Skip to main content
SpringerLink
Log in

Live FRET-FLIM Imaging to Study Metabotropic Signaling via the NMDA Receptor

  • Protocol
  • First Online:
New Technologies for Glutamate Interaction

Part of the book series: Neuromethods ((NM,volume 2780))

  • 170 Accesses

Abstract

Until recently, NMDA receptor (NMDAR) functions have been attributed to its ability to conduct calcium ions. However, growing evidence demonstrates that the NMDAR can induce synaptic depression without ion flux, suggesting that it has a metabotropic function. Our results show that glutamate binding or elevated amounts of beta-amyloid can trigger a conformational change in the NMDAR c-terminal domain. We have shown previously that this movement affects interactions between the NMDAR and signaling molecules, which results in synaptic depression. Here, we describe in detail how to monitor conformational movement in the NMDAR and its interactions with associated signaling molecules using FRET-FLIM live imaging in primary hippocampal neurons. A method to selectively block the NMDAR metabotropic function will also be explained. These approaches could be directly used to test the effect of novel NMDAR binding compounds on the NMDAR intracellular conformation or to study signaling proteins implicated in ion-flux-independent synaptic depression. Moreover, one could adapt these procedures to study any kind of protein-protein interaction and its dynamics in living cells.

This is a preview of subscription content, log in via an institution to check access.

Access this chapter

Log in via an institution
Protocol
USD 49.95
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
eBook
USD 189.00
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Hardcover Book
USD 249.99
Price excludes VAT (USA)
  • Durable hardcover edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info

Tax calculation will be finalised at checkout

Purchases are for personal use only

Institutional subscriptions

References

  1. Collingridge G (1987) Synaptic plasticity. The role of NMDA receptors in learning and memory. Nature 330(6149):604–605. https://doi.org/10.1038/330604a0

    Article  CAS  PubMed  Google Scholar 

  2. Riedel G, Platt B, Micheau J (2003) Glutamate receptor function in learning and memory. Behav Brain Res 140(1–2):1–47. https://doi.org/10.1016/s0166-4328(02)00272-3

    Article  CAS  PubMed  Google Scholar 

  3. Dore K, Labrecque S, Tardif C, De Koninck P (2014) FRET-FLIM investigation of PSD95-NMDA receptor interaction in dendritic spines; control by calpain, CaMKII and Src family kinase. PLoS One 9(11):e112170. https://doi.org/10.1371/journal.pone.0112170

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  4. Brocher S, Artola A, Singer W (1992) Intracellular injection of Ca2+ chelators blocks induction of long-term depression in rat visual cortex. Proc Natl Acad Sci U S A 89(1):123–127

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  5. Malenka RC (1994) Synaptic plasticity in the hippocampus: LTP and LTD. Cell Rev 78(4):535–538

    Article  CAS  Google Scholar 

  6. Dore K, Aow J, Malinow R (2015) Agonist binding to the NMDA receptor drives movement of its cytoplasmic domain without ion flow. Proc Natl Acad Sci U S A 112(47):14705–14710. https://doi.org/10.1073/pnas.1520023112

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  7. Dore K, Aow J, Malinow R (2016) The emergence of NMDA receptor metabotropic function: insights from imaging. Front Synap Neurosci 8:20. https://doi.org/10.3389/fnsyn.2016.00020

    Article  CAS  Google Scholar 

  8. Aow J, Dore K, Malinow R (2015) Conformational signaling required for synaptic plasticity by the NMDA receptor complex. Proc Natl Acad Sci U S A 112(47):14711–14716. https://doi.org/10.1073/pnas.1520029112

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. Carter BC, Jahr CE (2016) Postsynaptic, not presynaptic NMDA receptors are required for spike-timing-dependent LTD induction. Nat Neurosci. https://doi.org/10.1038/nn.4343

  10. Ferreira JS et al (2017) Co-agonists differentially tune GluN2B-NMDA receptor trafficking at hippocampal synapses. elife 6. https://doi.org/10.7554/eLife.25492

  11. Nabavi S, Kessels HW, Alfonso S, Aow J, Fox R, Malinow R (2013) Metabotropic NMDA receptor function is required for NMDA receptor-dependent long-term depression. Proc Natl Acad Sci U S A 110(10):4027–4032. https://doi.org/10.1073/pnas.1219454110

