2.6. Experimental design and data analysis

Data from previous experiments where respiratory depression or antinociception was measured following acute opioid administration in naïve mice were subjected to post hoc power analyses using G*Power (version 3.1.9, RRID:SCR_013726). Our calculations indicated that in acute respiration experiments, n = 6 for each individual group would produce a significant result if an actual effect occurred.

The results for each experiment were expressed as the means ± SEM. In the experiments performed in Germany, a cut‐off for respiratory rate was set at 60 and 360 breaths·min⁻¹ for baseline measurement and 40 and 270 breaths·min⁻¹ after morphine or fentanyl treatment.

Respiratory time course data from the United Kingdom and Australia were normalised to the pre‐drug baseline as 100%. Data are presented as percentage change from the pre‐drug baseline, calculated for each mouse individually before mean data were plotted. Presenting data as percentage change from the pre‐drug levels has been done to control for variation between treatment groups that may have different baseline levels of respiration. Normal distribution of the data was verified before performing parametric statistical analysis. Wherever appropriate, data were analysed using one‐way or two‐way ANOVA, followed by Bonferroni's post hoc tests. Statistical significance is assumed when P <0.05. All calculations were performed using GraphPad Prism 5, 6, or 7 software (GraphPad Software, Inc., San Diego, CA, RRID:SCR_002798). The data and statistical analysis comply with the recommendations of the British Journal of Pharmacology on experimental design and analysis in pharmacology (Curtis et al., 2015).