- Email: [email protected]
In vivo action of a new octadecaneuropeptide (ODN) antagonist on gonadotropin-releasing hormone gene expression in the male rat brain
Neuroscience 125 (2004) 411– 415
IN VIVO ACTION OF A NEW OCTADECANEUROPEPTIDE ANTAGONIST ON GONADOTROPIN-RELEASING HORMONE EXPRESSION IN THE MALE RAT BRAIN V. COMPE`RE,a S. LI,b J. LEPRINCE,a M. C. TONON,a H. VAUDRYa AND G. PELLETIERb*
(ODN) GENE
on the basis of its ability to displace diazepam from its binding sites (Guidotti et al., 1983). Proteolytic cleavage of DBI generates 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 discovered 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 GABAA 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 GABAA 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 concentration 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 neuronal activity, we have studied the in vivo effects of ODN as well as the influence of an new ODN antagonist to metabotropic receptor, cyclo1– 8[Dleu5]OP (Leprince et al., 2001), on GnRH mRNA levels in the castred male rat.
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 University Medical Center, Que´bec, Canada
Abstract—It has been reported that several of the effects induced 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 gonadotropinreleasing 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, cyclo1– 8[Dleu5]OP, on GnRH mRNA expression as evaluated by in situ hybridization in castrated male rats. The administration of ODN produced a decrease in the hybridization signal while the administration of cyclo1– 8[Dleu5]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 ODNtreated 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, cyclo1– 8[Dleu5]OP.
The hypothalamo–pituitary– gonadal axis is complexly regulated by sex steroids as well as by several neurotransmitters and neuromodulators including GABA (Kalra, 1986). Previous reports have established that GABA inhibits the secretion of gonadotropic hormones via activation of the GABAA 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 GABAA receptor complex by different GABAAergic 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
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 constant 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 approved 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 Experimental 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-ArgPro-Gly-Leu-Leu-Asp-Leu-Lysup-OH) and the ODN antagonist, cyclo1– 8[Dleu5]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 Laboratory, CHUL Research Center, 2705 Laurier Boulevard, Que´bec, G1V 4G2, Canada. Tel: ⫹1-418-654-2296; fax: ⫹1-418-654-2761. E-mail address: [email protected] (G. Pelletier). Abbreviations: ANT, antagonist; BZD, benzodiazepine; DBI, diazepam-binding inhibitor; GABA, ␥-aminobutyric acid; GnRH, gonadotropin-releasing hormone; MPAO, medial preoptic area; ODN, octadecaneuropeptide.
0306-4522/04$30.00⫹0.00 © 2004 IBRO. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.neuroscience.2004.02.016
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Twenty animals were castrated 5 days before treatment and equally divided in four groups. They were then stereotaxically implanted with a permanent stainless-steel canula into the left lateral ventricle of the brain, as described previously (Compe`re et al., 2003). The castrated animals received an i.c.v. injection of vehicle (0.9% NaCl), ODN (2 g), ODN antagonist alone (20 g) or a combination of these two drugs. One group of intact animals received only an i.c.v. injection of the vehicle. They were all fixed 4 h after injection as described below.
In situ hybridization For histological purposes, the rats were deeply anesthetized with an i.m. injection of a mixture of ketamine (80 mg/kg b.w.) and xylazine (10 mg/kg b.w.) and then rapidly perfused trans-
cardially with 4% paraformaldehyde in 0.2 M phosphate buffer 4 h after treatment. The brains were removed and postfixed in the same fixative overnight at 4 °C, and then placed in 15% sucrose in the 0.1 M phosphate buffer overnight at 4 °C. Thereafter, the tissues were frozen on dry ice in support medium (OCT; Bayer Corporation, Elkhart, IN, USA). Frontal sections through an area extending from the medial preoptic area (MPOA) to the anterior hypothalamus were serially cut at 10 m with a cryostat. The brain sections were then mounted on Superfrost/PLUS Microscope slides (Fisher Scientific, Montreal, Canada) and maintained at 80 °C until use. In situ hybridization was performed as described previously (Li and Pelletier, 1993). The probe chosen was a 35S-labeled 48-base of oligonucleotide complementary to the GnRH coding region of the
Fig. 1. Typical light microscope autoradiographs showing GnRH-labeled neurons (arrows) in the MPOA ⫻600. (A) Vehicle-treated rats. (B) Castrated rats. (C) ODN-treated castrated rats. (D) Cyclo1– 8 (Dleu5OP)-treated castrated rats. (E) Castrated rats treated with both ODN and cyclo1– 8 (Dleu5OP).
