2.4. Mouse respiration experiments
The changes in respiratory function were assessed in awake, freely moving mice using four parallel whole-body plethysmography chambers connected to a pressure transducer with the accompanying controller and software (Buxco, DSI Instruments). Experiments were conducted at room temperature (23–25°C). A continuous flow of fresh air (2 L/min) through the plethysmograph chamber was used to maintain constant temperature and humidity. Breathing variables were determined by Ponemah Analysis Modules (DSI). System software was used to calculate the respiratory frequency, tidal volume, minute ventilation (respiratory frequency × tidal volume), inspiratory time, and expiratory time. To measure respiratory functions, each mouse was allowed to acclimatize to the chamber for 1 h. A baseline plethysmographic recording was performed for 30 min. Following the baseline recordings, the mice were removed from the chamber, and a pharmacological agent or vehicle was administered. The mice were then returned to the chamber, and respiratory functions were recorded after morphine or fentanyl administration. The average f, TI, TE, and VE were calculated.