1 m sodium phosphate, pH 7.4) containing 0.01% heparin, followed by 400 mL of ice-cold fixative (4% paraformaldehyde and 0.18% picric acid in phosphate buffer). Paw withdrawal latencies were measured using a Plantar Analgesia Meter model 390G (IITC Life Sciences, Woodland Hills, CA, USA), consisting of an acrylic enclosure on an elevated warm glass surface (Cheppudira, 2006). Rats implanted with intrathecal catheters were acclimated to the instrument for 30 min for 3 days. The test consisted of heating the plantar surface of the hind paw from below with a radiant heat source. The intensity of the lamp was set at 30% of maximal power. Cut-off

time was 25 s to prevent tissue damage. Baseline paw withdrawal latencies were measured three times at 5-min intervals. Within 2 min of establishing the baseline, drugs were injected intrathecally. Ten minutes after the injection, paw withdrawal selleck chemicals llc latencies were measured again, four times at 5-min intervals. Results were calculated as percentage of MPE, the maximum possible selleck inhibitor response (Paronis & Holtzman, 1991): The NK1R antibody was rabbit antiserum no. 94168, made at CURE: Digestive Diseases Research Center, UCLA, under the sponsorship of Dr Nigel Bunnett, UCSF. It was generated in rabbits using a peptide corresponding to the C-terminus of the rat NK1R (amino acids 393-407, KTMTESSSFYSNMLA)

coupled to Keyhole limpet hemocyanin (KLH) (Grady et al., 1996). It labeled by immunofluorescence cells transfected with rat NK1R, and it did not label nontransfected cells. Staining of the transfected cells was eliminated by preadsorption with its SPTLC1 immunizing peptide. In Western blots from cells transfected with the NK1R, the antiserum produced a single band corresponding to a molecular weight of 100 kDa (Grady et al., 1996). Spinal cord slices were fixed, cryoprotected, frozen and re-sectioned at 25 μm in a cryostat as described (Marvizon et al.,

2003a; Adelson et al., 2009). Rats were fixed by aortic perfusion as described above, and lumbar spinal cord segments were similarly processed and sectioned at 25 μm in the coronal plane (Chen et al., 2007; Lao et al., 2008). Sections were washed four times and then incubated overnight with the NK1R antiserum diluted 1 : 3000 in phosphate-buffered saline containing 0.3% Triton X-100, 0.001% thimerosal and 10% normal goat serum. After three washes, the secondary antibody was applied at for 2 h at 1 : 2000 dilution. The secondary antibody was goat antirabbit IgG coupled to Alexa Fluor 488 (Invitrogen). Sections were washed four more times, mounted on glass slides, and coverslipped with Prolong Gold (Invitrogen). All incubations were done at room temperature. The amount of NK1R internalization was quantified using a standard method (Mantyh et al., 1995; Marvizon et al., 2003a).