Fig. 2. Effects of intrathecal 20 μg morphine on formalin-induced, paw-flinching behavior and neurokinin-1 receptor (NK-1r) internalization. Time-effect curves of intrathecal morphine and saline on formalin-induced paw flinching (A ). Histogram showing cumulative flinching during phase 1 and phase 2 (B ). Intrathecal morphine significantly reduced phase 2 of formalin-induced paw flinching. The percentage of NK-1r(+) neurons showing internalization versus spinal segment as a function of intrathecal treatment (saline vehicle ipsilateral, saline vehicle contralateral, and morphine treatment ipsilateral) (C ). Unilateral intraplantar injection of formalin (5%) produced a robust ipsilateral NK-1r internalization at L4–L6 levels of lumbar lamina I compared with the contralateral side. Intrathecal morphine significantly reduced formalin-induced NK-1r internalization in the spinal segments L5 and L6 compared with saline. Representative light microscopic images of NK-1r internalization induced by unilateral injection of formalin (5%) into the hind paw (D–F ). Image of formalin-induced NK-1r internalization in the ipsilateral spinal lamina I from a rat administered intrathecal saline (D ). Note the lack of a homogeneous cell membrane and the presence of NK-1–containing endosomes internalizing into the cytoplasm. Image of formalin-induced NK-1r internalization in the contralateral spinal lamina I (E ). Note the presence of a homogeneous cell membrane and the lack of NK-1–containing endosomes internalizing into the cytoplasm. Intrathecal morphine (20 μg) blocked formalin-induced NK-1r internalization (F ). Data are presented as mean number of paw flinching and percentage of NK-1r internalization with vertical bars showing SEM. * Significant difference between saline-treated and drug-treated animals, P < 0.05. Magnification, ×60. Scale bar is 10 μm. Saline, n = 6; morphine, n = 5 (A and B ). Saline ipsilateral, n = 5; contralateral, n = 5; morphine; n = 3 (C ).