Anatomical distribution and biochemical characterization of the novel RFamide peptide 26RFa in the human hypothalamus and spinal cord Federica Bruzzone,* , § Benoı ˆt Lectez,* He ´le `ne Tollemer,* Je ´ro ˆme Leprince,* Cynthia Dujardin,* Walid Rachidi,* David Chatenet,* Marc Baroncini, Jean-Claude Beauvillain,à Mauro Vallarino,§ Hubert Vaudry* and Nicolas Chartrel* *INSERM U413, Laboratory of Cellular and Molecular Neuroendocrinology, European Institute for Peptide Research (IFRMP 23), University of Rouen, Mont-Saint-Aignan, France  Laboratoire d’Anatomie CHRU de Lille, Lille, France àINSERM U 816, Unite ´ de Neuroendocrinologie et Physiopathologie Neuronale, Institut Fe ´de ´ratif de Recherches 114, Lille, France §Department of Experimental Biology, DIBISAA, University of Genova, Genova, Italy Abstract 26RFa is a novel RFamide peptide originally isolated in the amphibian brain. The 26RFa precursor has been subse- quently characterized in various mammalian species but, until now, the anatomical distribution and the molecular forms of 26RFa produced in the CNS of mammals, in particular in human, are unknown. In the present study, we have investigated the localization and the biochemical characteristics of 26RFa-like immunoreactivity (LI) in two regions of the human CNS – the hypothalamus and the spinal cord. Immunohistochemical labeling using specific antibodies against human 26RFa and in situ hybridization histochemistry revealed that in the human hypothalamus 26RFa-expressing neurons are located in the paraventricular and ventromedial nuclei. In the spinal cord, 26RFa-expres- sing neurons were observed in the dorsal and lateral horns. Characterization of 26RFa-related peptides showed that two distinct molecular forms of 26RFa are present in the human hypothalamus and spinal cord, i.e. 26RFa and an N-terminally elongated form of 43 amino acids designated 43RFa. These data provide the first evidence that 26RFa and 43RFa are actually produced in the human CNS. The distribution of 26RF-LI suggests that 26RFa and/or 43RFa may modulate feeding, sexual behavior and transmission of nociceptive stimuli. Keywords: hypothalamus, neuropeptides, processing, pre- cursor, spinal cord. J. Neurochem. (2006) 99, 616–627. Since the initial identification of the cardioexcitatory peptide FMRFamide from the ganglia of the venus clam Macrocal- lista nimbosa (Price and Greenberg 1977), a number of peptides possessing the Arg-Phe-NH 2 motif at their C- terminus (collectively termed RFamide peptides) have been characterized in various groups of invertebrates (for a review, see Chartrel et al. 2002). In comparison, the number of RFamide peptides identified in vertebrates remains quite modest. Indeed, until recently, only three genes encoding RFamide peptide precursors had been characterized in mammals (Chartrel et al. 2006a). One of these genes encodes the precursor for two RFamide peptides – neuropeptide FF (NPFF) and neuropeptide AF (Perry et al. 1997; Vilim et al. 1999). These two peptides modulate the action of morphine Received February 15, 2006; revised manuscript received May 24, 2006; accepted June 24, 2006. Address correspondence and reprint requests to Hubert Vaudry, IN- SERM U 413, Laboratory of Cellular and Molecular Neuroendocrinol- ogy, European Institute for Peptide Research (IFRMP 23), University of Rouen, 76821 Mont-Saint-Aignan, France. E-mail: hubert.vaudry@univ-rouen.fr Abbreviations used: BSA, bovine serum albumin; CRH, corticotropin- releasing hormone; LH, luteinizing hormone; NPFF, neuropeptide FF; NPY, neuropeptide Y; PBS, phosphate-buffered saline; PC, prohormone convertase; PrRP, prolactin-releasing peptide; PVN, paraventricular nucleus; 26RFa-LI, 26RFa-like immunoreactivity; SPN, sympathetic preganglionic neurons; TFA, trifluoroacetic acid; VMN, ventromedial hypothalamic nucleus. Journal of Neurochemistry , 2006, 99, 616–627 doi:10.1111/j.1471-4159.2006.04090.x 616 Journal Compilation Ó 2006 International Society for Neurochemistry, J. Neurochem. (2006) 99, 616–627 Ó 2006 The Authors