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NMDA Receptors pp 171–183Cite as

GluN2B Subunit Labeling with Fluorescent Probes and High-Resolution Live Imaging

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Part of the book series: Methods in Molecular Biology ((MIMB,volume 1677))

Abstract

Laser Scanning Confocal Microscopy (LSCM) imaging using an appropriate fluorescent probe enables the visualization of a molecular target with high resolution, and represents a method of choice for studying expression, subcellular location, and trafficking of receptors in living cells. The chemical, physical, and pharmacological properties of the probe remain essential. Here, we describe (1) the preparation of a specific probe for NMDAR GluN2B receptor by conjugation of fluorescein to an ifenprodil-based ligand, (2) an in vitro functional assay by calcium imaging for GluN2B binding and inhibition evaluation of the probe, and (3) the labeling and confocal imaging of GluN2B in DS-red labeled living cortical neurons.

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References

  1. Sanz-Clemente A, Nicoll RA, Roche KW (2013) Diversity in NMDA receptor composition: many regulators, many consequences. Neuroscientist 19:62–75

    Article  CAS  PubMed  Google Scholar 

  2. Traynelis SF, Wollmuth LP, McBain CJ, Menniti FS, Vance KM, Ogden KK, Hansen KB, Yuan H, Myers SJ, Dingledine R (2010) Glutamate receptor ion channels: structure, regulation, and function. Pharmacol Rev 62:405–496

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  3. Laube B, Kuhse J, Betz H (1998) Evidence for a tetrameric structure of recombinant NMDA receptors. J Neurosci 18:2954–2961

    CAS  PubMed  Google Scholar 

  4. Loftis JM, Janowsky A (2003) The N-methyl-D-aspartate receptor subunit NR2B: localization, functional properties, regulation, and clinical implications. Pharmacol Ther 97:55–85

    Article  CAS  PubMed  Google Scholar 

  5. Paoletti P (2011) Molecular basis of NMDA receptor functional diversity. Eur J Neurosci 33:1351–1365

    Article  PubMed  Google Scholar 

  6. Papouin T, Oliet SHR (2014) Organization, control and function of extrasynaptic NMDA receptors. Philos Trans R Soc Lond B Biol Sci 369(1654):20130601

    Article  PubMed  PubMed Central  Google Scholar 

  7. Cull-Candy SG, Leszkiewicz DN (2004) Role of distinct NMDA receptor subtypes at central synapses. Sci STKE 2004(255):re16

    PubMed  Google Scholar 

  8. Hardingham GE, Bading H (2010) Synaptic versus extrasynaptic NMDA receptor signalling: implications for neurodegenerative disorders. Nat Rev Neurosci 11:682–696

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. Parsons MP, Raymond LA (2014) Extrasynaptic NMDA receptor involvement in central nervous system disorders. Neuron 82:279–293

    Article  CAS  PubMed  Google Scholar 

  10. Sousa JB, Frescol P, Diniz C (2014) Imaging receptors with Laser Scanning Confocal Microscopy: qualitative and quantitative analysis. In: Méndes-Vilas A (ed) Microscopy: advances in scientific research and education, Microscopy book series #6, vol 1. Formatex Research Center Publisher, Badajoz, Spain, pp 201–208

    Google Scholar 

  11. McGrath JC, Arribas S, Daly CJ (1996) Fluorescent ligands for the study of receptors. Trends Pharmacol Sci 17:393–399

    Article  CAS  PubMed  Google Scholar 

  12. Masuko T, Kashiwagi K, Kuno T, Nguyen ND, Pahk AJ, Fukuchi J, Igarashi K, Williams K (1999) A regulatory domain (R1-R2) in the amino terminus of the N-methyl-D-aspartate receptor: effects of spermine, protons, and ifenprodil, and structural similarity to bacterial leucine/isoleucine/valine binding protein. Mol Pharmacol 55:957–969

    CAS  PubMed  Google Scholar 

  13. Perin-Dureau F, Rachline J, Neyton J, Paoletti P (2002) Mapping the binding site of the neuroprotectant ifenprodil on NMDA receptors. J Neurosci 22:5955–5965

    CAS  PubMed  Google Scholar 

  14. Mony L, Krzaczkowski L, Leonetti M, Le Goff A, Alarcon K, Neyton J, Bertrand H-O, Acher F, Paoletti P (2009) Structural basis of NR2B-selective antagonist recognition by N-methyl-D-aspartate receptors. Mol Pharmacol 75:60–74

    Article  CAS  PubMed  Google Scholar 

  15. Ng F-M, Geballe MT, Snyder JP, Traynelis SF, Low C-M (2008) Structural insights into phenylethanolamines highaffinity binding site in NR2B from binding and molecular modeling studies. Mol Brain 1:1–11

    Article  Google Scholar 

  16. Reynold IJ, Miller R (1989) Ifenprodil is a novel type of N-methyl-D-aspartate receptor antagonist: interaction with polyamines. Mol Pharmacol 36:758–765

    Google Scholar 

  17. Williams K (2001) Ifenprodil, a novel NMDA receptor antagonist: site and mechanism of action. Curr Drug Targets 2:285–298

    Article  CAS  PubMed  Google Scholar 

  18. Borza I, Domany G (2006) NR2B selective NMDA antagonists: the evolution of the ifenprodil-type pharmacophore. Curr Top Med Chem 6:687–695

