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Receptor-Receptor Interactions in the Central Nervous System pp 71–90Cite as

Electrophysiological Approach to GPCR–RTK Interaction Study in Hippocampus of Adult Rats

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Part of the book series: Neuromethods ((NM,volume 140))

Abstract

The allosteric receptor–receptor interactions in G protein-coupled receptor (GPCR) heteroreceptor complexes provided a new dimension for the integration of signaling at plasma membrane level of neurons in brain. Neuronal plasticity processes underlying brain functions, such as learning and memory, have been proposed to be based on rearrangement of heteroreceptor complexes and protomer interactions in the postsynaptic membrane. Among the different partners for GPCRs to form heteromers, receptor tyrosine kinases (RTKs) represent an intriguing combination due to their possible involvement in brain diseases. Different methodologies are available to study heteroreceptor complexes in brain tissue, but their functional role in modulating neuron electrical activity can be properly evaluated using an electrophysiological approach. Here, we describe patch clamp technique protocol for studying GPCR–RTK interaction in hippocampus CA1 pyramidal neurons of adult rat, paying particular attention to highlight major problems that can occur using this technique and providing useful troubleshooting steps to achieve reliable results.

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References

  1. Lefkowitz RJ (2013) A brief history of G-protein coupled receptors (Nobel Lecture). Angew Chem 52(25):6366–6378. https://doi.org/10.1002/anie.201301924

    Article  CAS  Google Scholar 

  2. Heng BC, Aubel D, Fussenegger M (2013) An overview of the diverse roles of G-protein coupled receptors (GPCRs) in the pathophysiology of various human diseases. Biotechnol Adv 31(8):1676–1694. https://doi.org/10.1016/j.biotechadv.2013.08.017

    Article  PubMed  CAS  Google Scholar 

  3. Katritch V, Cherezov V, Stevens RC (2013) Structure-function of the G protein-coupled receptor superfamily. Annu Rev Pharmacol Toxicol 53:531–556. https://doi.org/10.1146/annurev-pharmtox-032112-135923

    Article  PubMed  CAS  Google Scholar 

  4. Insel PA, Tang CM, Hahntow I, Michel MC (2007) Impact of GPCRs in clinical medicine: monogenic diseases, genetic variants and drug targets. Biochim Biophys Acta 1768(4):994–1005. https://doi.org/10.1016/j.bbamem.2006.09.029

    Article  PubMed  CAS  Google Scholar 

  5. Agnati LF, Fuxe K, Zini I, Lenzi P, Hokfelt T (1980) Aspects on receptor regulation and isoreceptor identification. Med Biol 58(4):182–187

    PubMed  CAS  Google Scholar 

  6. Agnati LF, Fuxe K, Zoli M, Rondanini C, Ogren SO (1982) New vistas on synaptic plasticity: the receptor mosaic hypothesis of the engram. Med Biol 60(4):183–190

    PubMed  CAS  Google Scholar 

  7. Maggio R, Vogel Z, Wess J (1993) Coexpression studies with mutant muscarinic/adrenergic receptors provide evidence for intermolecular “cross-talk” between G-protein-linked receptors. Proc Natl Acad Sci U S A 90(7):3103–3107

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. Guo W, Urizar E, Kralikova M, Mobarec JC, Shi L, Filizola M, Javitch JA (2008) Dopamine D2 receptors form higher order oligomers at physiological expression levels. EMBO J 27(17):2293–2304. https://doi.org/10.1038/emboj.2008.153

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  9. Fuxe K, Borroto-Escuela DO (2016) Heteroreceptor complexes and their allosteric receptor-receptor interactions as a novel biological principle for integration of communication in the CNS: targets for drug development. Neuropsychopharmacology 41(1):380–382. https://doi.org/10.1038/npp.2015.244

    Article  PubMed  CAS  Google Scholar 

  10. Cuppini C, Ambrogini P, Lattanzi D, Ciuffoli S, Cuppini R (2009) FGF2 modulates the voltage-dependent K+ current and changes excitability of rat dentate gyrus granule cells. Neurosci Lett 462(3):203–206. https://doi.org/10.1016/j.neulet.2009.07.029

    Article  PubMed  CAS  Google Scholar 

  11. Flajolet M, Wang Z, Futter M, Shen W, Nuangchamnong N, Bendor J, Wallach I, Nairn AC, Surmeier DJ, Greengard P (2008) FGF acts as a co-transmitter through adenosine A2A receptor to regulate synaptic plasticity. Nat Neurosci 11(12):1402–1409. https://doi.org/10.1038/nn.2216

