IN VIVO ACTION OF A NEW OCTADECANEUROPEPTIDE (ODN) ANTAGONIST ON GONADOTROPIN-RELEASING HORMONE GENE EXPRESSION IN THE MALE RAT BRAIN V. COMPE ` RE, a S. LI, b J. LEPRINCE, a M. C. TONON, a H. VAUDRY a AND G. PELLETIER b * a European Institute for Peptide Research (IFRMP 23), Laboratory of Cellular and Molecular Neuroendocrinology, INSERM U413, UA CNRS, University of Rouen, Mont-Saint-Aignan, France b Oncology and Molecular Endocrinology Research Center, Laval Uni- versity Medical Center, Que ´ bec, Canada Abstract—It has been reported that several of the effects in- duced by octadecaneuropeptide (ODN) could be mediated by an activation of a metabotropic receptor. In order to investigate the role and mechanism of action of ODN in gonadotropin- releasing hormone (GnRH) neuron regulation, we studied the effects of the acute i.c.v. administration of ODN and of a new ODN antagonist to metabotropic receptor, cyclo 1–8 [Dleu 5 ]OP, on GnRH mRNA expression as evaluated by in situ hybrid- ization in castrated male rats. The administration of ODN produced a decrease in the hybridization signal while the administration of cyclo 1–8 [Dleu 5 ]OP alone produced an 18% increase. When administrated concomitantly with ODN, the antagonist both inhibited the depressing effect of ODN and induced a 22% increase over the values detected in ODN- treated rats. The data suggest that the effect of ODN on GnRH mRNA expression might be mediated by interaction with metabotropic receptors. © 2004 IBRO. Published by Elsevier Ltd. All rights reserved. Key words: octadecaneuropeptide, metabotropic receptor, gonadotropin-releasing hormone, cyclo 1–8 [Dleu 5 ]OP. The hypothalamo–pituitary– gonadal axis is complexly reg- ulated by sex steroids as well as by several neurotrans- mitters and neuromodulators including GABA (Kalra, 1986). Previous reports have established that GABA inhib- its the secretion of gonadotropic hormones via activation of the GABA A receptor complex (Fuchs et al., 1984). We have previously demonstrated that castration induced an increase in gonadotropin-releasing hormone (GnRH) and that activation of the GABA A receptor complex by different GABA A ergic agonists has an inhibitory influence on GnRH gene expression (Li and Pelletier, 1993, 1995a; Vincens et al., 1994). Diazepam binding inhibitor (DBI) is an 86 amino-acid polypeptide that has been initially isolated from the rat brain on the basis of its ability to displace diazepam from its binding sites (Guidotti et al., 1983). Proteolytic cleavage of DBI gen- erates active fragments, including the octadecaneuropeptide ODN (DBI 33–50) (Ferrero et al., 1986). Pharmacological studies have shown that ODN interacts predominantly with central-type benzodiazepine (BZD) receptors (Ferrero et al., 1986; Slobodyansky et al., 1989). We have recently discov- ered that ODN administration could decrease GnRH mRNA levels in intact and castrated male rats (Li and Pelletier, 1995; Li et al., 1997). The inhibitory effect of ODN was completely prevented by the GABA A receptor antagonist, picrotoxin (Li and Pelletier, 1995b) and the specific antagonist to BZD receptors, flumazenil (Li and Pelletier, 1996). These studies suggest that ODN can negatively modulate GnRH neuronal activity by an activation of the BZD sites at the GABA A receptor complex. Recently, it has been shown that several of the effects induced by ODN could be mediated by an activation of a metabotropic receptor positively coupled to phospholipase C (Patte et al., 1995; Gandolfo et al., 1997). In in vitro studies, ODN has been shown to increase intracellular calcium con- centration in cultured rat astrocytes through activation of a metabotropic receptor positively coupled to phospholipase C (Patte et al., 1995; Gandolfo et al., 1997). In order to further investigate the mechanism of action of ODN on GnRH neu- ronal activity, we have studied the in vivo effects of ODN as well as the influence of an new ODN antagonist to metabo- tropic receptor, cyclo 1–8 [Dleu 5 ]OP (Leprince et al., 2001), on GnRH mRNA levels in the castred male rat. EXPERIMENTAL PROCEDURES Animals and treatment Twenty five adult male Sprague–Dawley rats (Charles River Inc., Saint-Constant, Quebec, Canada), weighing 200 –225 g at the beginning of the different experiments, were housed under con- stant temperature (21 °C) and a 14/10-h light/dark cycle (lights on 06:00 h). They had free access to standard rat chow and drinking tap water. The Laval University’s Animal Welfare Committee ap- proved all the protocols. The experiments were conducted in an animal facility approved by the Canadian Council on Animal Care (CCAC) and by the Association for Assessment and Accreditation of Laboratory Animal Care (AAALAC). The study was performed in accordance with the CCAC Guide for Care and Use of Exper- imental Animals. All efforts were made to minimize the number of animals used and their suffering. Rat ODN (H-Gln-Ala-Thr-Val-Gly-Asp-Val-Asn-Thr-Asp-Arg- Pro-Gly-Leu-Leu-Asp-Leu-Lysup-OH) and the ODN antagonist, cyclo 1–8 [Dleu 5 ]OP (cyclic Arg-Pro-Gly-Leu-DLeu-Asp-Leu-Lys) were synthesized using solid phase methodology, as described previously (Leprince et al., 2001). *Correspondence to: G. Pelletier, Molecular Endocrinology Labora- tory, CHUL Research Center, 2705 Laurier Boulevard, Que ´bec, G1V 4G2, Canada. Tel: 1-418-654-2296; fax: 1-418-654-2761. E-mail address: georges.pelletier@crchul.ulaval.ca (G. Pelletier). Abbreviations: ANT, antagonist; BZD, benzodiazepine; DBI, diaze- pam-binding inhibitor; GABA, -aminobutyric acid; GnRH, gonado- tropin-releasing hormone; MPAO, medial preoptic area; ODN, octadecaneuropeptide. Neuroscience 125 (2004) 411– 415 0306-4522/04$30.000.00 © 2004 IBRO. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.neuroscience.2004.02.016 411