Hypnorm

IVGTTs were performed on 5 h fasted mice after 8 weeks of diet treatment. Blood samples were collected from anaesthetized mice (Hypnorm, 0.5 mg fluanisone, 0.02 mg fentanyl per mouse; Janssen, Beerse, Belgium; 0.25 mg midazolam per mouse; Dormicum, Hoffman LaRoche, Basel, Switzerland) from the retrobulbar, intraorbital, capillary plexus before d‐glucose injection (0.35 g/kg bw) to the tail vein. Additional blood samples were collected at 1, 5, 10, 20, 30, and 50 min from each mouse. Plasma was separated and stored at −20°C until it was analyzed for insulin with a mouse insulin ELISA (Mercodia, Uppsala, Sweden) and glucose (glucose oxidase method).

Mice (female BALB/c; Harlan Nederland, Horst, The Netherlands) were kept in 12 h light/12 h dark cycle (lights on at 8:00 AM to 8:00 PM) under constant conditions of temperature (20±2°C) and humidity (55% humidity) with water and food ad libitum. Mice underwent surgical procedure at 11–13 weeks of age. Mice were anesthetized by FFM intraperitoneal injection, a mixture of fentanylcitrate/fluanisone (Hypnorm; Janssen, Beerse, Belgium) and midozolam (Dormicum; Roche, Mijdrecht, The Netherlands) in a ratio 1∶1∶2 (Hypnorm: Dormicum: H₂0).

The rats were anesthetized with a combination of fentanyl and fluanisone (Hypnorm^®, Janssen Pharmaceutica, Beerse, Belgium) and midazolam (Dormicum^®, F Hoffman-La Roche AG, Basel, Switzerland) at a dose of 0.01–0.05 mg per 100 g and 1.0–1.75 mg per 100 g, respectively.

All animal procedures were carried out according to the guidelines of the University of Zurich, and were approved by the Cantonal Veterinary Office. C57BL/6 mice (2–4 months old, of either sex) were either first sedated with chlorprothixene (Sigma; 0.2 mg/mouse) and anaesthetized with urethane (0.5–1.0 g/kg) or anaesthetized by 2.7 ml/kg of a solution containing one part fentanyl citrate and fluanisone (Hypnorm; Janssen-Cilag, UK) and one part midazolam (Hypnovel; Roche, Switzerland) in two parts of water, both delivered by intraperitoneal injections. Atropine (0.3 mg/kg) and dexamethasone (2 mg/kg) were administered subcutaneously to reduce secretions and oedema. Lactate-Ringer solution was regularly injected subcutaneously to prevent dehydration. Pinch reflexes were used to assess the depth of anaesthesia.

Prior to surgery, rats were anaesthetized by intramuscular fentanyl/fluanisone (0.315 mg/kg fentanyl, 10 mg/kg fluanisone, Hypnorm, Janssen Pharmaceutica, Belgium). Xylocaine was sprayed on the skull to provide local anesthesia (Lidocaine 100 mg/mL, AstraZeneca BV, the Netherlands). All rats received three daily perisurgical injections of carprofen (5 mg/kg, s.c. Carporal, AST Farma BV, the Netherlands), starting at the day of surgery. Rats (n = 3) were unilaterally injected with 0.3 μL of AAV‐hSyn‐ChR‐YFP (4.8*10¹² genomic copies/mL; UNC vector core) in the DMH (from bregma: anterior–posterior (AP): −2.30 mm, mediolateral (ML): +1.40 mm, dorsoventral (DV): −9.30 mm, at an angle of 5°), using a stereotactic apparatus and a microliter infusion system. Virus was injected through 34G needles, which were connected to a Hamilton microliter syringe with polyethylene tubing. By using a microinfusion pump, the injection speed of all injections was set at 0.1 μL/min. Following infusion, the needles were left in place for 10 min to prevent backflow. Prior to these surgeries, we performed pilot experiments to compare injections between the microliter infusion system and the nanojet (where glass capillaries are used). As no differences were observed in the targeting of the injections between the two systems, we decided to use the microliter infusion system for all our experiments.

A third group of rats (n = 16) underwent surgery under identical procedures as described for experiment 1, but were bilaterally injected with 0.3 μL of AAV‐hSyn‐DIO‐hM₃D(G_q)‐mCherry (3.8*10¹² genomic copies/mL; UNC vector core) in the DMH, and bilaterally injected with 0.3 μL of a mixture of CAV2Cre (final concentration in mixture 1.8*10¹² genomic copies/mL; IGMM, France) and AAV‐hSyn‐EYFP (final concentration in mixture 1.65*10¹² genomic copies/mL; UNC vector core) in the PAG. Rats were randomly divided into four groups of four and injected at four different coordinates in the PAG, respectively (from bregma: group 1: AP −5.30 mm; ML ± 1.40 mm/<10°; DV −5.70 mm; group 2: AP −6.30 mm; ML ±1.40 mm/<10°; DV −5.70 mm; group 3: AP −7.30 mm; ML ± 1.40 mm/<10°; DV −5.70 mm; group 4: AP −8.30 mm; ML ±1.40 mm/<10°; DV −5.70 mm). In addition, an intra‐abdominal dual transmitter (TL11M3F40‐TT, Data Science International, USA) with leads to the portal vein in the liver and interscapular brown adipose tissue was implanted under fentanyl/fluanisone (0.315 mg/kg fentanyl, 10 mg/kg fluanisone, Hypnorm, Janssen Pharmaceutica, Belgium) and midazolam (2.5 mg/kg, i.p., Actavis, the Netherlands) anesthesia in order to record core body temperature, BAT temperature, and activity.