Avertin

Six- to eight-week week-old miR137^flox/+ and miR-137^flox/+;Emx1-cre male mice were anesthetized with 200 mg/kg Avertin (Wako Pure Chemical Industries, Limited Osaka, Japan). One microliter concentrated lentivirus with titer of ~1 × 10⁹/μl was injected stereotaxically into the hippocampus with the following coordinates (stereotaxic coordinates from Bregma: 2.0 mm caudal, 1.2 mm lateral, 2.0 mm ventral; 2.8 mm caudal, 2.0 mm lateral, 1.7 mm ventral). One month after viral grafting, mice were subjected to behavioral tests.

Surgery and i.c.v. injection of FGF21 was performed as previously described^{39 (link)}. Briefly, male rats (9 weeks old, slc:Wistar; SLC Japan, Shizuoka, Japan) were anesthetized with Avertin (200 mg/ kg BW, i.p.; tribromoethanol; WAKO Pure Chemical Industries Ltd, Osaka, Japan) and placed in a stereotaxic frame. Stainless steel guide cannulae (23-gauge) were inserted into the right lateral cerebral ventricle (0.6 mm caudal to the bregma, 1.6 mm lateral to the midline and 4.5 mm below the skull) and secured to the skull with screws and dental cement. Rats were allowed to recover for 2 weeks. Rats were injected i.c.v. with FGF21 (0, 10, 100, or 1,000 ng/5 μL, 5–6 rats per dose) via inner cannulae (30-gauge).

Rats were anaesthetised with an i.p. injection of 200 mg kg^‐1 tribromoethanol (Avertin; Wako Pure Chemical Industries, Ltd, Osaka, Japan) and placed in a stereotactic frame. A stainless steel guide cannula of 23 gauge was inserted into the right lateral cerebral ventricle at coordinates of 0.6 mm caudal to the bregma, 1.6 mm lateral to the midline and 4.5 mm below the skull. The rats were allowed to recover in individual cages for 1‐2 weeks and were given an i.c.v. injection of mouse FGF21 at doses of 0, 10, 100 and 1000 ng per 5 μL with a 30‐gauge needle. The vehicle consisted of 147 mmol L^‐1 sodium chloride, 1.2 mmol L^‐1 calcium chloride, 0.9 mmol L^‐1 magnesium chloride, 4 mmol L^‐1 potassium chloride, 1.8 mmol L^‐1 HEPES (pH 7.4) and 0.1% rat serum albumin. Recombinant FGF21 was a generous gift from Dr Moosa Mohammadi (New York University School of Medicine, New York, NY, USA). Recombinant mouse FGF21 (residues 33‐209) was produced in Escherichia coli, refolded in vitro, and purified by affinity, ion exchange and size exclusion chromatography as described previously.²⁶ The number of animals injected with 0, 10, 100 and 1000 ng FGF21 was 6, 6, 6 and 5, respectively. The same brains were used in a previous study showing single immunocytochemical detection of c‐Fos protein.²⁴