Mechanisms of xenin-induced anorectic response in chicks (Gallus gallus) Wint Nandar a , Justin M. Milligan b , Mark A. Cline b, * a Department of Neuroscience and Anatomy, Pennsylvania State University, P.O. Box 850, Hershey, PA 17033, USA b Department of Biology, Radford University, P.O. Box 6931, Radford, VA 24142, USA article info Article history: Received 14 January 2008 Revised 5 March 2008 Accepted 6 March 2008 Available online 21 March 2008 Keywords: Behavior Chick Feed intake Hypothalamus Opioid Ventromedialis hypothalami Xenin abstract We recently reported that the 25 amino acid peptide xenin caused reduced feed intake when centrally injected in chicks. The present study was designed to explore possible mechanisms of the xenin-induced anorexigenic response in chicks. In Experiments 1 and 2, chicks were implanted with cannulas and xenin injections were made directly into the ventromedialis hypothalami (VMH). Chicks responded with reduced feed intake and increased c-Fos immunoreactivity at the VMH. In Experiment 3 chicks that received co-intracerebroventricular (ICV) injection of naloxone and a dose of xenin (100 pmol), that alone does not affect feed intake, had reduced feed intake. In Experiment 4, chicks responded to ICV xenin with reduced feed- but increased exploratory-pecking. Thus, we conclude that xenin may mediate its effect directly at the VMH and that the endogenous opioid system may counter anorexigenic effects of low xenin doses in chicks. Xenin also caused increased exploration of a novel environment, an effect that may be competitive with feeding. Taken together, these results suggest that xenin regulation of chick appetite is the result of several central and behavioral mechanisms acting in synergism. Ó 2008 Elsevier Inc. All rights reserved. 1. Introduction The perception of appetite is governed by numerous central and peripheral chemical messengers that through both synergistic and antagonistic relations regulate ingestion. One such messenger of both central and peripheral origin is xenin, a 25 amino acid pep- tide. Xenin may have appeared early in evolution as its amino acid sequence is conserved from yeast to mammals (Hamscher et al., 1996). Xenin (Cline et al., 2007a) has been isolated from the diges- tive systems of mammals (Feurle et al., 1992; Anlauf et al., 2000; Hamscher et al., 1995) and birds (Carraway and Feurle, 1985) where it influences gut dynamics (Feurle et al., 1997, 2001). Addi- tionally, xenin is found in the central nervous system (Hamscher et al., 1995) where it acts as a satiety signal in rats (Anlauf et al., 2000). In chicks, xenin also affects appetite-related processes. We re- ported that both central and peripheral xenin injection cause a reduction in feed intake with no effect on water intake (Cline et al., 2007a). Xenin injection was associated with increased c-Fos immunoreactivity in the nucleus ventromedialis hypotha- lami (VMH) but did not affect the regio lateralis hypothalami (LH; Cline et al., 2007a). Based on a comparison of our findings to rodent reports, we concluded that the xenin anorexigenic sys- tem appeared to be conserved between mammalian and avian species. However, the underlying mechanisms that mediate xenin-in- duced anorectic effects are poorly documented in any species. To our knowledge, behavioral effects unrelated to ingestion after xenin treatment are not reported. Therefore the aim of the present study was to explore possible mechanisms contributing to xenin- induced satiety in chicks. Xenin was microinjected into the VMH and feed and water intake, in addition to hypothalamic c-Fos immunoreactivity were measured. The interaction of xenin with the chick’s endogenous opioid system was also studied. Finally, a comprehensive behavior analysis was conducted to determine if xenin caused behaviors that may be competitive with ingestion. 2. Materials and methods 2.1. Animals Unsexed Cobb-500 broiler chicks (Gallus gallus) from breeders 30 to 40 weeks of age were obtained from a commercial hatchery on the morning of hatch. They were caged individually in a room at 30 ± 2 °C and 50 ± 5% relative humidity with ad libi- tum access to a mash diet (20% crude protein and 2685 kcal ME/kg) and tap water. All experimental procedures were performed according to the National Research Council publication, Guide for Care and Use of Laboratory Animals and were approved by the Radford University Institutional Animal Care and Use committee. 2.2. Cannulation procedure Chicks, 14 d post hatch, were anesthetized with 30 mg/kg body mass sodium pentobarbital via the brachial vein. Chicks were then unilaterally implanted with a 22 gauge stainless steel guide cannula stereotaxically. Stereotaxic coordinates were 0.3 mm lateral to midline (L); 7.5 mm below the skull surface (Y); 7.0 mm anterior to interaural line (A) based on Kuenzel and Masson (1988). Using these coordinates the guide cannula’s bevel point was positioned 1 mm 0016-6480/$ - see front matter Ó 2008 Elsevier Inc. All rights reserved. doi:10.1016/j.ygcen.2008.03.012 * Corresponding author. E-mail address: mcline@radford.edu (M.A. Cline). General and Comparative Endocrinology 157 (2008) 58–62 Contents lists available at ScienceDirect General and Comparative Endocrinology journal homepage: www.elsevier.com/locate/ygcen