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Visualization of Functional Neuropeptide Y Receptors in the Mouse Hippocampus and Neocortex Using [35S]GTPγS Binding

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Abstract

The peptide transmitter neuropeptide Y (NPY) has been implicated in a plethora of actions in the central nervous system, including the hippocampus and neocortex (NeoCx). Previous studies using traditional receptor autoradiography show that NPY receptor binding is altered under various pathophysiological conditions. However, these do not provide information about downstream signal transduction. To this means, agonist-stimulated [35S]GTPγS binding has in recent years been introduced as a method for measuring receptor activation of intracellular G-proteins. In the present study, this method was further optimized to visualize the distribution of individual NPY receptor-mediated [35S]GTPγS binding in the mouse hippocampus and NeoCx using the endogenous ligand NPY in combination with optimized concentrations of selective antagonists for Y1, Y2, and Y5 NPY receptors. Consistent with previous studies, Y1 receptor-mediated [35S]GTPγS binding was mainly found in the dentate gyrus and NeoCx while Y2 receptor-mediated [35S]GTPγS binding predominated in the hippocampal CA3 and CA1. Only very low levels of Y5 receptor-mediated [35S]GTPγS binding appeared to be present in the hippocampus and NeoCx. Furthermore, the effect of NPY receptor antagonists per se was also studied. Both BIIE0246 and L-152,804 significantly attenuated the basal [35S]GTPγS binding response, suggesting inverse agonism.

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References

  • Agasse F, Bernardino L, Kristiansen H, Christiansen SH, Ferreira R, Silva B, Grade S, Woldbye DP, Malva JO (2008) Neuropeptide Y promotes neurogenesis in murine subventricular zone. Stem Cells 26(6):1636–1645

    Article  CAS  PubMed  Google Scholar 

  • Berglund MM, Hipskind PA, Gehlert DR (2003) Recent developments in our understanding of the physiological role of PP-fold peptide receptor subtypes. Exp Biol Med 228(3):217–244

    CAS  Google Scholar 

  • Castro ME, Diaz A, del Olmo E, Pazos A (2003) Chronic fluoxetine induces opposite changes in G protein coupling at pre and postsynaptic 5-HT1A receptors in rat brain. Neuropharmacology 44(1):93–101

  • Chen G, Way J, Armour S, Watson C, Queen K, Jayawickreme CK, Chen WJ, Kenakin T (2000) Use of constitutive G protein-coupled receptor activity for drug discovery. Mol Pharmacol 57(1):125–134

    CAS  PubMed  Google Scholar 

  • Christensen DZ, Olesen MV, Kristiansen H, Mikkelsen JD, Woldbye DP (2006) Unaltered neuropeptide Y (NPY)-stimulated [35S]GTPgammaS binding suggests a net increase in NPY signalling after repeated electroconvulsive seizures in mice. J Neurosci Res 84(6):1282–1291

    Article  CAS  PubMed  Google Scholar 

  • Dautzenberg FM, Higelin J, Pflieger P, Neidhart W, Guba W (2005) Establishment of robust functional assays for the characterization of neuropeptide Y (NPY) receptors: identification of 3-(5-benzoyl-thiazol-2-ylamino)-benzonitrile as selective NPY type 5 receptor antagonist. Neuropharmacology 48(7):1043–1055

    Article  CAS  PubMed  Google Scholar 

  • Doods H, Gaida W, Wieland HA, Dollinger H, Schnorrenberg G, Esser F, Engel W, Eberlein W, Rudolf K (1999) BIIE0246: a selective and high affinity neuropeptide Y Y(2) receptor antagonist. Eur J Pharmacol 384(2–3):R3–R5

    Article  CAS  PubMed  Google Scholar 

  • Dumont Y, Jacques D, Bouchard P, Quirion R (1998) Species differences in the expression and distribution of the neuropeptide Y Y1, Y2, Y4, and Y5 receptors in rodents, guinea pig, and primates brains. J Comp Neurol 402(3):372–384

