Cell culture Nodose ganglia were dissected under aseptic conditions, desheathed, and digested for 120 min at 37uC in 3 ml of Ca2+ and Mg2+ free HBSS containing 6 mg collagenase type Ia (Roche Diagnostics, Indianapolis, IM) as previously described [46]. Cells were plated onto four-well chamber slides, and maintained in HEPES-buffered DMEM with 10% fetal calf serum at 37uC in 5.5% CO2, and media changed every 24 h. Cells were maintained in culture for 72 h, at which time they were transferred to serum- free medium for 1 h before treatment with CCK (0.01–10 nM) either alone or in combination with leptin (10 ng/ml) for 2 h. Doses were selected based on previous publications [23]. Immunohistochemistry Cryostat sections of fixed nodose ganglia (5 mm) were mounted on polysine-coated slides (Polysine; MenzelGlaser, Braunschweig, Germany) and processed for immunohistochemistry with an antibody raised against Y2, MCH1R and CB1 (Santa Cruz Biotechnology, Santa Cruz, CA). Cultured neurons were fixed in 4% paraformaldehyde in PBS (30 min RT) and processed for immunocytochemistry with antibodies raised EGR1 (Cell Signal- ing Technology, Danvers, MA). Secondary antibodies were used as appropriate and included donkey anti-rabbit immunoglobulin conjugated with AlexaFluor 488 and donkey anti-goat immuno- globulin conjugated with AlexaFluor 546 (Molecular Probes, Eugene, OR). Specificity of immunostaining was determined by omitting the primary antibody and by pre-incubation with an excess of appropriate peptide where available. Cultured neurons were mounted in Vectashield with 49,6-diamidino-2-phenylindole (DAPI; Vector Laboratories, Peterborough, UK) for nuclear localization. Images were collected using an Olympus spinning disc confocal microscope (BX61 System, Olympus, Melville, NY). The quantification of neurons expressing CB1, MCH1R, or Y2 was made by counting 200–300 immunoreactive cell profiles in four sections per ganglion, selecting sections separated by 90 mm that passed through the full length of the caudal and mid regions of the ganglia. Results are expressed as immunopositive cells as a proportion of all neurons in the relevant region. References 1. Feinle C, O’Donovan D, Doran S, Andrews JM, Wishart J, et al. (2003) Effects of fat digestion on appetite, APD motility, and gut hormones in response to duodenal fat infusion in humans. Am J Physiol Gastrointest Liver Physiol 284: G798–807. 2. Stoeckel LE, Weller RE, Giddings M, Cox JE (2008) Peptide YY levels are associated with appetite suppression in response to long-chain fatty acids. Physiol Behav 93: 289–295. 3. Heddle R, Collins PJ, Dent J, Horowitz M, Read NW, et al. (1989) Motor mechanisms associated with slowing of the gastric emptying of a solid meal by an intraduodenal lipid infusion. J Gastroenterol Hepatol 4: 437–447. 4. De Lartigue G, Barbier de la Serre C, Espero E, Lee J, Raybould HE (2011) Diet-induced obesity leads to the development of leptin resistance in vagal afferent neurons. Am J Physiol Endocrinol Metab. 5. Lawton CL, Burley VJ, Wales JK, Blundell JE (1993) Dietary fat and appetite control in obese subjects: weak effects on satiation and satiety. Int J Obes Relat Metab Disord 17: 409–416. 6. Stewart JE, Seimon RV, Otto B, Keast RS, Clifton PM, et al. (2011) Marked differences in gustatory and gastrointestinal sensitivity to oleic acid between lean and obese men. Am J Clin Nutr 93: 703–711. 7. Swartz TD, Duca FA, Covasa M (2010) Differential feeding behavior and neuronal responses to CCK in obesity-prone and -resistant rats. Brain Res 1308: 79–86. 8. Covasa M, Ritter RC (1998) Rats maintained on high-fat diets exhibit reduced satiety in response to CCK and bombesin. Peptides 19: 1407–1415. 9. Torregrossa AM, Smith GP (2003) Two effects of high-fat diets on the satiating potency of cholecystokinin-8. Physiol Behav 78: 19–25. 