Strain-Specific Respiratory Responses

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in baseline respiration levels do not influence the extent of opioid depression of respiration (Hill and Henderson, unpublished data).