Plethysmography chambers

Respiration was measured in freely moving mice using plethysmography chambers (EMKA Technologies, Paris, France) supplied with room air as described previously (Hill et al., 2016 (link), 2018 (link)). Mice were habituated to respiratory chambers for 30 min, on the day prior to experimentation. Respiratory parameters were recorded (IOX software ‐ EMKA Technologies, Paris, France) and averaged over 5 min periods.
Data are presented both as minute volume (MV) and as percentage change from the pre‐drug MV baseline, calculated from data for each individual mouse before collating and plotting as a mean. Mice within cohorts may vary in size and this varies their individual MVs, accordingly; data are presented as a percentage change from each mouse's pre‐drug baseline controls for these inherent variations.

To study respiratory function, awake unrestricted mice were placed inside plethysmography chambers (EMKA Technologies) following a procedure adapted to our laboratory^{69 (link)}. Chambers were perfused with normal air (21% O₂, normoxia), 10% O₂ (hypoxia), or 5% CO₂ (hypercapnia). The hypoxic stimulus was maintained during 5 minutes once O₂ percentage reached 10% and the hypercapnic stimulus was maintained during 1 min when CO₂ percentage reached 5% CO₂. Both O₂ and CO₂ tensions were continuously monitored and recorded during the experiments.

Awake unrestricted mice were placed inside plethysmography chambers (EMKA Technologies) to study respiratory function^{45 (link)}. Data acquisition was performed using the IOX2 software (EMKA Technologies; RRID:SCR_022973). Chambers were filled with either air (21% O₂, normoxia) or a gas mixture: 10% O₂ (hypoxia, maintained for 5 min once O₂ percentage reached 10%); 5% CO₂ (hypercapnia, maintained during 1 min when CO₂ percentage reached 5%). Both O₂ and CO₂ tensions were continuously recorded during the experiments. To calculate changes in respiratory frequency, basal, hypoxic and hypercapnic respiratory frequency was estimated in each animal. Basal respiratory frequency was calculated by averaging the values of 80 digital points (160 s of recording) previous to hypoxia. Peak respiratory frequency during hypoxia was calculated in each animal by averaging the values of 20 points (40 s) at the peak of the hypoxic response. Respiratory frequency during exposure to hypoxia was estimated by averaging either 40 or 150 digital points (80 s or 300 s, respectively) after reaching 10% O₂ tension in the chamber before returning to normoxia. Respiratory frequency during exposure to hypercapnia was estimated by averaging 45 digital points (90 s) after reaching ~5% CO₂ in the chamber before returning to normoxia^{45 (link)}.

We have used both C57BL mice, the strain used by Manglik et al. (2016), and CD‐1 mice, the strain we have used in previous studies of opioid depression of respiration (Hill et al.,2016; Lyndon et al.,2017; Withey et al.,2017) to ensure that any responses observed were not strain‐specific. Respiration was measured in freely moving mice using plethysmography chambers (EMKA Technologies, Paris, France) supplied with either air or a 5% CO₂ in air mixture (BOC Gas Supplies, Manchester, UK) as described previously (Hill et al.,2016). Rate and volume of respiration were recorded and averaged over 5 min periods. Breathing 5% CO₂ in air increases minute volume (MV) but does not induce stress in mice (Hill et al.,2016).
Data are presented both as MV and as percentage change from the pre‐drug MV 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. In our experience, variations in baseline respiration levels do not influence the extent of opioid depression of respiration (Hill and Henderson, unpublished data).

Respiration was measured in freely moving mice using plethysmography chambers (EMKA Technologies, France) supplied with a 5% CO₂ in air mixture (BOC Gas Supplies, United Kingdom) as described previously (17 (link)). Rate and depth of respiration were recorded and averaged over 5 min periods (except immediately after drug injection when the time period was approximately 3 min) and converted to minute volume (rate × tidal volume).

Respiration was measured in freely moving mice using plethysmography chambers (EMKA Technologies, Paris, France) supplied with room air or a 5% CO₂ in air mixture (Figure S5) as described previously (Hill et al., 2016 (link)). Mice were habituated to respiratory chambers for 30 min the day prior to experimentation. Respiratory parameters were recorded (IOX software—EMKA Technologies, Paris, France) and averaged over 5‐min periods. On the day of the experiment, respiration was measured for 20 min prior to drug administration, of which the last 10 min were taken as the baseline.
Data are presented as percentage change from the pre‐drug minute volume baseline, calculated from data for each individual mouse before data being collated and plotted as a mean. Data are presented as a percentage change from each mouse's pre‐drug baseline controls to account for variations in individual minute volume due to variations in mouse size. Raw minute volume data are presented in Figure S1.