Figure 6. Formalin- and capsaicin-induced NK1R internalization. NK1R internalization in the spinal cord of male Sprague Dawley rats was observed by immunofluorescence. A, In control rats, NK1R-like immunostaining was clearly visible throughout the superficial laminae of the spinal cord. Immunolabeling of NK1R appeared at the cell surface. Ten minutes following intradermal injection of formalin, NK1R immunostaining in the ipsilateral side appeared as being inside the cells in vesicle-like structures (C). By contrast, NK1R labeling in the contralateral side of the spinal cord remained at the cell surface (B). When Dlt II (30 μg; 38.1 nmol) was injected intrathecally 5 min before formalin, a significant reduction in NK1R internalization was observed (D). This effect was blocked by NTI (150 nmol) (E). A similar effect was seen when DAMGO (10 μg; 19.4 nmol) was injected intrathecally 5 min before formalin (F), an effect blocked by CTOP (30 nmol) (G). Similarly, capsaicin induced a robust NK1R internalization in the ipsilateral side of the spinal cord (I) but not on the contralateral side (H). Injection of Dlt II (J) or DAMGO (L) 5 min before capsaicin injection prevented NK1R internalization. Again, the effects of Dlt and DAMGO were blocked by preinjection of NTI and CTOP, respectively (K, M). N, Graphic representation of the percentage of neurons with internalized NK1R induced by formalin and capsaicin injections for ipsilateral and contralateral sides of the lumbar spinal cord. *p < 0.05, **p < 0.01, and ***p < 0.001, one-way ANOVA with Bonferroni's post hoc test. The numbers in parentheses represent the number of animals per group. Scale bar: B, 30 μm. Error bars indicate SEM.