    Article  PubMed  PubMed Central  Google Scholar 

  12. Wong JM, Gray JA (2018) Long-term depression is independent of GluN2 subunit composition. J Neurosci 38(19):4462–4470. https://doi.org/10.1523/JNEUROSCI.0394-18.2018

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. Mayford M, Wang J, Kandel ER, O'Dell TJ (1995) CaMKII regulates the frequency-response function of hippocampal synapses for the production of both LTD and LTP. Cell 81(6):891–904

    Article  CAS  PubMed  Google Scholar 

  14. Scanziani M, Malenka RC, Nicoll RA (1996) Role of intercellular interactions in heterosynaptic long-term depression. Nature 380(6573):446–450. https://doi.org/10.1038/380446a0

    Article  CAS  PubMed  Google Scholar 

  15. Dore K, Malinow R (2021) Elevated PSD-95 blocks ion-flux independent LTD: a potential new role for PSD-95 in synaptic plasticity. Neuroscience 456:43–49. https://doi.org/10.1016/j.neuroscience.2020.02.020

    Article  CAS  PubMed  Google Scholar 

  16. Gray NW, Weimer RM, Bureau I, Svoboda K (2006) Rapid redistribution of synaptic PSD-95 in the neocortex in vivo. PLoS Biol 4(11):e370. https://doi.org/10.1371/journal.pbio.0040370

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  17. Zhou Q, Homma KJ, Poo MM (2004) Shrinkage of dendritic spines associated with long-term depression of hippocampal synapses. Neuron 44(5):749–757. https://doi.org/10.1016/j.neuron.2004.11.011

    Article  CAS  PubMed  Google Scholar 

  18. Stein IS, Gray JA, Zito K (2015) Non-ionotropic NMDA receptor signaling drives activity-induced dendritic spine shrinkage. J Neurosci 35(35):12303–12308. https://doi.org/10.1523/JNEUROSCI.4289-14.2015

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. Birnbaum JH, Bali J, Rajendran L, Nitsch RM, Tackenberg C (2015) Calcium flux-independent NMDA receptor activity is required for A beta oligomer-induced synaptic loss. Cell Death Dis 6:ARTN e1791. https://doi.org/10.1038/cddis.2015.160

    Article  CAS  Google Scholar 

  20. Dore K et al (2021) PSD-95 protects synapses from beta-amyloid. Cell Rep 35(9):109194. https://doi.org/10.1016/j.celrep.2021.109194

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. Kessels HW, Nabavi S, Malinow R (2013) Metabotropic NMDA receptor function is required for beta-amyloid-induced synaptic depression. Proc Natl Acad Sci U S A 110(10):4033-8. https://doi.org/10.1073/pnas.1219605110

    Article  PubMed  Google Scholar 

  22. Tamburri A, Dudilot A, Licea S, Bourgeois C, Boehm J (2013) NMDA-receptor activation but not ion flux is required for amyloid-beta induced synaptic depression. PLoS One 8(6):e65350. https://doi.org/10.1371/journal.pone.0065350

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. Weilinger NL et al (2016) Metabotropic NMDA receptor signaling couples Src family kinases to pannexin-1 during excitotoxicity. Nat Neurosci 19(3):432–442. https://doi.org/10.1038/nn.4236

    Article  CAS  PubMed  Google Scholar 

  24. Park DK et al (2022) Reduced d-serine levels drive enhanced non-ionotropic NMDA receptor signaling and destabilization of dendritic spines in a mouse model for studying schizophrenia. Neurobiol Dis 170:105772. https://doi.org/10.1016/j.nbd.2022.105772

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  25. Becker W, Bergmann A, Hink MA, Konig K, Benndorf K, Biskup C (2004) Fluorescence lifetime imaging by time-correlated single-photon counting. Microsc Res Tech 63(1):58–66

    Article  CAS  PubMed  Google Scholar 

  26. Lakowicz JR (2006) Principles of fluorescence spectroscopy, 3rd edn. Springer, p 954

    Book  Google Scholar 

  27. Hille C, Lahn M, Lohmannsroben HG, Dosche C (2009) Two-photon fluorescence lifetime imaging of intracellular chloride in cockroach salivary glands. Photochem Photobiol Sci 8(3):319–327. https://doi.org/10.1039/b813797h