    Article  CAS  PubMed  Google Scholar 

  • Dumont Y, Cadieux A, Doods H, Pheng LH, Abounader R, Hamel E, Jacques D, Regoli D, Quirion R (2000) BIIE0246, a potent and highly selective non-peptide neuropeptide Y Y(2) receptor antagonist. Br J Pharmacol 129(6):1075–1088

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Elbrønd-Bek H, Olling JD, Gøtzsche CR, Waterfield A, Wörtwein G, Woldbye DPD (2014) Neuropeptide Y-stimulated [35S]GTPγS functional binding is reduced in the hippocampus after kainate-induced seizure in mice. Synapse 68:427–436

    Article  PubMed  Google Scholar 

  • Falenski KW, Blair RE, Sim-Selley LJ, Martin BR, DeLorenzo RJ (2007) Status epilepticus causes a long-lasting redistribution of hippocampal cannabinoid type 1 receptor expression and function in the rat pilocarpine model of acquired epilepsy. Neuroscience 146(3):1232–1244

  • Gackenheimer SL, Schober DA, Gehlert DR (2001) Characterization of neuropeptide Y Y1-like and Y2-like receptor subtypes in the mouse brain. Peptides 22(3):335–341

    Article  CAS  PubMed  Google Scholar 

  • Gehlert DR, Shaw JL (2007) Increased brain neuropeptide Y1 and Y2 receptor binding in NPY knock-out mice does not result in increased receptor function. Peptides 28(2):241–249

    Article  CAS  PubMed  Google Scholar 

  • Gerald C, Walker MW, Criscione L, Gustafson EL, Batzl-Hartmann C, Smith KE, Vaysse P, Durkin MM, Laz TM, Linemeyer DL, Schaffhauser AO, Whitebread S, Hofbauer KG, Taber RI, Branchek TA, Weinshank RL (1996) A receptor subtype involved in neuropeptide-Y-induced food intake. Nature 382(6587):168–171

    Article  CAS  PubMed  Google Scholar 

  • Gøtzsche CR, Nikitidou L, Sørensen AT, Olesen MV, Sørensen G, Christiansen SHO, Ängehagen M, Woldbye DPD, Kokaia M (2012) Combined gene overexpression of neuropeptide Y and its receptor Y5 in the hippocampus suppresses seizures. Neurobiol Dis 45:288–296

    Article  PubMed  Google Scholar 

  • Harrison C, Traynor JR (2003) The [35S]GTPgammaS binding assay: approaches and applications in pharmacology. Life Sci 74(4):489–508

    Article  CAS  PubMed  Google Scholar 

  • Hong W, Werling L (2001) Lack of effects by sigma ligands on neuropeptide Y-induced G-protein activation in rat hippocampus and cerebellum. Brain Res 901(1–2):208–218

    Article  CAS  PubMed  Google Scholar 

  • Howell OW, Doyle K, Goodman JH, Scharfman HE, Herzog H, Pringle A, Beck-Sickinger AG, Gray WP (2005) Neuropeptide Y stimulates neuronal precursor proliferation in the post-natal and adult dentate gyrus. J Neurochem 93(3):560–570

    Article  CAS  PubMed  Google Scholar 

  • Hungund BL, Vinod KY, Kassir SA, Basavarajappa BS, Yalamanchili R, Cooper TB, Mann JJ, Arango V (2004) Upregulation of CB1 receptors and agonist-stimulated [35S]GTPgammaS binding in the prefrontal cortex of depressed suicide victims. Mol Psychiatry 9(2):184–190

  • Olesen MV, Christiansen SH, Gøtzsche CR, Holst B, Kokaia M, Woldbye DPD (2012a) Y5 neuropeptide Y receptor overexpression in mice neither affects anxiety- and depression-like behaviours nor seizures but confers moderate hyperactivity. Neuropeptides 46:71–79

    Article  CAS  PubMed  Google Scholar 

  • Olesen MV, Christiansen SH, Gøtzsche CR, Nikitidou L, Kokaia M, Woldbye DPD (2012b) Neuropeptide Y Y1 receptor hippocampal overexpression via viral vectors is associated with modest anxiolytic-like and proconvulsant effects in mice. J Neurosci Res 90(2):498–507

