Norepinephrine bitartrate

In 4 mice under general isoflurane anesthesia, the glass coverslip previously implanted over the visual cortex was removed and replaced with a coverslip of the same size but with a small hole near the edge. The mouse was head-fixed under the microscope where it remained anesthetized for the duration of the experiment. The following agents were diluted with cortex buffer and Alexa 568 dye to achieve specific molecular concentrations: acetylcholine chloride (crystals/powder, Sigma-Aldrich), norepinephrine bitartrate (crystalline, Sigma-Aldrich). A borosilicate patching pipette was fitted to a syringe and backfilled with the mixed pharmacological agent and inserted into the cortex through the hole in the coverslip. 200 mBar pressure was applied to the syringe, which was released with a button press.

The induction of AF was performed as described previously by us^{22 (link),33 (link),34 (link)}. Briefly, mice were anesthetized with isoflurane (1.5–2.0% vol/vol) to maintain adequate anesthesia, and norepinephrine bitartrate (Sigma-Aldrich) dissolved in natural saline (Otsuka Pharmaceutical, Tokyo, Japan) was intraperitoneally injected 10 min before AF induction. While a surface ECG was recorded using electrode in a lead-II configuration, a 1.1-French catheter electrode (EPR800; Millar Instruments, Houston, TX, USA) was carefully advanced and fixed at the esophageal position dorsal to the left atrium. Induction of AF was conducted by applying transesophageal atrial burst pacing for 10 s at a stimulation amplitude of 1.5 mA with 10 ms cycle lengths and a pulse width of 3 ms. AF was defined according to the following criteria: (i) loss of P wave, (ii) irregular R-R interval, and (iii) duration greater than 2 s, which have already been adopted for AF in mice induced by rapid transesophageal atrial pacing by us^{33 (link)–35 (link)} and other groups^{58 (link),59 (link)}. Further, after the screening of AF induction based on these criteria by a single technician who was blind to information about the experimental groups, the diagnosis was always re-checked by an expert on cardiac physiology.

Medium 199 containing Earle’s salt and HEPES (Sigma-Aldrich, St. Louis, MO, USA) was prepared from a powder according to the manufacturer’s instructions (pH adjusted to 7.2 with NaOH) and sterilized by filtration. Before use, the sterile solution of ascorbic acid (Sigma-Aldrich, St. Louis, MO, USA) in Medium 199 and Antibiotic-Antimycotic Solution (Sigma-Aldrich, St. Louis, MO, USA) were added to give a final concentration 300 mg/L of ascorbic acid, 100 IU/mL of penicillin, 100 μg/mL of streptomycin, and 0.25 μg/mL of amphotericin B. A 1 mM solution (freshly prepared) of norepinephrine bitartrate (Sigma-Aldrich, St. Louis, MO, USA) was sterilized by filtration and diluted in the culture medium to 10 μM.

The following drugs and standards were procured from the source specified: acetylcholine chloride ≥98% (Alfa Aesar Gmbh & Co, Karlsruhe, Germany), atropine sulphate monohydrate >98% (Fluka-AG, Buchs, Switzerland), potassium chloride, phenylephrine (PE) hydrochloride ≥98%, norepinephrine bitartrate ≥98%, Nω-nitro-l-arginine methyl ester (l-NAME) hydrochloride ≥98%, verapamil hydrochloride ≥99%, indomethacin hydrochloride (Sigma-Aldrich Inc., St. Louis, MO) and ethylene glycol-O-O′-bis(2-aminoethyl)-N,N,N′,N′-tetraacetic acid (EGTA) ≥ 97% (Alfa Aesar, Heysham, UK). Stock solutions of all the chemicals were made in distilled water and diluted freshly when required.