    Article  CAS  PubMed  Google Scholar 

  28. Yasuda R (2006) Imaging spatiotemporal dynamics of neuronal signaling using fluorescence resonance energy transfer and fluorescence lifetime imaging microscopy. Curr Opin Neurobiol 16(5):551–561

    Article  CAS  PubMed  Google Scholar 

  29. Gustiananda M, Liggins JR, Cummins PL, Gready JE (2004) Conformation of prion protein repeat peptides probed by FRET measurements and molecular dynamics simulations. Biophys J 86(4):2467–2483. https://doi.org/10.1016/S0006-3495(04)74303-9

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  30. Piston DW, Kremers GJ (2007) Fluorescent protein FRET: the good, the bad and the ugly. Trends Biochem Sci 32(9):407–414. https://doi.org/10.1016/j.tibs.2007.08.003

    Article  CAS  PubMed  Google Scholar 

  31. Selvin PR (2000) The renaissance of fluorescence resonance energy transfer. Nat Struct Biol, Research Support 7(9):730–734. https://doi.org/10.1038/78948

    Article  CAS  PubMed  Google Scholar 

  32. Sapkota K et al (2019) The NMDA receptor intracellular C-terminal domains reciprocally interact with allosteric modulators. Biochem Pharmacol 159:140–153. https://doi.org/10.1016/j.bcp.2018.11.018

    Article  CAS  PubMed  Google Scholar 

  33. Kornau HC, Schenker LT, Kennedy MB, Seeburg PH (1995) Domain interaction between NMDA receptor subunits and the postsynaptic density protein PSD-95. Science 269(5231):1737–1740

    Article  CAS  PubMed  Google Scholar 

  34. Mulkey RM, Herron CE, Malenka RC (1993) An essential role for protein phosphatases in hippocampal long-term depression. Science 261(5124):1051–1055

    Article  CAS  PubMed  Google Scholar 

  35. Westphal RS et al (1999) Regulation of NMDA receptors by an associated phosphatase-kinase signaling complex. Science 285(5424):93–96

    Article  CAS  PubMed  Google Scholar 

  36. Malenka RC et al (1989) An essential role for postsynaptic calmodulin and protein kinase activity in long-term potentiation. Nature 340(6234):554–557. https://doi.org/10.1038/340554a0

    Article  CAS  PubMed  Google Scholar 

  37. Malinow R, Schulman H, Tsien RW (1989) Inhibition of postsynaptic PKC or CaMKII blocks induction but not expression of LTP. Science 245(4920):862–866

    Article  CAS  PubMed  Google Scholar 

  38. Coultrap SJ et al (2014) Autonomous CaMKII mediates both LTP and LTD using a mechanism for differential substrate site selection. Cell Rep 6(3):431–437. https://doi.org/10.1016/j.celrep.2014.01.005

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  39. Bayer KU, De Koninck P, Leonard AS, Hell JW, Schulman H (2001) Interaction with the NMDA receptor locks CaMKII in an active conformation. Nature 411(6839):801–805. https://doi.org/10.1038/35081080

    Article  CAS  PubMed  Google Scholar 

  40. Lee SJ, Escobedo-Lozoya Y, Szatmari EM, Yasuda R (2009) Activation of CaMKII in single dendritic spines during long-term potentiation. Nature 458(7236):299–304

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  41. Koushik SV, Vogel SS (2008) Energy migration alters the fluorescence lifetime of Cerulean: implications for fluorescence lifetime imaging Forster resonance energy transfer measurements. J Biomed Opt 13(3):031204. https://doi.org/10.1117/1.2940367

    Article  CAS  PubMed  Google Scholar 

  42. Joosen L, Hink MA, Gadella TW Jr, Goedhart J (2014) Effect of fixation procedures on the fluorescence lifetimes of Aequorea victoria derived fluorescent proteins. J Microsc 256(3):166–176. https://doi.org/10.1111/jmi.12168

    Article  CAS  PubMed  Google Scholar 

  43. Tang S, Yasuda R (2017) Imaging ERK and PKA activation in single dendritic spines during structural plasticity. Neuron 93(6):1315–1324 e3. https://doi.org/10.1016/j.neuron.2017.02.032

