Ketamine hydrochloride

Ketamine-hydrochloride (Sigma-Aldrich), MRK-016 (Tocris), and fluoxetine hydrochloride (FLX; National Institute of Mental Health Chemical Synthesis and Drug Supply Program) were dissolved in DMSO and administered intraperitoneally in a volume of 1.125 ml/kg of body mass. 2,3-Dioxo-6-nitro-1,2,3,4-tetrahydrobenzo[f]quinoxaline-7-sulfonamide (NBQX; National Institute of Mental Health Chemical Synthesis and Drug Supply Program) was dissolved in 0.9% saline and administered intraperitoneally in a volume of 7.5 ml/kg of body mass. Ketamine was administered at 10 mg/kg, a widely used sub-anesthetic dose widely used in rodent studies to induce antidepressant effects (Maeng et al., 2008 (link); Zanos et al., 2016 (link)). Unless stated otherwise, MRK-016 was administered at 3 mg/kg, a dose chosen to produce 70% receptor occupancy (Atack et al., 2009 (link)). MRK-016 has equivalent affinity for α1-, α2-, α3-, and α5-containing GABA_ARs but has a 5- to 10-fold greater efficacy at inhibiting GABA_ARs containing α5 subunits (Atack et al., 2009).

Step 1 The adult mice were euthanized by i.p. injection of a mixture of 10 mg/kg xylazine and 100 mg/kg ketamine hydrochloride (Sigma, St. Louis, Missouri, USA).
Step 2 The skin and abdominal muscle were split and moved to the mid-axillary line to expose the liver, and the intestine was poked with a cotton swab to expose the common bile duct.
Step 3 A total of 150 μl PBS containing 5 µg anti-LYVE-1^A488 antibody was injected from the common bile duct in the retrograde direction into the bile flow in 3 s.
Step 4 Ten minutes later, the mice were perfused with 0.1 M PBS for 2 min via the portal vein using a perfusion pump at 35 rpm.
Step 5 Low-glucose DMEM containing 0.5 mg/ml collagenase IV (37°C water bath) was perfused via the portal vein for 20 min using a perfusion pump at 9 rpm.
Step 6 The mouse livers were carefully excised, and the gallbladders were removed. Then, the livers were placed into Petri dishes containing 2 ml of DMEM.
Step 7 We carefully peeled off the liver capsule with sharp tweezers. Then, liver parenchymal cells were carefully knocked off using the inner core of the insulin syringe.
Step 8 Thus, the liver vascular portions were successfully detached and mounted with glass slides under coverslips and imaged with an LSM 710 confocal laser scanning microscope (Zeiss, Germany).

Three-week-old mice were anaesthetized with an intraperitoneal injection of ketamine hydrochloride (50 mg/kg, Sigma-Aldrich, Burlington, MA, USA) and xylazine hydrochloride (10 mg/kg, Sigma-Aldrich, Burlington, MA, USA) and maintained at 37°C on a hot pad during the procedure. ABR recording was performed on both ears as described previously (Chen et al., 2003 (link)). Under anesthetic conditions, 25 μL 50 mg/mL gentamicin solution was injected through the tympanic membrane using a 30G needle into the right ear and the mice were kept lying on their left side until awake. Two weeks after gentamicin injection, recovery of the ear drum was confirmed under microscope after administering anesthesia, and ABR was performed on both injected ears and the contralateral control ears.

Ketamine hydrochloride (Sigma-Aldrich, St. Louis, MO, USA) was dissolved in physiologic saline and injected i.p. at doses expressed as salt weight per kilogram of body weight. The injection volume was 10 mL/kg.