    Article  CAS  PubMed  Google Scholar 

  19. Layton ME, Kelly MJ, Rodzinak KJ (2006) Recent advances in the development of NR2B subtype-selective NMDA receptor antagonists. Curr Top Med Chem 6:697–709

    Article  CAS  PubMed  Google Scholar 

  20. MacCauley JA (2006) Amide-containing NR2B/NMDA receptor antagonists. Expert Opin Ther Patents 16:863–870

    Article  Google Scholar 

  21. MacCauley JA (2005) NR2B subtype-selective NMDA receptor antagonists: 2001−2004. Expert Opin Ther Patents 15:389–407

    Article  Google Scholar 

  22. Nikam SS, Meltzer LT (2002) NR2B selective NMDA receptor antagonists. Curr Pharm Des 8:845–855

    Article  CAS  PubMed  Google Scholar 

  23. Rosahl TW, Wingrove PB, Hunt V, Fradley RL, Lawrence JMK, Heavens RP, Treacey P, Usala M, Macauley A, Bonnert TP, Whiting PJ, Wafford KA (2006) A genetically modified mouse model probing the selective action of ifenprodil at the N-methyl-D-aspartate type 2B receptor. Mol Cell Neurosci 33:47–56

    Article  CAS  PubMed  Google Scholar 

  24. Grimwood S, Richards P, Murray F, Harrison N, Wingrove PB, Hutson PH (2000) Characterisation of N-methyl-Daspartate receptor-specific [3H]ifenprodil binding to recombinant human NR1a/NR2B receptors compared with native receptors in rodent brain membranes. J Neurochem 75:2455–2463

    Article  CAS  PubMed  Google Scholar 

  25. Avenet P, Leonardon J, Besnard F, Graham D, Frost J, Depoortere H, Langer SZ, Scatton B (1996) Antagonist properties of the stereoisomers of ifenprodil at NR1A/NR2A and NR1A/NR2B subtypes of the NMDA receptor expressed in Xenopus oocytes. Eur J Pharmacol 296:209–213

    Article  CAS  PubMed  Google Scholar 

  26. Marchand P, Becerril-Ortega J, Mony L, Bouteiller C, Paoletti P, Nicole O, Barré L, Buisson A, Perrio C (2012) Confocal microscopy imaging of NR2B containing NMDA receptor using fluorescent ifenprodil-based conjugates. Bioconjug Chem 23:2126

    Article  Google Scholar 

  27. Dhilly M, Becerril-Ortega J, Colloc’h N, MacKenzie ET, Barré L, Buisson A, Nicole O, Perrio C (2013) Synthesis and in vitro characterization of ifenprodil-based fluorescein conjugates as GluN1/GluN2B N-Methyl-D-Aspartate receptor antagonists. Chembiochem 14:759769

    Article  Google Scholar 

  28. Dingledine R, Borges K, Bowie D, Traynelis SF (1999) The glutamate receptor ion channels. Pharmacol Rev 51:7–61

    CAS  PubMed  Google Scholar 

  29. Sim N, Gottschalk S, Pal R, Delbianco M, Degtyaruk O, Razansky D, Westmeyer GG, Ntziachristos V, Parker D, Mishra A (2015) Wavelength-dependent optoacoustic imaging probes for NMDA receptor visualization. Chem Commun 51:15149–15152

    Article  CAS  Google Scholar 

  30. Léveillé F, El Gaamouch F, Gouix E, Lecocq M, Lobner D, Nicole O, Buisson A (2008) Neuronal viability is controlled by a functional relation between synaptic and extrasynaptic NMDA receptors. FASEB J 22:4258–4271

    Article  PubMed  Google Scholar 

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Acknowledgment

This work was supported by the CNRS, CEA and UNICAEN for financial support.

Author information

Authors and Affiliations

  1. Normandie University, UNICAEN, CEA, CNRS, UMR6301-ISTCT, LDM-TEP, Cyceron, Caen, France

    Cécile Perrio

  2. Université de Bordeaux, CNRS UMR 5293, Institut des Maladies Neurodégénératives, Bordeaux, France

    Olivier Nicole

  3. Grenoble Institut des Neurosciences, Centre Inserm U1216, Equipe Neuropathologies et Dysfonctions Synaptiques, Université Grenoble Alpes, Grenoble, France

    Alain Buisson

Authors
  1. Cécile Perrio
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  2. Olivier Nicole
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  3. Alain Buisson
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Corresponding author

Correspondence to Cécile Perrio .

Editor information

Editors and Affiliations

  1. Mediterranean Institute of Neurobiology (INMED), Aix-Marseille University, INSERM U901, Marseille, France

    Nail Burnashev

  2. Mediterranean Institute of Neurobiology (INMED), Aix-Marseille University, INSERM U901, Marseille, France

    Pierre Szepetowski

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Perrio, C., Nicole, O., Buisson, A. (2017). GluN2B Subunit Labeling with Fluorescent Probes and High-Resolution Live Imaging. In: Burnashev, N., Szepetowski, P. (eds) NMDA Receptors. Methods in Molecular Biology, vol 1677. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-7321-7_9

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  • DOI: https://doi.org/10.1007/978-1-4939-7321-7_9

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  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-7320-0

  • Online ISBN: 978-1-4939-7321-7

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