1700 differential ac amplifier

Electrical signals were recorded using X series multifunction data acquisition device (National instruments) and 1700 Differential AC amplifier (A‐M Systems). The samples were placed on a warm surface and connected to the signal recording system. The data was processed to filter the noise by moving average using the Excel software (Microsoft).
Stimulation was performed using an SP‐150 potentiostat (BioLogic, Science Instruments) with a RE‐1B reference electrode (Ag/AgCl). The pacing was performed by applying 7 V for 50 ms‐long pulses at 1–2Hz. Calcium imaging was used to visualize signal propagation.

Adult mice of each genotype were anesthetized with isoflurane (1.5%) and positioned on a heating pad to maintain body temperature. The skin covering the lateral intercostal muscle to the rectus abdominus muscle was resected and bathed with sterile saline. Silver wire electrodes were inserted into the diaphragm and the lateral portion of the rectus abdominus; these were connected to an A-M Systems 1700 differential AC amplifier (gain 10k, no filter). Raw diaphragm (Dia_EMG) and abdominal (Abd_EMG) activity was recorded using PowerLab 26T and LabChart 8 (ADInstruments). Raw diaphragm and abdominal EMG recordings included an electrocardiogram (ECG) artifact that was removed through thresholding in LabChart. Integrated diaphragm (∫Dia_EMG) and abdominal (∫Abd_EMG) muscle activities were obtained after removal of ECG contaminating signal and smoothened using a triangular (Barlett) window of 101 samples. Amplitude and frequency of each EMG signal was evaluated from a 20 s section of data acquired during exposure to 0, 5, and 7% CO₂ before and 1.5 h after systemic application of saline (30 µL) or PF-04531083 (40 mg/kg).