Data and statistical analysis
The data and statistical analysis comply with the recommendations on experimental design and analysis in pharmacology (Curtis et al. , 2018). All data were analyzed using GraphPad Prism 8.4.3 (GraphPad; LaJolla, CA, USA). Since all data but those presented in Fig. 8 panel B passed the normality tests (implemented on the GraphPad software), parametric statistics was run on all data but those reported in Fig. 7B. cAMP data in Fig. 1-3 were calculated as percentage of SKF-38393 stimulation. Concentration-response curves for N/OFQ and AT-403 in cells co-transfected with NOP, NOP/RGS4 and NOP/RGS19 were fitted to non-linear least-square regression to the log (inhibitor) vs response (three parameters) in GraphPad Prism. Potency was expressed as pIC50 accompanied by 95% confidence interval in parenthesis. pIC50 and Emax values in Fig.1 were compared by the Student t-test, two-tailed for unpaired data. pERK data in striatal slices (Fig. 3) were analysed using a 3-way ANOVA followed by Bonferroni’s post hoc test. Immobility time and number of steps (Fig. 4) were expressed in absolute values and analysed using 2-way repeated measure (RM) ANOVA followed by the Tukey test. Dyskinesia values (Fig. 5A) were expressed as ALO score (frequency x amplitude) and were treated using two-way RM ANOVA followed by the Tukey test. Rotarod performance (Fig. 5B) was expressed as time on rod (in seconds) and was analysed using the Student t-test (two-tailed for unpaired data). Western blot values in Fig. 6-7 were analysed using the Student t-test, two-tailed for unpaired data, comparing the signal in the lesioned vs unlesioned striatum. RGS4 protein levels (Fig. 8) were expressed as a ratio to tubulin as housekeeper in absolute values (Fig. 8A) or as percentage of unlesioned striatum (Fig. 8B). Data in Fig. 8A were analysed using one-way ANOVA followed by the Newman-Keuls test whereas data in Fig. 8B with the Kruskal-Wallis test for non-parametric ANOVA followed by the Dunn test. Overall, two outliers were removed using the ROUT test implemented on the GraphPad Prism software. Outliers were indicated in figure legends. P values <0.05 were considered statistically significant.