V. Compe`re et al. / Neuroscience 125 (2004) 411– 415
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Fig. 2. Response of GnRH mRNA levels to acute i.c.v. injection of ODN and ODN antagonist cyclo1– 8 (Dleu5OP) alone or in combination in castrated male rat. ANT, antagonist; INT, intact animals; VEH, vehicle. *** P⬍0.001 vehicle-treated castrated rats versus all the other experimental groups.
rat complementary DNA (bases 102–149). After hybridization, the sections were dehydrated and coated with liquid photographic emulsion (Kodak NTB-2). They were processed after 7 days of exposure. To assess the specificity of the hybridization signal, consecutive sections were alternatively hybridized with the labeled oligonucleotide probe encoding GnRH and a labeled sense oligomer directed to the complementary DNA strand. For each experimental group, the mean number of silver grains per cell was calculated from at least 225 cells (45 cells/animal) located in the MPAO. Comparison of the mRNA levels between experimental groups was performed by an analysis of variance (Statview; Abacus Concepts, Berkeley, CA, USA). P⬍0.05 was considered statistically significant.
RESULTS GnRH neurons are widely distributed in the septal and preoptic areas (Toranzo et al., 1989; Li and Pelletier, 1993). Analysis of the autoradiograms indicated that a strong signal could be obtained after 7 days of exposure. Hybridization with the labeled sense oligomer produced no labeling (data not shown). It was observed (Figs. 1 and 2) that castration enhanced GnRH mRNA levels in male rat
(23% over values observed in intact animals). In castrated rats, the acute i.c.v. administration of ODN (4 h before kill) produced a significant decrease (P⬍0.001) in the hybridization signal which was almost identical to that observed in the intact animals (Figs. 1 and 2). The administration of the ODN antagonist alone produced an 18% increase over the values observed in vehicle-injected castrated animals (P⬍0.001; Figs. 1 and 2). When administered concomitantly with ODN, the antagonist not only reversed the depressing effect of ODN but induced a 22% increase (P⬍0.001) over values detected in ODN-treated rats (Figs. 1 and 2).
DISCUSSION An increase in GnRH mRNA was observed 5 days after castration. This observation is in agreement with previous results indicating an increase in GnRH mRNA 5 and 21 days following castration (Toranzo et al., 1989; Li et al., 1997). The i.c.v. injection of ODN at the dose of 2 g induced a decrease in GnRH m RNA levels in the cas-
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trated rat MPOA, an effect which was completely prevented by the concomitant administration of the ODN antagonist cyclo1– 8[Dleu5]OP. The depressing effect of ODN is in agreement with our previous results clearly demonstrating that ODN decreased GnRH mRNA expression in castrated male rat (Li and Pelletier, 1995b, 1997). We have also reported that the effect of ODN was mediated by activation of the GABAA BZD receptor (Li and Pelletier, 1996). On the other hand, ODN has been also shown to increase intracellular calcium concentration in cultured rat astrocytes through activation of a metabotropic receptor positively coupled to phospholipase C (Patte et al., 1995; Gandolfo et al., 1997). Leprince et al. (1998, 2001) synthesized an ODN antagonist to metabotropic receptors, cyclo1– 8[Dleu5]OP, which exhibits potent antagonistic activities both on calcium mobilization and on polyphosphoinositide metabolism in rat astroglial cells. On the other hand, BZD antagonists