Frozen coronal sections (25 μm thick, typically at a frequency of 1 in 3) from each brain were then cut using a cryostat, and every section was mounted onto gelatin-subbed, poly--lysine-coated microscope slides and stored in antifreeze at −20 C until use. Briefly, the slides were slow boiled for 10 min, incubated in 0.01 KPBS for 15 min.The sections were processed for double immunofluorescence after washing in KPBS as described previously (19, 24). The sections were placed in 2% normal serum 0.3% Triton X-100 overnight before incubation with either rat anti-Brd U (0; Abcam, Inc., Cambridge, MA) and mouse anti-human neuronal protein Hu C/Hu D (Hu C/D) antibodies (neuronal marker; 0; Invitrogen, Carlsbad, CA) or rat anti-Brd U (0; Abcam) and rabbit anti-c Fos antibodies (00; Calbiochem, La Jolla, CA) for 48 h at 4 C in 2% normal serum 0.3% Triton X-100.For the experiments combining Brd U and c Fos stainings, pregnant mice received ip injections of Brd U (50 m/kg) on E12, E14, or E16 three times a day (at 0800, 1000, and 1200 h).

Surprisingly little is known regarding the embryonic development of hypothalamic neurons despite a tremendous increase in the interest in the influence of prenatal factors on lifelong metabolic regulation and hypothalamic development.ARH neurons that coproduce neuropeptide Y (NPY) and agouti-related peptide (Ag RP) or that contain proopiomelanocortin (POMC)-derived peptides directly respond to peripheral hormonal signals, such as the adipocyte-derived hormone leptin.Both NPY/Ag RP- and POMC-containing neurons project extensively to other key hypothalamic nuclei, including the PVH, DMH, and LHA.These offspring were perfused 2 h later as described below.Male offspring of the Brd U-injected dams were examined on P10 in most experiments.

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