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  12. Fuxe K, Dahlstrom A, Hoistad M, Marcellino D, Jansson A, Rivera A, Diaz-Cabiale Z, Jacobsen K, Tinner-Staines B, Hagman B, Leo G, Staines W, Guidolin D, Kehr J, Genedani S, Belluardo N, Agnati LF (2007) From the Golgi-Cajal mapping to the transmitter-based characterization of the neuronal networks leading to two modes of brain communication: wiring and volume transmission. Brain Res Rev 55(1):17–54. https://doi.org/10.1016/j.brainresrev.2007.02.009

    Article  PubMed  CAS  Google Scholar 

  13. Borroto-Escuela DO, Romero-Fernandez W, Mudo G, Perez-Alea M, Ciruela F, Tarakanov AO, Narvaez M, Di Liberto V, Agnati LF, Belluardo N, Fuxe K (2012) Fibroblast growth factor receptor 1- 5-hydroxytryptamine 1A heteroreceptor complexes and their enhancement of hippocampal plasticity. Biol Psychiatry 71(1):84–91. https://doi.org/10.1016/j.biopsych.2011.09.012

    Article  PubMed  CAS  Google Scholar 

  14. Borroto-Escuela DO, Brito I, Di Palma M, Jiménez-Beristain A, Narvaez M, Corrales F, Pita-Rodríguez M, Sartini S, Ambrogini P, Lattanzi D, Cuppini R, Agnati LF, Fuxe K (2015) On the role of the balance of GPCR homo/heteroreceptor complexes in the brain. J Adv Neurosci Res 2:36–44

    Article  Google Scholar 

  15. Borroto-Escuela DO, Narvaez M, Perez-Alea M, Tarakanov AO, Jimenez-Beristain A, Mudo G, Agnati LF, Ciruela F, Belluardo N, Fuxe K (2015) Evidence for the existence of FGFR1-5-HT1A heteroreceptor complexes in the midbrain raphe 5-HT system. Biochem Biophys Res Commun 456(1):489–493. https://doi.org/10.1016/j.bbrc.2014.11.112

    Article  PubMed  CAS  Google Scholar 

  16. Borroto-Escuela DO, Tarakanov AO, Fuxe K (2016) FGFR1-5-HT1A heteroreceptor complexes: implications for understanding and treating major depression. Trends Neurosci 39(1):5–15. https://doi.org/10.1016/j.tins.2015.11.003

    Article  PubMed  CAS  Google Scholar 

  17. Di Liberto V, Borroto-Escuela DO, Frinchi M, Verdi V, Fuxe K, Belluardo N, Mudò G (2017) Existence of muscarinic acetylcholine receptor (mAChR) and fibroblast growth factor receptor (FGFR) heteroreceptor complexes and their enhancement of neurite outgrowth in neural hippocampal cultures. Biochim Biophys Acta Gen Subj 1861(2):235–245. https://doi.org/10.1016/j.bbagen.2016.10.026

    Article  CAS  PubMed  Google Scholar 

  18. Hubbard SR (2004) Juxtamembrane autoinhibition in receptor tyrosine kinases. Nat Rev Mol Cell Biol 5(6):464–471. https://doi.org/10.1038/nrm1399

    Article  PubMed  CAS  Google Scholar 

  19. Luttrell LM, Daaka Y, Lefkowitz RJ (1999) Regulation of tyrosine kinase cascades by G-protein-coupled receptors. Curr Opin Cell Biol 11(2):177–183

    Article  CAS  PubMed  Google Scholar 

  20. Borroto-Escuela DO, Carlsson J, Ambrogini P, Narváez M, Wydra K, Tarakanov AO, Li X, Millón C, Ferraro L, Cuppini R, Tanganelli S, Liu F, Filip M, Diaz-Cabiale Z, Fuxe K (2017) Understanding the role of GPCR heteroreceptor complexes in modulating the brain networks in health and disease. Front Cell Neurosci 11:37. https://doi.org/10.3389/fncel.2017.00037

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  21. Fuxe K, Agnati LF, Borroto-Escuela DO (2014) The impact of receptor-receptor interactions in heteroreceptor complexes on brain plasticity. Expert Rev Neurother 14(7):719–721. https://doi.org/10.1586/14737175.2014.922878