did not block those ODN-induced effects (Leprince et al., 2001). This cyclo1– 8[Dleu5]OP then appears to be a potent metabotropic receptor antagonist in in vitro condition. In the present study, the complete abolition of the effect of ODN by the metabotropic receptor antagonist strongly suggests that ODN is interacting with metabotropic receptor to negatively modulate GnRH neurons. The observation that cyclo1– 8[Dleu5]OP can not only reverse inhibitory effect of ODN but also increase GnRH mRNA levels was not unexpected since an antagonist can induce by itself an effect opposite to that produced by the agonist. Since cyclo1– 8[Dleu5]OP can by itself exert a positive influence on GnRH mRNA levels, it might be suggested that ODN is exerting a tonic inhibitory influence on GnRH neuronal activity. The effect of ODN on GnRH expression might then be not related only to an interaction with the GABAA/BZD receptor complex as previously reported (Li and Pelletier, 1996). The present data coupled to previous observations from this laboratory suggest that the effect of ODN on GnRH mRNA might be mediated by interaction with either BZD receptors or metabotropic receptors or both. Previous studies have shown that ODN provoked behavioral actions which might be relayed by the metabotropic receptor. In the rat, the anorexigenic effect of ODN was reversed by cyclo1– 8[Dleu5]OP but not by BZD receptors antagonists (De Mateos-Verchere et al., 2001; Orta et al., 2003). Moreover, cyclo1– 8[Dleu5]OP alone increased food intake (Orta et al., 2003). In human studies, levels of DBI (the precursor of ODN) in cerebrospinal fluid were found to be elevated in depressed patients (Roy, 1991). It might be suggested that DBI or ODN plays a role in the pathophysiology of depression, since, among the clinical signs of depression, there are anorexia and a decrease in gonadotropin secretion (Meller et al., 2001) which might be well related to an increase of ODN. The development of specific ODN antagonists might be of great interest for the treatment of disorders associated with depression.
Acknowledgements—We thank Ms Louise De´sy and Johanne Ouellet for their expert technical assistance. This work was partly supported by a FRSQ-INSERM exchange program (to G.P. and H.V.).
REFERENCES Compe`re V, Li S, Leprince J, Tonon MC, Vaudry H, Pelletier G (2003) Effect of intracerebroventricular administration of the octadecaneuropeptide on the expression of pro-opiomelanocortin, neuropeptide y and corticotropin-releasing hormone mRNAs in rat hypothalamus. J Neuroendocrinol 15:197–203. De Mateos-Verchere JG, Leprince J, Tonon MC, Vaudry H, Costentin J (2001) The octadecaneuropeptide [diazepam-inhibitor (33–50)] exerts potent anorexigenic effects in rodents. Eur J Pharmacol 414:225–231. Ferrero P, Santi MR, Conti-Tronconi B, Costa E, Guidotti A (1986) Study of an octadecaneuropeptide derived from diazepam binding inhibitor (DBI): biological activity and presence in rat brain. Proc Natl Acad Sci USA 83:827–831. Fuchs E, Mansky T, Stock KW, Vijayan E, Wuttke W (1984) Involvement of catecholamines and glutamate in GABAergic mechanism regulatory to luteinizing hormone and prolactin secretion. Neuroendocrinology 38:484 –489. Gandolfo P, Patte C, Leprince J, Thoumas JL, Vaudry H, Tonon MC (1997) The stimulatory effect of the octadecaneuropeptide (ODN) on cytosolic Ca2⫹ in rat astrocytes is not mediated through classical benzodiazepine receptors. Eur J Pharmacol 322:275–281. Guidotti A, Forchetti CM, Corda MG, Kondel D, Bennett CD, Costa E (1983) Isolation, characterization