    Article  CAS  PubMed  Google Scholar 

  • Olling JD, Ulrichsen J, Christensen DZ, Woldbye DP (2009) Complex plastic changes in the neuropeptide Y system during ethanol intoxication and withdrawal in the rat brain. J Neurosci Res 87(10):2386–2397

    Article  CAS  PubMed  Google Scholar 

  • Paxinos G, Franklin KBJ (2001) The mouse brain in stereotaxic coordinates. Academic Press, San Diego

    Google Scholar 

  • Primus RJ, Yevich E, Gallager DW (1998) In vitro autoradiography of GTPgamma[35S] binding at activated NPY receptor subtypes in adult rat brain. Mol Brain Res 58(1–2):74–82

    Article  CAS  PubMed  Google Scholar 

  • Redrobe JP, Dumont Y, St-Pierre JA, Quirion R (1999) Multiple receptors for neuropeptide Y in the hippocampus: putative roles in seizures and cognition. Brain Res 848(1–2):153–166

    Article  CAS  PubMed  Google Scholar 

  • Shaw JL, Gackenheimer SL, Gehlert DR (2003) Functional autoradiography of neuropeptide Y Y1 and Y2 receptor subtypes in rat brain using agonist stimulated [35S]GTPgammaS binding. J Chem Neuroanat 26(3):179–193

    Article  CAS  PubMed  Google Scholar 

  • Silva AP, Lourenco J, Xapelli S, Ferreira R, Kristiansen H, Woldbye DP, Oliveira CR, Malva JO (2007) Protein kinase C activity blocks neuropeptide Y-mediated inhibition of glutamate release and contributes to excitability of the hippocampus in status epilepticus. FASEB J 21(3):671–681

    Article  CAS  PubMed  Google Scholar 

  • Sim LJ, Selley DE, Childers SR (1995) In vitro autoradiography of receptor-activated G proteins in rat brain by agonist-stimulated guanylyl 5′-[gamma-[35S]thio]-triphosphate binding. Proc Natl Acad Sci USA 92(16):7242–7246

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Sim LJ, Selley DE, Xiao R, Childers SR (1996) Differences in G-protein activation by mu- and delta-opioid, and cannabinoid, receptors in rat striatum. Eur J Pharmacol 307(1):97–105

    Article  CAS  PubMed  Google Scholar 

  • Sim LJ, Selly DE, Childers SR (1997) In vitro autoradiographic localization of receptor-stimulated 35S-GTPgammaS binding in brain. In: Mishra RK, Baker GB, Boulton AA (eds) G-protein methods and protocols. Humana Press, Totawa, pp 1–27

    Chapter  Google Scholar 

  • Sorensen AT, Kanter-Schlifke I, Carli M, Balducci C, Noe F, During MJ, Vezzani A, Kokaia M (2008) NPY gene transfer in the hippocampus attenuates synaptic plasticity and learning. Hippocampus 18(6):564–574

    Article  CAS  PubMed  Google Scholar 

  • Sovago J, Dupuis DS, Gulyas B, Hall H (2001) An overview on functional receptor autoradiography using [35S]GTPgammaS. Brain Res Brain Res Rev 38(1–2):149–164

    Article  CAS  PubMed  Google Scholar 

  • Strange PG (2002a) Mechanisms of inverse agonism at G-protein-coupled receptors. Trends Pharmacol Sci 23(2):89–95

    Article  CAS  PubMed  Google Scholar 

  • Strange PG (2002b) Use of the GTPγS ([35S]GTPγS and Eu-GTPγS) binding assay for analysis of ligand potency and efficacy at G protein-coupled receptors. Br J Pharmacol 161(6):1238–1249

    Article  Google Scholar 

  • Trivedi PG, Yu H, Trumbauer M, Chen H, Van der Ploeg LH, Guan X (2001) Differential regulation of neuropeptide Y receptors in the brains of NPY knock-out mice. Peptides 22(3):395–403

