Whole body plethysmography system

Respiration data was collected using a whole-body plethysmography system (Data Sciences International (DSI), St. Paul, MN). Mice were placed in the chambers after DSI airflow transducers were attached to each plethysmography chamber to maintain a constant flow rate. Each chamber was calibrated to its attached transducer before recording. Mice were habituated to the clear plexiglass chambers for 20 min per day for two days. On Day 3, mice were first habituated to the chambers for 10 min. Respiratory parameters were then recorded for 10 min to establish a baseline before injection of vehicle or drugs and were collected for 80 min post drug injection at 10 min intervals. Opioid drugs were given to mice at their AD₈₀ analgesic dose to measure their respiratory depressive effects. To measure respiratory tolerance, mice were administered the AD₈₀ dose for 6 days following the first testing day. On Day 8, mice were given the AD_80, and their respiratory rates were measured as on Day 1. Both area under the curve (AUC) and maximum respiratory depression were  calculated (minimum respiratory rate/baseline rate x 100 for each mouse.)