Methods for probe synthesis, hybridization and autoradiography as described previously 46. In situ hybridization was conducted using 35S-labelled cRNA probes generated from a rat melanin concentrating hormone (MCH) cDNA 48, a partial β-galactosidase (β-Gal) cDNA (681bp), and full-length mouse CART cDNA. The partial β-Gal cDNA was PCR amplified and cloned from the gene-trap cassette isolated from TORC1 −/− mice. The full-length mouse CART cDNA was cloned from hypothalamic mouse RNA. Brain sections were mounted onto poly-L-lysine coated slides, dried under vacuum, and postfixed with 4% paraformaldehyde for 30 min at 4°C. Tissue sections were digested with proteinase K (10μg/mL) for 30 min at 37°C, acetylated and dehydrated prior to hybridization. Labeled probes (1–3×109 dpm/μg) were diluted in hybridization solution, applied to sections and allowed to hybridize overnight at 60°C. Sections were subsequently treated with ribonuclease A (20μg/ml) for 30 min at 37°C, washed with 0.1× SSC buffer (65–85°C), dehydrated and exposed to x-ray films (β-max; Eastman Kodak) for 1–2 d. Slides were then coated with Kodak NTB-2 liquid emulsion, and exposed at 4°C for 3–4 weeks.