    Article  PubMed  CAS  Google Scholar 

  22. Fuxe K, Borroto-Escuela DO, Ciruela F, Guidolin D, Agnati LF (2014) Receptor-receptor interactions in heteroreceptor complexes: a new principle in biology. Focus on their role in learning and memory. Neurosci Discov 2:6

    Article  Google Scholar 

  23. Borroto-Escuela DO, Pérez-Alea M, Narvaez M, Tarakanov AO, Mudó G, Jiménez-Beristain A, Agnati LF, Ciruela F, Belluardo N, Fuxe K (2015) Enhancement of the FGFR1 signaling in the FGFR1-5-HT1A heteroreceptor complex in midbrain raphe 5-HT neuron systems. Relevance for neuroplasticity and depression. Biochem Biophys Res Commun 463(3):180–186. https://doi.org/10.1016/j.bbrc.2015.04.133

    Article  PubMed  CAS  Google Scholar 

  24. Hansen JL, Hansen JT, Speerschneider T, Lyngso C, Erikstrup N, Burstein ES, Weiner DM, Walther T, Makita N, Iiri T, Merten N, Kostenis E, Sheikh SP (2009) Lack of evidence for AT1R/B2R heterodimerization in COS-7, HEK293, and NIH3T3 cells: how common is the AT1R/B2R heterodimer? J Biol Chem 284(3):1831–1839. https://doi.org/10.1074/jbc.M804607200

    Article  PubMed  CAS  Google Scholar 

  25. Frederick AL, Yano H, Trifilieff P, Vishwasrao HD, Biezonski D, Meszaros J, Urizar E, Sibley DR, Kellendonk C, Sonntag KC, Graham DL, Colbran RJ, Stanwood GD, Javitch JA (2015) Evidence against dopamine D1/D2 receptor heteromers. Mol Psychiatry 20(11):1373–1385. https://doi.org/10.1038/mp.2014.166

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  26. Gomes I, Ayoub MA, Fujita W, Jaeger WC, Pfleger KD, Devi LA (2016) G protein-coupled receptor heteromers. Annu Rev Pharmacol Toxicol 56:403–425. https://doi.org/10.1146/annurev-pharmtox-011613-135952

    Article  PubMed  CAS  Google Scholar 

  27. Liu XY, Chu XP, Mao LM, Wang M, Lan HX, Li MH, Zhang GC, Parelkar NK, Fibuch EE, Haines M, Neve KA, Liu F, Xiong ZG, Wang JQ (2006) Modulation of D2R-NR2B interactions in response to cocaine. Neuron 52(5):897–909. https://doi.org/10.1016/j.neuron.2006.10.011

    Article  PubMed  CAS  Google Scholar 

  28. Weiss M, Blier P, de Montigny C (2007) Effect of long-term administration of the antidepressant drug milnacipran on serotonergic and noradrenergic neurotransmission in the rat hippocampus. Life Sci 81(2):166–176. https://doi.org/10.1016/j.lfs.2007.04.039

    Article  PubMed  CAS  Google Scholar 

  29. Coetzee WA, Amarillo Y, Chiu J, Chow A, Lau D, McCormack T, Moreno H, Nadal MS, Ozaita A, Pountney D, Saganich M, Vega-Saenz de Miera E, Rudy B (1999) Molecular diversity of K+ channels. Ann N Y Acad Sci 868:233–285

    Article  CAS  PubMed  Google Scholar 

  30. Nicoll RA (1988) The coupling of neurotransmitter receptors to ion channels in the brain. Science 241(4865):545–551

    Article  CAS  PubMed  Google Scholar 

  31. Montalbano A, Corradetti R, Mlinar B (2015) Pharmacological characterization of 5-HT1A autoreceptor-coupled GIRK channels in rat dorsal raphe 5-HT neurons. PLoS One 10(10):e0140369. https://doi.org/10.1371/journal.pone.0140369

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  32. Borin M, Fogli Iseppe A, Pignatelli A, Belluzzi O (2014) Inward rectifier potassium (Kir) current in dopaminergic periglomerular neurons of the mouse olfactory bulb. Front Cell Neurosci 8:223. https://doi.org/10.3389/fncel.2014.00223

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  33. Luscher C, Jan LY, Stoffel M, Malenka RC, Nicoll RA (1997) G protein-coupled inwardly rectifying K+ channels (GIRKs) mediate postsynaptic but not presynaptic transmitter actions in hippocampal neurons. Neuron 19(3):687–695