10. Savastano DM, Covasa M (2005) Adaptation to a high-fat diet leads to hyperphagia and diminished sensitivity to cholecystokinin in rats. J Nutr 135: 1953–1959. Mechanism of Onset of Hyperphagia in Obese Rats Western blotting As previously described _ENREF_47 [47], briefly, 10 mg of proteins were used, unless otherwise stated in the legends, samples were loaded into precast 10% BisTris gel and the gel was run for 50 min at 200 V. The proteins were transferred 1 h. Primary antibodies were left to incubate overnight (STAT3 phosphorylated at tyrosine 705 (D3A7) Ab and EGR1 Ab, Cell Signaling Technology). GAPDH was used as a loading control (14C10) rabbit mAb, Cell Signaling Technology). The film was analyzed by Imagequant v 5.1 software (Amersham, Biosciences, Amer- sham, UK). Statistics Statistical analysis was performed using Prism software (Prism 5.0, GraphPad Software Inc., La Jolla, CA, USA). Two way ANOVA was used to analyze energy intake data and leptin feeding studies (time and diet were used as the variables). One ANOVA was used to analyze immunohistochemical data. One way ANOVA was also performed to analyze western blot and CCK feeding studies with diet used as a variable and differences among groups were analyzed using multiple comparison proce- dure (Bonferroni). Differences were considered significant if p,0.05. Data are mean 6 SEM. a,b,c different letters denote significant differences between groups. Unpaired one-tailed student’s t-test was used to compare lean vs. obese Zucker rats’ body weight and pSTAT3 expression in hypothalamus of these rats. Paired one-tailed student’s t-test was used to compare the effects of CCK in LF, DR, and DIO rats. Significant differences were represented as * for p,0.05; ** for p,0.01; and *** for p,0.001. Author Contributions Conceived and designed the experiments: GdL HR. Performed the experiments: GdL CBdlS EE JL. Analyzed the data: GdL CBdlS. Contributed reagents/materials/analysis tools: EE. Wrote the paper: GdL HR. 11. Paulino G, Barbier de la Serre C, Knotts TA, Oort PJ, Newman JW, et al. (2009) Increased expression of receptors for orexigenic factors in nodose ganglion of diet-induced obese rats. Am J Physiol Endocrinol Metab 296: E898–903. 12. Burdyga G, de Lartigue G, Raybould HE, Morris R, Dimaline R, et al. (2008) Cholecystokinin regulates expression of Y2 receptors in vagal afferent neurons serving the stomach. J Neurosci 28: 11583–11592. 13. de Lartigue G, Dimaline R, Varro A, Dockray GJ (2007) Cocaine- and amphetamine-regulated transcript: stimulation of expression in rat vagal afferent neurons by cholecystokinin and suppression by ghrelin. J Neurosci 27: 2876–2882. 14. Burdyga G, Varro A, Dimaline R, Thompson DG, Dockray GJ (2006) Feeding- dependent depression of melanin-concentrating hormone and melanin-concen- trating hormone receptor-1 expression in vagal afferent neurones. Neuroscience 137: 1405–1415. 15. Burdyga G, Lal S, Varro A, Dimaline R, Thompson DG, et al. (2004) Expression of cannabinoid CB1 receptors by vagal afferent neurons is inhibited by cholecystokinin. J Neurosci 24: 2708–2715. 16. Burdyga G, Spiller D, Morris R, Lal S, Thompson DG, et al. (2002) Expression of the leptin receptor in rat and human nodose ganglion neurones. Neuroscience 109: 339–347. 17. Buyse M, Ovesjo ML, Goiot H, Guilmeau S, Peranzi G, et al. (2001) Expression and regulation of leptin receptor proteins in afferent and efferent neurons of the vagus nerve. Eur J Neurosci 14: 64–72. 18. Peiser C, Springer J, Groneberg DA, McGregor GP, Fischer A, et al. (2002) Leptin receptor expression in nodose ganglion cells projecting to the rat gastric fundus. Neurosci Lett 320: 41–44. 19. Gaige S, Abysique A, Bouvier M (2002) Effects of leptin on cat intestinal vagal mechanoreceptors. J Physiol 543: 679–689. PLoS ONE | www.plosone.org 9 March 2012 | Volume 7 | Issue 3 | e32967