and purification to homogeneity of an endogenous polypeptide with agonistic action on benzodiazepine receptors. Proc Natl Acad Sci USA 80:3531–3533. Kalra SP (1986) Neural circuitry involved in the control of LHRH secretion: a model for preovilatory LH release. Front Neuroendocrinol 9:31–35. Leprince J, Gandolfo P, Thoumas JL, Patte C, Fauchere JL, Vaudry H, Tonon MC (1998) Structure-activity relationships of a series of analogues or the octadecaneuropeptide ODN on calcium mobilization in rat astrocytes. J Med Chem 41:4433–4438. Leprince J, Oulyadi H, Vaudry D, Masmoudi O, Gandolfo P, Patte C, Costentin J, Fauchere JL, Davoust D, Vaudry H, Tonon MC (2001) Synthesis, conformational analysis and biological activity of cyclic analogs of the octadecaneuropeptide ODN: design of a potent endozepine antagonist. Eur J Biochem 268:6045–6057. Li S, Pelletier G (1993) Chronic administration of muscimol and pentobarbital decreases gonadotropin-releasing hormone mRNA levels in the male rat hypothalamus determined by quantitative in situ hybridization. Neuroendocrinology 58:136 –139. Li S, Pelletier G (1995a) Effet de l’activation du site des benzodiazepines du complexe des re´cepteurs GABAA sur l’expression ge´ne´tique de la GnRH dans le cerveau de rat. XXIVe Colloque de la Socie´te´ de Neuroendocrinologie Expe´rimentale Ann Endocrinol, Magog-Oxford (Que´bec):414 (abs. P37). Li S, Pelletier G (1995b) Inhibitory effect of the endogenous benzodiazepine receptor ligand, octadecaneuropeptide (ODN), on gonadotropin-releasing hormone gene expression in the male rat brain. Neuroreport 6:1354 –1356. Li S, Pelletier G (1996) Further studies on the mechanism of action of the endogenous benzodiazepine ligand octadecaneuropeptide (ODN) on gonadotropin-releasing hormone gene expression in rat brain. Neuroendocrinology 64:79 –84. Li S, Givalois L, Pelletier G (1997) Role of adrenal an gonadal steroids in the response of GnRH gene expression to the endogenous benzodiazepine receptor ligand octadecaneuropeptide in the male rat brain. Neuropeptides 31:463–468. Meller WH, Grambsch PL, Bingham C, Tagatz GE (2001) Hypothalamic pituitary gonadal axis dysregulation in depressed women. Psychoneuroendocrinology 26:253–259.
V. Compe`re et al. / Neuroscience 125 (2004) 411– 415 Orta, MH, Do Rego, JC, Leprince, J, Vaudry, H, Costentin, J (2003) Effets de l’octadecaneuropeptide (ODN) sur la prise alimentaire: caracte´risation du re´cepteur implique´. 6e colloque de la Socie´te´ des neurosciences I-51. Patte C, Vaudry H, Desrue L, Gandolpho P, Strijveen I, Lamacz M, Tonon MC (1995) The endozepine ODN stimulates polyphosphoinositide metabolism in rat astrocyte. FESB Lett 362:106 –110. Roy A (1991) Cerebrospinal fluid diazepam binding inhibitor in depressed patients and normal controls. Neuropharmacology 30: 1441–1444. Slobodyansky E, Guidotti A, Wambebe C, Berkovich A, Costa E
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(1989) Isolation and characterization of a rat brain triakontatetraneuropeptide, a posttranslational product of diazepam binding inhibitor: specific action at the Ro5– 4864 recognition site. J Neurochem 53:1276 –1284. Toranzo D, Dupont E, Simard J, Labrie C, Couet J, Labrie F, Pelletier G (1989) Regulation of pro-gonadotropin-releasing hormone gene expression by sex steroids in the brain of male and female rats. Mol Endocrinol 3:1748 –1756. Vincens M, Li S, Pelletier G (1994) Inhibitory effect of 5-pregnan-3␣ol-1 on gonadotropin-releasing hormone gene expression in the male rat. Eur J Pharmacol 260:157–162.