    Article  CAS  PubMed  Google Scholar 

  34. Lujan R, Maylie J, Adelman JP (2009) New sites of action for GIRK and SK channels. Nat Rev Neurosci 10(7):475–480. https://doi.org/10.1038/nrn2668

    Article  PubMed  CAS  Google Scholar 

  35. Fernandez-Alacid L, Watanabe M, Molnar E, Wickman K, Lujan R (2011) Developmental regulation of G protein-gated inwardly-rectifying K+ (GIRK/Kir3) channel subunits in the brain. Eur J Neurosci 34(11):1724–1736. https://doi.org/10.1111/j.1460-9568.2011.07886.x

    Article  PubMed  PubMed Central  Google Scholar 

  36. Ambrogini P, Lattanzi D, Ciuffoli S, Agostini D, Bertini L, Stocchi V, Santi S, Cuppini R (2004) Morpho-functional characterization of neuronal cells at different stages of maturation in granule cell layer of adult rat dentate gyrus. Brain Res 1017(1-2):21–31. https://doi.org/10.1016/j.brainres.2004.05.039

    Article  PubMed  CAS  Google Scholar 

  37. Ambrogini P, Minelli A, Lattanzi D, Ciuffoli S, Fanelli M, Cuppini R (2006) Synaptically-silent immature neurons show gaba and glutamate receptor-mediated currents in adult rat dentate gyrus. Arch Ital Biol 144(2):115–126

    PubMed  CAS  Google Scholar 

  38. Ambrogini P, Cuppini R, Lattanzi D, Ciuffoli S, Frontini A, Fanelli M (2010) Synaptogenesis in adult-generated hippocampal granule cells is affected by behavioral experiences. Hippocampus 20(7):799–810. https://doi.org/10.1002/hipo.20679

    Article  PubMed  CAS  Google Scholar 

  39. Ambrogini P, Lattanzi D, Ciuffoli S, Betti M, Fanelli M, Cuppini R (2013) Physical exercise and environment exploration affect synaptogenesis in adult-generated neurons in the rat dentate gyrus: possible role of BDNF. Brain Res 1534:1–12. https://doi.org/10.1016/j.brainres.2013.08.023

    Article  PubMed  CAS  Google Scholar 

  40. Betti M, Ambrogini P, Minelli A, Floridi A, Lattanzi D, Ciuffoli S, Bucherelli C, Prospero E, Frontini A, Santarelli L, Baldi E, Benetti F, Galli F, Cuppini R (2011) Maternal dietary loads of alpha-tocopherol depress protein kinase C signaling and synaptic plasticity in rat postnatal developing hippocampus and promote permanent deficits in adult offspring. J Nutr Biochem 22(1):60–70. https://doi.org/10.1016/j.jnutbio.2009.11.014

    Article  PubMed  CAS  Google Scholar 

  41. Sartini S, Lattanzi D, Ambrogini P, Di Palma M, Galati C, Savelli D, Polidori E, Calcabrini C, Rocchi MB, Sestili P, Cuppini R (2016) Maternal creatine supplementation affects the morpho-functional development of hippocampal neurons in rat offspring. Neuroscience 312:120–129. https://doi.org/10.1016/j.neuroscience.2015.11.017

    Article  PubMed  CAS  Google Scholar 

  42. Ting JT, Daigle TL, Chen Q, Feng G (2014) Acute brain slice methods for adult and aging animals: application of targeted patch clamp analysis and optogenetics. Methods Mol Biol 1183:221–242. https://doi.org/10.1007/978-1-4939-1096-0_14

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  43. Traystman RJ, Kirsch JR, Koehler RC (1991) Oxygen radical mechanisms of brain injury following ischemia and reperfusion. J Appl Physiol 71(4):1185–1195

    Article  CAS  PubMed  Google Scholar 

  44. Siesjo BK, Agardh CD, Bengtsson F (1989) Free radicals and brain damage. Cerebrovasc Brain Metab Rev 1(3):165–211

    PubMed  CAS  Google Scholar 

  45. Desagher S, Cordier J, Glowinski J, Tencé M (1997) Endothelin stimulates phospholipase D in striatal astrocytes. J Neurochem 68(1):78–87. https://doi.org/10.1046/j.1471-4159.1997.68010078.x

    Article  PubMed  CAS  Google Scholar 

  46. Brahma B, Forman RE, Stewart EE, Nicholson C, Rice ME (2000) Ascorbate inhibits edema in brain slices. J Neurochem 74(3):1263–1270

