Data analysis and statistics
Sample sizes were calculated using the GPower 3.1.2 program with α < 0.05 and a power of 80% based on a defined effect size derived from pilot experiments. Experiments were randomized to compensate for the position effects on plates or filter apparatus and unequal sample processing time. All experiments were performed by an investigator blinded to the sample assignments. The codes were broken after the completion of the experiments. Statistical analyses were performed using GraphPad Prism software (version 5 and 9 for Windows; GraphPad Software Inc., San Diego, USA). Grubbs’ test was performed to identify potential outliers. No samples were excluded from the analysis. All data were assessed for normal distribution and equal variances by D’Agostino and Pearson or Kolmogorov–Smirnov tests. Analysis of concentration-response relationships was performed with simple linear regression using Prism 9 (where y = [35S]-GTPγS bound and x = [NFEPP] or [fentanyl]). Ca2+imaging time series of single cells were extracted from stacks of ratio images as averages over the entire cell area in excel‑sheets, which were then plotted as time vs. ratio 340/380 graphs using Prism 5. For quantitative analyses, the first 10 data points (corresponding to the first 20 s of the experiment) were averaged to form the baseline which was then subtracted from all high potassium (high K+) responding cells to obtain Δ ratio 340/380 values. During each recording, we typically were able to record at least from 10 cells, meaning that each trace in Ca2+ imaging recordings represents the mean of more than 20 cells. The precise number of cells is stated in the figures or in the figure legends. Data are represented as means ± standard error of the mean (SEM). Detailed statistical analyses are presented in figure legends.