Minidigi 1a

Unilateral femoral artery pre-catheterized pregnant rats were purchased from Charles River Laboratory (Charles River Laboratories, Inc.). The artery catheter (volume ~ 60 μl) was filled with saline containing heparin (50–100 IU/ml) and was connected to a pressure transducer (NL 108T2, Neurolog^TM System, Digitimer Limited, Letchworth Garden City, UK) with tubing filled with saline. The transducer was connected to a pressure amplifier (NL 108 A, Digitimer Limited) with a gain of 1 V/100 mmHg.
ECG was recorded with two subcutaneous needle electrodes placed on the left front and rear limbs (lead III). ECG signal was amplified (x10,000) and band-pass filtered at 1–300 Hz with an AC amplifier (Model LP511, Grass/Natus Neurology Inc. Warwick, RI, USA).
Blood flow, BP and ECG signals were acquired at a sampling frequency of 1 K Hz using a digitizer MiniDigi 1 A and software Axoscope 10.2 (Axon/Molecular Devices, LLC. Sunnyvale, CA. USA).

Before recording, oocytes were either Na⁺-loaded, to saturate intracellular-facing Na⁺-binding sites, or Na⁺-depleted (to remove most intracellular Na⁺). Na⁺ depletion was done in either a K⁺-loading or an NMG⁺-loading solution. These procedures were done by a 1-h incubation in a solution containing (in mM) 90 cation [either NaOH (Na⁺-loading), KOH (K⁺-loading), or NMG⁺ (NMG⁺-loading)], 20 tetraethylammonium-OH, 0.2 EGTA, and 40 HEPES (pH 7.2 with sulfamic acid), supplemented with 10 μM ouabain. The extracellular solution contained (also in mM): 133 methane-sulfonic acid (MS), 10 HEPES, 5 Ba(OH)₂, 1 Mg(OH)₂, 0.5 Ca(OH)₂, 125 NaOH (Na⁺ solution). External K⁺ was added from a 450 mM K⁺-MS stock. Ouabain was directly added to the extracellular solution. Its solubility above 10 mM was achieved by warming the solution & vortexing on the day of the experiment. These solutions were allowed to cool to room temperature before oocyte perfusion.
Two-electrode voltage clamp was performed at room temperature (21–23 °C), with an OC-725C amplifier (Warner Instruments), a Digidata 1440 A/D board, a Minidigi 1A, and pClamp 10 software (Molecular Devices). Signals were filtered at 2 kHz and digitized at 10 kHz. Resistance of both microelectrodes (filled with 3M KCl) was 0.5–1 MΩ.

Ventilation was measured by UWBP, measuring pressure changes resulting from the warming of the inspired air and cooling during expiration10 (link). Mice were placed in a plexiglas chamber (1.2 L volume, custom made) that was connected to a differential low-pressure transducer (model DP1 03, Validyne Engineering, Northridge, CA). The second channel of the pressure transducer was connected to a reference chamber. The bias flow in the chamber was 200 ml/min, introduced by the suction of a CO₂-sensor (Dräger). The pressure transducer was connected to a sine wave carrier demodulator (CD-15, Validyne Engineering). A MiniDigi1A (Molecular Devices) was used for digitization (1 kHz sampling rate) and storage on a PC Notebook (Fujitsu) using AxoScope Software (Molecular Devices). For analysis, pressure fluctuations were imported into LabChart 8 Software (ADInstruments) and offline band-pass filtered (0.5–20 Hz). The Peak Analysis Module was used to detect pressure maxima and calculate peak amplitude [a.u.], respiratory rate [BPM] and relative inspiratory time (t_in[%]). Max- and MinSlope parameter, were derived as measure for chamber flow as the first derivative from pressure trace. Because animals were allowed to explore the chamber freely, and thus, some pressure changes resulted from sniffing behavior, we analyzed a period of 2 min after an initial period of adaptation (3 min).