    Article  CAS  PubMed  Google Scholar 

  47. Neher E (1992) Correction for liquid junction potentials in patch clamp experiments. Methods Enzymol 207:123–131

    Article  CAS  PubMed  Google Scholar 

  48. Jeong HJ, Han SH, Min BI, Cho YW (2001) 5-HT1A receptor-mediated activation of G-protein-gated inwardly rectifying K+ current in rat periaqueductal gray neurons. Neuropharmacology 41(2):175–185

    Article  CAS  PubMed  Google Scholar 

  49. Luscher C, Slesinger PA (2010) Emerging roles for G protein-gated inwardly rectifying potassium (GIRK) channels in health and disease. Nat Rev Neurosci 11(5):301–315. https://doi.org/10.1038/nrn2834

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  50. Signorini S, Liao YJ, Duncan SA, Jan LY, Stoffel M (1997) Normal cerebellar development but susceptibility to seizures in mice lacking G protein-coupled, inwardly rectifying K+ channel GIRK2. Proc Natl Acad Sci U S A 94(3):923–927

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  51. Sago H, Carlson EJ, Smith DJ, Kilbridge J, Rubin EM, Mobley WC, Epstein CJ, Huang TT (1998) Ts1Cje, a partial trisomy 16 mouse model for Down syndrome, exhibits learning and behavioral abnormalities. Proc Natl Acad Sci U S A 95(11):6256–6261

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  52. Harkins AB, Fox AP (2002) Cell death in weaver mouse cerebellum. Cerebellum 1(3):201–206. https://doi.org/10.1080/14734220260418420

    Article  PubMed  Google Scholar 

  53. Llamosas N, Bruzos-Cidon C, Rodriguez JJ, Ugedo L, Torrecilla M (2015) Deletion of GIRK2 subunit of GIRK channels alters the 5-HT1A receptor-mediated signaling and results in a depression-resistant behavior. Int J Neuropsychopharmacol 18(11):pyv051. https://doi.org/10.1093/ijnp/pyv051

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  54. Borroto-Escuela DO, Narvaez M, Marcellino D, Parrado C, Narvaez JA, Tarakanov AO, Agnati LF, Diaz-Cabiale Z, Fuxe K (2010) Galanin receptor-1 modulates 5-hydroxtryptamine-1A signaling via heterodimerization. Biochem Biophys Res Commun 393(4):767–772. https://doi.org/10.1016/j.bbrc.2010.02.078

    Article  PubMed  CAS  Google Scholar 

  55. Tena-Campos M, Ramon E, Borroto-Escuela DO, Fuxe K, Garriga P (2015) The zinc binding receptor GPR39 interacts with 5-HT1A and GalR1 to form dynamic heteroreceptor complexes with signaling diversity. Biochim Biophys Acta 1852(12):2585–2592. https://doi.org/10.1016/j.bbadis.2015.09.003

    Article  PubMed  CAS  Google Scholar 

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Author notes

  1. David Savelli and Michael Di Palma contributed equally.

Authors and Affiliations

  1. Department of Biomolecular Sciences, Section of Physiology, University of Urbino Carlo Bo, Urbino, Italy

    Davide Lattanzi, David Savelli, Michael Di Palma, Stefano Sartini, Silvia Eusebi, Dasiel O. Borroto-Escuela, Riccardo Cuppini, Kjell Fuxe & Patrizia Ambrogini

  2. Department of Neuroscience, Karolinska Institutet, Stockholm, Sweden

    Dasiel O. Borroto-Escuela

  3. Department of Neuroscience, Karolinska Institutet, Stockholm, Sweden

    Kjell Fuxe

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  1. Davide Lattanzi
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  2. David Savelli
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  4. Stefano Sartini
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  9. Patrizia Ambrogini
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Correspondence to Patrizia Ambrogini .

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Editors and Affiliations

  1. Department of Neuroscience, Karolinska Institutet, Stockholm, Sweden

    KJELL FUXE

  2. Department of Neuroscience, Karolinska Institutet, Stockholm, Sweden

    Dasiel O. Borroto-Escuela

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Lattanzi, D. et al. (2018). Electrophysiological Approach to GPCR–RTK Interaction Study in Hippocampus of Adult Rats. In: FUXE, K., Borroto-Escuela, D. (eds) Receptor-Receptor Interactions in the Central Nervous System. Neuromethods, vol 140. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-8576-0_6

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  • DOI: https://doi.org/10.1007/978-1-4939-8576-0_6

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