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.