(Accepted 5 February 2004)
IN VIVO ACTION OF A NEW OCTADECANEUROPEPTIDE ANTAGONIST ON GONADOTROPIN-RELEASING HORMONE EXPRESSION IN THE MALE RAT BRAIN V. COMPE`RE,a S. LI,b J. LEPRINCE,a M. C. TONON,a H. VAUDRYa AND G. PELLETIERb*
(ODN) GENE
on the basis of its ability to displace diazepam from its binding sites (Guidotti et al., 1983). Proteolytic cleavage of DBI generates 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 discovered 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 GABAA 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 GABAA 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 concentration 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 neuronal activity, we have studied the in vivo effects of ODN as well as the influence of an new ODN antagonist to metabotropic receptor, cyclo1– 8[Dleu5]OP (Leprince et al., 2001), on GnRH mRNA levels in the castred male rat.
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 University Medical Center, Que´bec, Canada
Abstract—It has been reported that several of the effects induced 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 gonadotropinreleasing 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, cyclo1– 8[Dleu5]OP, on GnRH mRNA expression as evaluated by in situ hybridization in castrated male rats. The administration of ODN produced a decrease in the hybridization signal while the administration of cyclo1– 8[Dleu5]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 ODNtreated 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, cyclo1– 8[Dleu5]OP.
The hypothalamo–pituitary– gonadal axis is complexly regulated by sex steroids as well as by several neurotransmitters and neuromodulators including GABA (Kalra, 1986). Previous reports have established that GABA inhibits the secretion of gonadotropic hormones via activation of the GABAA 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 GABAA receptor complex by different GABAAergic 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
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 constant 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 approved 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 Experimental 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-ArgPro-Gly-Leu-Leu-Asp-Leu-Lysup-OH) and the ODN antagonist, cyclo1– 8[Dleu5]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 Laboratory, CHUL Research Center, 2705 Laurier Boulevard, Que´bec, G1V 4G2, Canada. Tel: ⫹1-418-654-2296; fax: ⫹1-418-654-2761. E-mail address: [email protected] (G. Pelletier). Abbreviations: ANT, antagonist; BZD, benzodiazepine; DBI, diazepam-binding inhibitor; GABA, ␥-aminobutyric acid; GnRH, gonadotropin-releasing hormone; MPAO, medial preoptic area; ODN, octadecaneuropeptide.
0306-4522/04$30.00⫹0.00 © 2004 IBRO. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.neuroscience.2004.02.016
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Twenty animals were castrated 5 days before treatment and equally divided in four groups. They were then stereotaxically implanted with a permanent stainless-steel canula into the left lateral ventricle of the brain, as described previously (Compe`re et al., 2003). The castrated animals received an i.c.v. injection of vehicle (0.9% NaCl), ODN (2 g), ODN antagonist alone (20 g) or a combination of these two drugs. One group of intact animals received only an i.c.v. injection of the vehicle. They were all fixed 4 h after injection as described below.
In situ hybridization For histological purposes, the rats were deeply anesthetized with an i.m. injection of a mixture of ketamine (80 mg/kg b.w.) and xylazine (10 mg/kg b.w.) and then rapidly perfused trans-
cardially with 4% paraformaldehyde in 0.2 M phosphate buffer 4 h after treatment. The brains were removed and postfixed in the same fixative overnight at 4 °C, and then placed in 15% sucrose in the 0.1 M phosphate buffer overnight at 4 °C. Thereafter, the tissues were frozen on dry ice in support medium (OCT; Bayer Corporation, Elkhart, IN, USA). Frontal sections through an area extending from the medial preoptic area (MPOA) to the anterior hypothalamus were serially cut at 10 m with a cryostat. The brain sections were then mounted on Superfrost/PLUS Microscope slides (Fisher Scientific, Montreal, Canada) and maintained at 80 °C until use. In situ hybridization was performed as described previously (Li and Pelletier, 1993). The probe chosen was a 35S-labeled 48-base of oligonucleotide complementary to the GnRH coding region of the
Fig. 1. Typical light microscope autoradiographs showing GnRH-labeled neurons (arrows) in the MPOA ⫻600. (A) Vehicle-treated rats. (B) Castrated rats. (C) ODN-treated castrated rats. (D) Cyclo1– 8 (Dleu5OP)-treated castrated rats. (E) Castrated rats treated with both ODN and cyclo1– 8 (Dleu5OP).
