin was injected into the hind paw, and c-Fos induction in lateral hypothalamic orexin neurons was quantified together with orexin A/B fiber density and orexin receptor OX1R/OX2R mRNA expression in the lumbar cord. Orexin A and B were fully colocalized, allowing orexin B to serve as a reliable marker. The total number of orexin-immunoreactive neurons did not differ between the sexes or across estrous stages, and spinal orexinergic innervation and OX1R/OX2R mRNA levels were comparable across groups. In contrast, formalin induced a significantly higher proportion of c-Fos-positive orexin neurons in males, indicating male-biased recruitment of orexin neurons during nociception, despite equivalent anatomical substrates. These findings suggest that sex differences in orexin involvement in nociception are not attributable to structural disparities in the number of orexin neurons, spinal projections, or receptor expression, but instead arise from sex- and context-dependent differences in the functional activation of orexin neurons in response to painful stimuli.