Mice were kept at 21 ± 2 °C under a reversed 12 : 12-h light/dark cycle (lights off at 08:30 h, < 0.5 lux; lights on at 20 : 30 h; 170 lux at the bottom of the cage, unless otherwise stated). Standard food pellets (Scanbur, Sollentuna, Sweden) and water were available ad libitum. All experiments were performed according to the Finnish Act on the Use of Animals for Experimental Purposes, and were approved by the Animal Experiment Committee of the State Provincial Office of Southern Finland. All
experiments were carried out in accordance with GSK-3 signaling pathway the European Communities Council Directive of 24 November 1986 (86/609/EEC), and with the Guidelines laid down by the NIH in the USA regarding the care and use of animals for experimental procedures. The total number of animals used in this study was 116. For the enzyme activity studies, histamine and 1-methylhistamine assays, and the in situ
hybridization assay, the mice were housed in groups of three, which may have potentially confounded the results. Although this issue has not been addressed specifically, we interpreted the data of Mistlberger & Skene (2004) and Paul & Schwartz (2007) to mean that, in the presence of light the effect of social cues should be small or absent. Importantly, the housing conditions were identical for all mice in the analysis. The chemical reagents used were as follows: Ketalar, Domitor, and Antisedane (Pfizer Animal Health, New York, USA); dental cement (Candulor, Wangen, Germany); buprenorphine (Temgesic; Reckitt Benckiser, Slough, UK); deoxyadenosine 5′-triphosphate, [α-33P] Pexidartinib supplier (NEG312H; NEN Research Products, PerkinElmer, Waltham, MS, USA); 3-methylhistamine, CaCl2, NaCl, KCl, KH2PO4, and methanol (Merck, Whitehouse Station, NJ, USA); 1-methylhistamine, aminoguanidine hydrochloride, citric acid, Denhardt’s solution, dithiothreitol, Cyclin-dependent kinase 3 histamine dihydrochloride,
H3PO4, l-histidine, NaH2PO4, NaN3, NaOH, N-lauroylsarcosine, o-phthalaldehyde, polyethylene glycol 300, pyridoxal phosphate, pargyline hydrochloride, HClO4, β-mercaptoethanol, phenylmethanesulfonyl fluoride, S-adenosyl-methionine, and sodium salt of octanesulphonic acid (Sigma, St Louis, MO, USA); MgCl2 and H3BO3 (Riedel-deHaёn, Seelze, Germany); formamide (Amresco, Solon, OH, USA); RNA (Roche, Basel, Switzerland); and dextran sulphate (Amersham, Amersham, UK). Thirty male 8–9-week-old C57BL/6J mice were kept in groups of three under a 12 : 12-h light/dark cycle. Mice were killed by decapitation at zeitgeber time (ZT) 4, 8, 12 (lights on), 16, 20 and 24 (lights off). The brains were removed, and rapidly frozen at −80 °C. Coronal 20-μm sections were cut with a Leica CM3050S cryostat (Leica, Wetzlar, Germany), and mounted onto SuperFrost slides (ThermoScientific, Portsmouth, USA). The section levels corresponding to the TMN region (−2.2 mm to −2.8 mm relative to bregma) were determined according to the mouse brain stereotaxic atlas (Paxinos & Franklin, 2004). The sections were stored at −20 °C until analysis.