V. Compe`re et al. / Neuroscience 125 (2004) 411– 415
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Fig. 2. Response of GnRH mRNA levels to acute i.c.v. injection of ODN and ODN antagonist cyclo1– 8 (Dleu5OP) alone or in combination in castrated male rat. ANT, antagonist; INT, intact animals; VEH, vehicle. *** P⬍0.001 vehicle-treated castrated rats versus all the other experimental groups.
rat complementary DNA (bases 102–149). After hybridization, the sections were dehydrated and coated with liquid photographic emulsion (Kodak NTB-2). They were processed after 7 days of exposure. To assess the specificity of the hybridization signal, consecutive sections were alternatively hybridized with the labeled oligonucleotide probe encoding GnRH and a labeled sense oligomer directed to the complementary DNA strand. For each experimental group, the mean number of silver grains per cell was calculated from at least 225 cells (45 cells/animal) located in the MPAO. Comparison of the mRNA levels between experimental groups was performed by an analysis of variance (Statview; Abacus Concepts, Berkeley, CA, USA). P⬍0.05 was considered statistically significant.
RESULTS GnRH neurons are widely distributed in the septal and preoptic areas (Toranzo et al., 1989; Li and Pelletier, 1993). Analysis of the autoradiograms indicated that a strong signal could be obtained after 7 days of exposure. Hybridization with the labeled sense oligomer produced no labeling (data not shown). It was observed (Figs. 1 and 2) that castration enhanced GnRH mRNA levels in male rat
(23% over values observed in intact animals). In castrated rats, the acute i.c.v. administration of ODN (4 h before kill) produced a significant decrease (P⬍0.001) in the hybridization signal which was almost identical to that observed in the intact animals (Figs. 1 and 2). The administration of the ODN antagonist alone produced an 18% increase over the values observed in vehicle-injected castrated animals (P⬍0.001; Figs. 1 and 2). When administered concomitantly with ODN, the antagonist not only reversed the depressing effect of ODN but induced a 22% increase (P⬍0.001) over values detected in ODN-treated rats (Figs. 1 and 2).
DISCUSSION An increase in GnRH mRNA was observed 5 days after castration. This observation is in agreement with previous results indicating an increase in GnRH mRNA 5 and 21 days following castration (Toranzo et al., 1989; Li et al., 1997). The i.c.v. injection of ODN at the dose of 2 g induced a decrease in GnRH m RNA levels in the cas-
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trated rat MPOA, an effect which was completely prevented by the concomitant administration of the ODN antagonist cyclo1– 8[Dleu5]OP. The depressing effect of ODN is in agreement with our previous results clearly demonstrating that ODN decreased GnRH mRNA expression in castrated male rat (Li and Pelletier, 1995b, 1997). We have also reported that the effect of ODN was mediated by activation of the GABAA BZD receptor (Li and Pelletier, 1996). On the other hand, ODN has been also shown to increase intracellular calcium concentration in cultured rat astrocytes through activation of a metabotropic receptor positively coupled to phospholipase C (Patte et al., 1995; Gandolfo et al., 1997). Leprince et al. (1998, 2001) synthesized an ODN antagonist to metabotropic receptors, cyclo1– 8[Dleu5]OP, which exhibits potent antagonistic activities both on calcium mobilization and on polyphosphoinositide metabolism in rat astroglial cells. On the other hand, BZD