These mice were generated using a mixed C57BL/6J and DBA strain as background and the coding region of the ghsr locus was precisely deleted and replaced with an in-frame lacZ reporter gene (Abizaid et al., 2006; Diano et al., 2006). Epacadostat All animals had free access to tap water at all times and to food unless otherwise specified. Prior to the beginning of the experiments, animals were group-housed under an LD cycle with the onset of light set at 08:00 h [zeitgeber time (ZT) 0], with light intensity ranging between 120 and 180 lux at cage level. Research was conducted according to the guidelines of the Canadian Council on Animal Care and approved by Carleton
University’s Animal Care Committee. GHSR-KO mice (n = 2) living on an LD schedule were taken from their home cage at ≈ ZT 4–6, overdosed with sodium pentobarbital and perfused using a 2% paraformaldehyde solution. The B-Raf assay brains were postfixed overnight in 2% paraformaldehyde, sliced into 50-μm sections on a Vibratome, and stained using the beta-galactocidase staining method described previously (Diano et al., 2006). Briefly, sections were thoroughly rinsed with 10 mm phosphate-buffered saline (PBS; in mm: NaCl, 137; KCl, 2.7; Na2HPO4, 8; KH2PO4, 2.6), rinsed once quickly in cold PBS plus 2 mm MgCl2 (PBS-MgCl2), then incubated in
PBS-MgCl2 for 10 min at 4 °C. Permeability was then increased by incubating in cold PBS with detergent (0.01% sodium desoxycholate and 0.02% NP40) for 10 min at 4 °C, and placed in
acetylcholine staining solution for 4 h at 37 °C in the staining solution containing (in mm) K3Fe(CN)6, 25; K4Fe(CN)6, 25; MgCl2, 2 in PBS with 1 mg/mL of X-Gal. For the LD condition, WT and GHSR-KO mice (n = 4 per group per time point) were taken from their home cage at ZT 0, ZT 6, ZT 12 or ZT 18. Pairs of animals consisting of one WT and one KO were injected with an overdose of sodium pentobarbital and perfused with 100 mL of saline (0.9%) followed by 100 mL of 4% paraformaldehyde. Brains were postfixed in 4% paraformaldehyde overnight and transferred to a 1% sodium azide solution until being sectioned at a thickness of 60 μm using a Vibratome. Sections were then cryoprotected in Watson’s solution and frozen. One out of four 60-μm sections containing the hypothalamus were processed for cFos immunocytochemistry as described previously (Abizaid et al., 2005). Separate sections through the SCN were processed for PERIOD1 (PER1) or PERIOD2 (PER2) as described previously (Amir et al., 2004). Images from different hypothalamic nuclei were captured with a digital camera connected to an Olympus microscope (Olympus Canada, Markham, ON, Canada), and analysed using Image XSM software (v. 1.91, 2010,http://www.liv.ac.uk/~sdb/ImageSXM/).