1, P = 0.0001), ‘stimulus’ (F1 = 336, P < 0.0001) and a significant interaction between them (F3 = 12, P < 0.0001). Bonferroni’s post hoc test showed that the effects of ACEA and AM251 were check details significant and significantly reversed when combined (Fig. 2A). Dorsal root stimulation at 100 Hz produced higher NK1R internalization (Fig 2B). The increase produced by ACEA was less pronounced and the inhibition by AM251 more pronounced than with 1 Hz stimulation. Combining ACEA and AM251 cancelled their effects, but this time the inhibition by AM251 predominated. Other CB1 antagonists, AM281 (100 nm) and rimonabant (SR141716A, 100 nm), also decreased the evoked NK1R internalization. However, the inhibition by

rimonabant was less pronounced than the inhibition by AM251 and AM281 (P < 0.001). Two-way anova of the data in Fig. 2B yielded significant effects of the two variables ‘drugs’ (F7 = 524, P < 0.0001), ‘stimulus’ (F1 = 25749, P < 0.0001) and a significant interaction between them (F7 = 455, P < 0.0001). The decrease in the number

of lamina I neurons with NK1R internalization produced by AM281 is illustrated in Fig. 1C, corresponding to the dorsal horn ipsilateral to the stimulated root. As AM251 is also an agonist of the putative new cannabinoid receptor GPR55 (Lauckner Epacadostat purchase et al., 2008; Kano et al., 2009; Ross, 2009), it is possible that its inhibition of NK1R internalization was mediated by GPR55 and not CB1 receptors. To explore this possibility, we determined whether the selective GPR55 agonist O-1640 (Johns et al., 2007; Oka et al., 2007; Waldeck-Weiermair et al., 2008) inhibited the evoked NK1R internalization. O-1640 produced no effect (Fig. 2B; P > 0.05, Bonferroni’s post hoc test), consistent with the idea that the inhibition produced by AM251 was caused by blockade of CB1 receptors. To confirm that AM251 inhibited substance P release and not NK1R internalization itself, we determined whether 100 nm AM251 and AM281 inhibited NK1R internalization induced by incubating spinal cord slices with substance P (1 μm). AM251 and AM281 produced no effect in this case (Fig. 3;

one-way anova: F2 = 1.65, P = 0.27). To further characterize the inhibition of substance P release by CB1 receptor antagonists, we obtained Casein kinase 1 concentration–response curves of the CB1 antagonists AM251 (Fig. 4A) and AM281 (Fig. 4B). NK1R internalization was evoked by stimulating the dorsal root at 100 Hz. AM251 and AM281 dose-dependently inhibited the evoked NK1R internalization, except that an outlier was found with the highest concentration of AM281, 1 μm. This data point was excluded by the outlier detection feature of the nonlinear regression program (see Data Analysis in Materials and methods) (Motulsky & Brown, 2006). We attributed this outlier to the interaction of AM281 at high concentrations with receptors other than CB1.