antagonists did not block those ODN-induced effects (Leprince et al., 2001). This cyclo1– 8[Dleu5]OP then appears to be a potent metabotropic receptor antagonist in in vitro condition. In the present study, the complete abolition of the effect of ODN by the metabotropic receptor antagonist strongly suggests that ODN is interacting with metabotropic receptor to negatively modulate GnRH neurons. The observation that cyclo1– 8[Dleu5]OP can not only reverse inhibitory effect of ODN but also increase GnRH mRNA levels was not unexpected since an antagonist can induce by itself an effect opposite to that produced by the agonist. Since cyclo1– 8[Dleu5]OP can by itself exert a positive influence on GnRH mRNA levels, it might be suggested that ODN is exerting a tonic inhibitory influence on GnRH neuronal activity. The effect of ODN on GnRH expression might then be not related only to an interaction with the GABAA/BZD receptor complex as previously reported (Li and Pelletier, 1996). The present data coupled to previous observations from this laboratory suggest that the effect of ODN on GnRH mRNA might be mediated by interaction with either BZD receptors or metabotropic receptors or both. Previous studies have shown that ODN provoked behavioral actions which might be relayed by the metabotropic receptor. In the rat, the anorexigenic effect of ODN was reversed by cyclo1– 8[Dleu5]OP but not by BZD receptors antagonists (De Mateos-Verchere et al., 2001; Orta et al., 2003). Moreover, cyclo1– 8[Dleu5]OP alone increased food intake (Orta et al., 2003). In human studies, levels of DBI (the precursor of ODN) in cerebrospinal fluid were found to be elevated in depressed patients (Roy, 1991). It might be suggested that DBI or ODN plays a role in the pathophysiology of depression, since, among the clinical signs of depression, there are anorexia and a decrease in gonadotropin secretion (Meller et al., 2001) which might be well related to an increase of ODN. The development of specific ODN antagonists might be of great interest for the treatment of disorders associated with depression.
Acknowledgements—We thank Ms Louise De´sy and Johanne Ouellet for their expert technical assistance. This work was partly supported by a FRSQ-INSERM exchange program (to G.P. and H.V.).
REFERENCES Compe`re V, Li S, Leprince J, Tonon MC, Vaudry H, Pelletier G (2003) Effect of intracerebroventricular administration of the octadecaneuropeptide on the expression of pro-opiomelanocortin, neuropeptide y and corticotropin-releasing hormone mRNAs in rat hypothalamus. J Neuroendocrinol 15:197–203. De Mateos-Verchere JG, Leprince J, Tonon MC, Vaudry H, Costentin J (2001) The octadecaneuropeptide [diazepam-inhibitor (33–50)] exerts potent anorexigenic effects in rodents. Eur J Pharmacol 414:225–231. Ferrero P, Santi MR, Conti-Tronconi B, Costa E, Guidotti A (1986) Study of an octadecaneuropeptide derived from diazepam binding inhibitor (DBI): biological activity and presence in rat brain. Proc Natl Acad Sci USA 83:827–831. Fuchs E, Mansky T, Stock KW, Vijayan E, Wuttke W (1984) Involvement of catecholamines and glutamate in GABAergic mechanism regulatory to luteinizing hormone and prolactin secretion. Neuroendocrinology 38:484 –489. Gandolfo P, Patte C, Leprince J, Thoumas JL, Vaudry H, Tonon MC (1997) The stimulatory effect of the octadecaneuropeptide (ODN) on cytosolic Ca2⫹ in rat astrocytes is not mediated through classical benzodiazepine receptors. Eur J Pharmacol 322:275–281. Guidotti A, Forchetti CM, Corda MG, Kondel D, Bennett CD, Costa E (1983) Isolation, characterization and purification to homogeneity of an endogenous polypeptide with agonistic action on benzodiazepine receptors. Proc Natl Acad Sci USA 80:3531–3533. Kalra SP (1986) Neural circuitry involved in the control of LHRH secretion: a model for preovilatory LH release. Front Neuroendocrinol 9:31–35. Leprince J, Gandolfo P, Thoumas JL, Patte C, Fauchere JL, Vaudry H, Tonon MC (1998) Structure-activity relationships of a series of analogues or the octadecaneuropeptide ODN on calcium mobilization in rat astrocytes. J Med Chem 41:4433–4438. Leprince J, Oulyadi H, Vaudry D, Masmoudi O, Gandolfo P, Patte C, Costentin J, Fauchere JL, Davoust D, Vaudry H, Tonon MC (2001) Synthesis, conformational analysis and biological activity of cyclic analogs of the octadecaneuropeptide ODN: design of a potent endozepine antagonist. Eur J Biochem 268:6045–6057. Li S, Pelletier G (1993) Chronic administration of muscimol and pentobarbital decreases gonadotropin-releasing hormone mRNA levels in the male rat hypothalamus determined by quantitative in situ hybridization. Neuroendocrinology 58:136 –139. Li S, Pelletier G (1995a) Effet de l’activation du site des benzodiazepines du complexe des re´cepteurs GABAA sur l’expression ge´ne´tique de la GnRH dans le cerveau de rat. XXIVe Colloque de la Socie´te´ de Neuroendocrinologie Expe´rimentale Ann Endocrinol, Magog-Oxford (Que´bec):414 (abs. P37). Li S, Pelletier G (1995b) Inhibitory effect of the endogenous benzodiazepine receptor ligand, octadecaneuropeptide (ODN), on gonadotropin-releasing hormone gene expression in the male rat brain. Neuroreport 6:1354 –1356. Li S, Pelletier G (1996) Further studies on the mechanism of action of the endogenous benzodiazepine ligand octadecaneuropeptide (ODN) on gonadotropin-releasing hormone gene expression in rat brain. Neuroendocrinology 64:79 –84. Li S, Givalois L, Pelletier G (1997) Role of adrenal an gonadal steroids in the response of GnRH gene expression to the endogenous benzodiazepine receptor ligand octadecaneuropeptide in the male rat brain. Neuropeptides 31:463–468. Meller WH, Grambsch PL, Bingham C, Tagatz GE (2001) Hypothalamic pituitary gonadal axis dysregulation in depressed women. Psychoneuroendocrinology 26:253–259.
V. Compe`re et al. / Neuroscience 125 (2004) 411– 415 Orta, MH, Do Rego, JC, Leprince, J, Vaudry, H, Costentin, J (2003) Effets de l’octadecaneuropeptide (ODN) sur la prise alimentaire: caracte´risation du re´cepteur implique´. 6e colloque de la Socie´te´ des neurosciences I-51. Patte C, Vaudry H, Desrue L, Gandolpho P, Strijveen I, Lamacz M, Tonon MC (1995) The endozepine ODN stimulates polyphosphoinositide metabolism in rat astrocyte. FESB Lett 362:106 –110. Roy A (1991) Cerebrospinal fluid diazepam binding inhibitor in depressed patients and normal controls. Neuropharmacology 30: 1441–1444. Slobodyansky E, Guidotti A, Wambebe C, Berkovich A, Costa E
415
(1989) Isolation and characterization of a rat brain triakontatetraneuropeptide, a posttranslational product of diazepam binding inhibitor: specific action at the Ro5– 4864 recognition site. J Neurochem 53:1276 –1284. Toranzo D, Dupont E, Simard J, Labrie C, Couet J, Labrie F, Pelletier G (1989) Regulation of pro-gonadotropin-releasing hormone gene expression by sex steroids in the brain of male and female rats. Mol Endocrinol 3:1748 –1756. Vincens M, Li S, Pelletier G (1994) Inhibitory effect of 5-pregnan-3␣ol-1 on gonadotropin-releasing hormone gene expression in the male rat. Eur J Pharmacol 260:157–162.
(Accepted 5 February 2004)