    Article  CAS  PubMed  Google Scholar 

  • Vanderheyden PM, Van Liefde I, De Backer JP, Vauquelin G (1997) Non-competitive binding of the nonpeptide antagonist BIBP3226 to rat forebrain neuropeptide Y1 receptors. Eur J Pharmacol 331(2–3):275–284

    Article  CAS  PubMed  Google Scholar 

  • Vezzani A, Sperk G, Colmers WF (1999) Neuropeptide Y: emerging evidence for a functional role in seizure modulation. Trends Neurosci 22(1):25–30

    Article  CAS  PubMed  Google Scholar 

  • Waeber C, Moskowitz MA (1997) 5-Hydroxytryptamine1A and 5-hydroxytryptamine1B receptors stimulate [35S]guanosine-5'-O-(3-thio)triphosphate binding to rodent brain sections as visualized by in vitro autoradiography. Mol Pharmacol 52(4):623–631

  • Woldbye DP, Kokaia M (2004) Neuropeptide Y and seizures: effects of exogenously applied ligands. Neuropeptides 38(4):253–260

    Article  CAS  PubMed  Google Scholar 

  • Woldbye DP, Nanobashvili A, Sorensen AT, Husum H, Bolwig TG, Sorensen G, Ernfors P, Kokaia M (2005) Differential suppression of seizures via Y2 and Y5 neuropeptide Y receptors. Neurobiol Dis 20(3):760–772

    Article  CAS  PubMed  Google Scholar 

  • Woldbye DP, Ängehagen M, Gotzsche CR, Elbrond-Bek H, Sorensen AT, Christiansen SH, Olesen MV, Nikitidou L, Hansen TV, Kanter-Schlifke I, Kokaia M (2010) Adeno-associated viral vector-induced overexpression of neuropeptide Y Y2 receptors in the hippocampus suppresses seizures. Brain 133(9):2778–2788

    Article  PubMed  Google Scholar 

  • Xapelli S, Agasse F, Ferreira R, Silva AP, Malva JO (2006) Neuropeptide Y as an endogenous antiepileptic, neuroprotective and pro-neurogenic peptide. Recent Pat CNS Drug Discov 1(3):315–324

    Article  CAS  PubMed  Google Scholar 

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Acknowledgments

This research was supported by Grants from the Danish Research Council for Health and Disease [64750], Lundbeck Foundation, Foundation of Doctor Sofus Carl Emil Friis and His Wife Olga Doris Friis, Elsass Foundation, Master Joiner Sophus Jacobsen and His Wife Astrid Jacobsen’s Foundation, Aase and Ejnar Danielsen’s Foundation, Hede Nielsen Family Foundation, Ivan Nielsen Foundation, Danish Research Foundation. The technical assistance of Birgitte H. Hansen is greatly appreciated.

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Authors and Affiliations

  1. Laboratory of Neural Plasticity, Department of Neuroscience and Pharmacology, University of Copenhagen, Symbion, 3 Fruebjergvej, 2100, Copenhagen, Denmark

    Heidi Elbrønd-Bek, Casper R. Gøtzsche & David P. D. Woldbye

  2. Department of Neuroscience and Pharmacology, Laboratory of Neuropsychiatry, Psychiatric Centre Copenhagen University Hospital, University of Copenhagen, Copenhagen, Denmark

    Heidi Elbrønd-Bek, Casper R. Gøtzsche, Mette Skinbjerg, Ditte Z. Christensen, Per Plenge & David P. D. Woldbye

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  1. Heidi Elbrønd-Bek
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  2. Casper R. Gøtzsche
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Correspondence to David P. D. Woldbye.

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Elbrønd-Bek, H., Gøtzsche, C.R., Skinbjerg, M. et al. Visualization of Functional Neuropeptide Y Receptors in the Mouse Hippocampus and Neocortex Using [35S]GTPγS Binding. Int J Pept Res Ther 21, 269–278 (2015). https://doi.org/10.1007/s10989-015-9455-y

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  • DOI: https://doi.org/10.1007/s10989-015-9455-y

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