Thus, blockade of ghrelin receptors by [D-Lys3]-GHRP-6 may be attenuating meal initiating events via a vagal afferent pathway or within the central nervous system. CCK1R?/? mice ingesting HF diet is usually reversed by blockade of the ghrelin receptor, suggesting that in the absence of the CCK1R, there is an increased ghrelin-dependent drive to feed. The site of action of ghrelin receptors is usually unclear, but may involve an increase in expression of CART peptide in VAN in HF-fed CCK1R?/? mice. Vectastain ABC Kit (Vector Labs) for 1.5 hours at 37C. Ni-33-diaminobenzidine (Cat.No. D-8000-5G, Sigma Aldrich Chemicals) was dissolved in PBS (30mg/100mL) and added to sections for 5 minutes followed by the addition of 30% H2O2 EPHB2 to each section with the reaction stopped after 5 minutes with three serial washes using chilly PBS. All reagents contained penicillin streptomycin (Cat.No. 15140-122, Gibco, Carlsbad, CA) antibiotic treatment to prevent bacterial growth on sections. Sections were then mounted onto Fisher Superfrost/Plus slides and dehydrated in six serial Whatman jars of distilled water/ethanol/xylene solutions. Coverslips were mounted using permanent mounting media (Cat. No. 6419, Tissue-Tek-Glas, Torrance, CA) and allowed to dry overnight. c-Fos images were taken in rostrocaudal order (rostral (bregma ?8.00 to ?7.92 mm), mid-NTS (?7.76 to ?7.32 mm) and caudal to the area postrema (?7.08 to ?6.48 mm) using an Olympus Provis AX70 light microscope (Olympus Optical Co., Tokyo, Japan) at 20 oil objective and analyzed by Scion Image (Beta 4.0.2, Scion Corporation, 2000). For each mouse brain, 4C8 sections were imaged to include bilateral dorsal vagal complex (2 images per section, one left side and one right side, for a total of 8C12 analyzed values). Using the Scion Image analysis software, the margins of the NTS were defined using a dashed outline and the number of fos positive neurons in that area counted. The total quantity of pixels in that same area was quantified and used to normalize the number of neurons to the area of the NTS. Data are therefore expressed as fos density (ie the number of fos (+) nuclei divided by total number of pixels in the ipsilateral NTS). Thus, 8C12 values per mouse brain were obtained and an average of these values was calculated. This common was then used to provide the imply and standard Dasatinib (BMS-354825) error of the imply per group. 2.6 Statistics and data analysis Data are presented as means SE. Meal pattern data were recorded using EZ count number software and analyzed using Spike2 (version 5.07, Cambridge Electronic Design 1988C2004). Statistical analyses were performed using GraphPad Prism version 3.02 (GraphPad Software, San Diego, CA) and SigmaStat (version3.11, Systat Software Inc. 2004). Protein expression data in nodose neurons was quantified by Scion Image version 4.02 (Scion, Frederick, MD) using set fluorescence threshold values (average of 8C10 analyzed images used as the mean value per animal) and compared by Students t-test. CART peptide expression in nodose neurons, c-fos-IR in hindbrain and arcuate nucleus were compared by two-way ANOVA followed by post hoc analysis with Holm-Sidaks multiple comparison test for the effects of genotype, diet or treatment. Differences in values Dasatinib (BMS-354825) were considered significant at em P /em 0.05. 3.0 Results 3.1 Effect of GHSR1a receptor antagonist on meal patterns As previously explained (16), there was no difference in time to first meal between wild-type and CCK1R?/? mice ingesting LF diet (Fig. 1A); when ingesting HF diets, there was a decrease in the time to the first meal in CCK1R?/? mice compared to wild-type controls (Fig 1B, p 0.05). Administration of the ghrelin receptor antagonist D-(Lys3)-GHRP-6 (2.8g/kg, IP15 min, Fig. 1B) experienced no significant effect on time to first meal in HF fed wild-type or CCK1R mice. However, administration of D-(Lys3)-GHRP-6 (8g/kg, Fig. 1D) significantly delayed the time to first meal in CCK1R null mice ingesting Dasatinib (BMS-354825) HF diets Dasatinib (BMS-354825) (p 0.05). Open in a separate window Physique 1 Effect of administration of the GHSR1a antagonist on meal patterns in wild-type and CCK1R?/? miceTime to first meal following a 6-hr fast in wild-type and CCK1R?/? mice.