Barium chloride

In anesthetized adult male rats, we used reverse-microdialysis to unilaterally microperfuse selected agents into the preBötC. The experimental procedures were as described previously^{12 (link),22 (link)}. Briefly, we recorded diaphragm muscle activity in isoflurane-anesthetized (2–2.5%), tracheotomised and spontaneously breathing (50% oxygen gas mixture, balance nitrogen) adult rats (average body weight: 305g). Diaphragm muscle activity was recorded using stainless steel bipolar electrodes positioned and sutured on the right side of the crural diaphragm. Genioglossus muscle activity was monitored during experiment. Electromyography signals were amplified (CWE Inc, Ardmore, Pennsylvania, USA), band-pass filtered (100-1000Hz), integrated and digitized at a sampling rate of 1000 Hz using CED acquisition system and Spike v6 software (Cambridge Electronic Design Limited, Cambridge, England). Rats were kept warm with a heating pad during all experiments. Using a dorsal approach, a microdialysis probe (CX-I-12-01) of 200 μm diameter, length of diffusing membrane 1 mm (Eicom, Kyoto, Japan) was inserted into the preBötC using a stereotaxic frame and micromanipulator (ASI Instruments, Warren, Michigan, USA). The probe was placed 12.2 mm posterior, 2 mm lateral, and 10.5 mm ventral to bregma. To accurately target the preBötC, we used several criteria to better position and to confirm the probe location as described previously^{12 (link),22 (link)}. (i) When the probe was inserted in the brain, genioglossus muscle activity showed a reduction of about 30% as it reached the vicinity of the preBötC. (ii) Post-mortem histology was used to confirm the probe location in the preBötC using standard anatomical markers such as the nucleus ambiguus, the caudal part of the facial nucleus, and the inferior olive, and immunohistochemistry of NK-1R. (iii) We created correlation maps associating the latency for breathing to respond to drug perfusion and the distances from the preBötC to probe locations, therefore identifying the region of the medulla highly sensitive to the drug perfused. We used these three anatomical and functional criteria and experience from our previous studies, to confirm that the probes were positioned in the region of the preBötC. On rare occasions (1/20 experiments), the probes damaged the preBötC and respiratory rhythm was irregular and unstable. In such an event, we did not continue the experiment. We perfused the probe with artificial cerebrospinal fluid (aCSF) and pH was adjusted to 7.4 by bubbling carbon dioxide in aCSF. Baseline levels of the physiological variables were recorded for at least 30 min while perfusing aCSF into the preBötC. Following this control period, the μ-opioid receptor agonist [D-Ala², N-MePhe^{4 (link)}, Gly-ol]-enkephalin (DAMGO, 5 μM) or the GIRK channel activator flupirtine (300 μM) were added to the aCSF perfusing the preBötC. The responses to DAMGO or flupirtine were recorded for the next 30 min. For the DAMGO experiments in rats, a solution of DAMGO and the potassium blocker barium chloride, or GIRK channel blocker Tertiapin Q (TQ), were added to the solution for another 30 min. All drugs were obtained from Tocris (Minneapolis, Minnesota, USA).
In anesthetized (isoflurane, 1.5-2%), spontaneously breathing (50% oxygen, balance nitrogen) adult mice, we used reverse microdialysis to perfuse agents into the preBötC of wild-type and GIRK2^−/− animals, while recording diaphragm muscle activity using a similar approach to the rats. The mice were also kept warm with a heating pad. We inserted the microdialysis probe into the brainstem 6.7 mm posterior, 1.2 mm lateral, and 5.7 mm ventral to bregma. For wild-type or GIRK2^−/− mice, baseline levels were recorded for at least 30 min followed by DAMGO (5 μM), the GABA_A receptor agonist 4,5,6,7-tetrahydroisoxazolo[5,4-c]pyridin-3-ol THIP (50 μM), or flupirtine (300 μM) for at least 30 min. The anatomical and functional criteria defined in rats were also used for experiments in mice. In addition, we used he GABA_A receptor agonist 4,5,6,7-Tetrahydroisoxazolo[5,4-c]pyridin-3-ol hydrochloride (THIP) in experiments where DAMGO was not expected to reduce respiratory rate (in GIRK2^−/− mice) to ensure that drugs were functional modulating respiratory rhythm. Because of the random production of GIRK2^−/− knockout animals, we did not randomize wild-type and GIRK2−/− mice. However, as standard practice in the lab, we alternated recordings from wild-type and knockout mice to avoid order effects or experimental conditions that may affect one group temporarily. To avoid experimenter bias, similar standard procedures, timelines for dosing, and automated analyses were used for both animal groups.
We also performed a separate set of experiments with systemic injection of the opioid analgesic fentanyl while recording diaphragm activity in wild-type and GIRK2^−/− mice. Data in mice were normalized as percentage of baseline respiratory rate and diaphragm amplitude to remove potential variability between mice.