Kisspeptin Neurons Co-Express Met-Enkephalin and Galanin in the Rostral Periventricular Region of the Female Mouse Hypothalamus Robert Porteous, 1 Sandra L. Petersen, 2 Shel Hwa Yeo, 1 Janardhan P. Bhattarai, 3 Philippe Ciofi, 4 Xavier D’anglemont de Tassigny, 5 William H. Colledge, 5 Alain Caraty, 6 and Allan E. Herbison 1 * 1 Centre for Neuroendocrinology and Department of Physiology, University of Otago School of Medical Sciences, Dunedin 9054, New Zealand 2 Department of Veterinary and Animal Sciences, University of Massachusetts Amherst, Amherst, Massachusetts 01003 3 Department of Oral Physiology & BK21 Program, School of Dentistry & Institute of Oral Bioscience, Chonbuk National University, Jeonju, Republic of Korea 4 Institut National de la Sante´ et de la Recherche Me ´dicale, Neurocentre Magendie-U862, Institut Franc ¸ois Magendie, and University of Bordeaux, F-33077, Bordeaux, France 5 Reproductive Physiology Group, Department of Physiology, Development, and Neuroscience, University of Cambridge, Cambridge CB2 3EG, United Kingdom 6 Physiologie de la Reproduction et des Comportements, Institut National de la Recherche Agronomique, F-37380 Nouzilly, France ABSTRACT It is now well established that the kisspeptin neurons of the hypothalamus play a key role in regulating the activity of gonadotropin-releasing hormone (GnRH) neu- rons. The population of kisspeptin neurons residing in the rostral periventricular region of the third ventricle (RP3V), encompassing the anteroventral periventricular (AVPV) and periventricular preoptic nuclei (PVpo), are implicated in the generation of the preovulatory GnRH surge mechanism and puberty onset in female rodents. The present study examined whether these kisspeptin neurons may express other neuropeptides in the adult female mouse. Initially, the distribution of galanin, neu- rotensin, met-enkephalin (mENK), and cholecystokinin (CCK)-immunoreactive cells was determined within the RP3V of colchicine-treated mice. Subsequent experi- ments, using a new kisspeptin-10 antibody raised in sheep, examined the relationship of these neuropepti- des to kisspeptin neurons. No evidence was found for expression of neurotensin or CCK by RP3V kisspeptin neurons, but subpopulations of kisspeptin neurons were observed to express galanin and mENK. Dual-labeled RP3V kisspeptin/galanin cells represented 7% of all kisspeptin and 21% of all galanin neurons whereas dual-labeled kisspeptin/mENK cells represented 28–38% of kisspeptin neurons and 58–68% of the mENK population, depending on location within the AVPV or PVpo. Kisspeptin neurons in the arcuate nu- cleus were also found to express galanin but not mENK. These observations indicate that, like the kiss- peptin population of the arcuate nucleus, kisspeptin neurons in the RP3V also co-express a range of neuro- peptides. This pattern of co-expression should greatly increase the dynamic range with which kisspeptin neu- rons can modulate the activity of their afferent neurons. J. Comp. Neurol. 519:3456–3469, 2011. V C 2011 Wiley-Liss, Inc. INDEXING TERMS: AVPV; RP3V; galanin; enkephalin; cholecystokinin; neurotensin; kisspeptin; GnRH It has been established that the kisspeptin neurons of the hypothalamus have a key role in the regulation of the reproductive axis. In all mammalian species examined to date, two major populations of kisspeptin neurons have been identified, one in the arcuate/infundibular nucleus and another in the preoptic area (Mikkelsen and Simonneaux, 2009; Oakley et al., 2009). The roles of these two kisspeptin populations are currently under Grant sponsor: National Institutes of Health; Grant number: HD027305 (to S.L.P.); Grant sponsor: Health Research Council of New Zealand. *CORRESPONDENCE TO: Allan E. Herbison, Centre for Neuroendocrinology, Department of Physiology, University of Otago School of Medical Sciences, P.O. Box 913, Dunedin, New Zealand. E-mail: allan.herbison@stonebow.otago.ac.nz V C 2011 Wiley-Liss, Inc. Received August 27, 2010; Revised May 11, 2011; Accepted July 5, 2011 DOI 10.1002/cne.22716 Published online July 28, 2011 in Wiley Online Library (wileyonlinelibrary. com) 3456 The Journal of Comparative Neurology | Research in Systems Neuroscience 129:3456–3469 (2011) RESEARCH ARTICLE