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  44. Oliveira AF, Yasuda R (2013) An improved Ras sensor for highly sensitive and quantitative FRET-FLIM imaging. PLoS One 8(1):e52874. https://doi.org/10.1371/journal.pone.0052874

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  45. Murakoshi H, Wang H, Yasuda R (2011) Local, persistent activation of Rho GTPases during plasticity of single dendritic spines. Nature 472(7341):100–104. https://doi.org/10.1038/nature09823

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  46. Laviv T et al (2020) In vivo imaging of the coupling between neuronal and CREB activity in the mouse brain. Neuron 105(5):799–812 e5. https://doi.org/10.1016/j.neuron.2019.11.028

    Article  CAS  PubMed  Google Scholar 

  47. Jongbloets BC, Ma L, Mao T, Zhong H (2019) Visualizing protein kinase A activity in head-fixed behaving mice using in vivo two-photon fluorescence lifetime imaging microscopy. J Vis Exp 148. https://doi.org/10.3791/59526

  48. Lodder B, Lee SJ, Sabatini BL (2021) Real-time, in vivo measurement of protein kinase a activity in deep brain structures using Fluorescence Lifetime Photometry (FLiP). Curr Protoc 1(10):e265. https://doi.org/10.1002/cpz1.265

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  49. Takahashi N et al (2015) Two-photon fluorescence lifetime imaging of primed SNARE complexes in presynaptic terminals and beta cells. Nat Commun 6:8531. https://doi.org/10.1038/ncomms9531

    Article  CAS  PubMed  Google Scholar 

  50. Sharda N, Pengo T, Wang Z, Kandimalla KK (2020) Amyloid-beta peptides disrupt interactions between VAMP-2 and SNAP-25 in neuronal cells as determined by FRET/FLIM. J Alzheimers Dis 77(1):423–435. https://doi.org/10.3233/JAD-200065

    Article  CAS  PubMed  Google Scholar 

  51. Marquer C et al (2011) Local cholesterol increase triggers amyloid precursor protein-Bace1 clustering in lipid rafts and rapid endocytosis. FASEB J 25(4):1295–1305. https://doi.org/10.1096/fj.10-168633

    Article  CAS  PubMed  Google Scholar 

  52. Winslow AR et al (2014) Convergence of pathology in dementia with Lewy bodies and Alzheimer's disease: a role for the novel interaction of alpha-synuclein and presenilin 1 in disease. Brain 137(Pt 7):1958–1970. https://doi.org/10.1093/brain/awu119

    Article  PubMed  PubMed Central  Google Scholar 

  53. Zachariassen LG, Katchan L, Jensen AG, Pickering DS, Plested AJ, Kristensen AS (2016) Structural rearrangement of the intracellular domains during AMPA receptor activation. Proc Natl Acad Sci U S A 113(27):E3950–E3959. https://doi.org/10.1073/pnas.1601747113

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  54. Rodriguez-Moreno A, Sihra TS (2007) Metabotropic actions of kainate receptors in the CNS. J Neurochem 103(6):2121–2135. https://doi.org/10.1111/j.1471-4159.2007.04924.x

    Article  CAS  PubMed  Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Department of Neurosciences, Center for Neural Circuits and Behavior, University of California San Diego, La Jolla, CA, USA

    Mehreen Manikkoth & Kim Dore

Authors
  1. Mehreen Manikkoth
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Kim Dore
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Kim Dore .

Editor information

Editors and Affiliations

  1. Science Park of the UPV/EHU, Achucarro Basque Center for Neuroscience, Leioa, Spain

    Maria Kukley

Rights and permissions

Reprints and permissions

Copyright information

© 2024 The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature

About this protocol

Check for updates. Verify currency and authenticity via CrossMark

Cite this protocol

Manikkoth, M., Dore, K. (2024). Live FRET-FLIM Imaging to Study Metabotropic Signaling via the NMDA Receptor. In: Kukley, M. (eds) New Technologies for Glutamate Interaction. Neuromethods, vol 2780. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-3742-5_4

Download citation

  • DOI: https://doi.org/10.1007/978-1-0716-3742-5_4

  • Published:

  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-0716-3741-8

  • Online ISBN: 978-1-0716-3742-5

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics

Search

Navigation