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Imaging Neuronal Signal Transduction Using Multiphoton FRET-FLIM

  • Paul R. Evans
  • Long Yan
  • Ryohei YasudaEmail author
Protocol
Part of the Neuromethods book series (NM, volume 148)

Abstract

Synaptic plasticity, the ability of neurons to modulate the strength of specific inputs, is critical for neural circuits to adapt to experience throughout life. In excitatory pyramidal neurons, plasticity is induced by coincident neuronal activity and glutamate release at tiny postsynaptic protrusions called dendritic spines, which initiate the coordinated activity of hundreds of different proteins located in spines and throughout the neuron at distinct temporal phases. Thus, elucidating the spatiotemporal dynamics of individual signaling proteins is critical to refine our understanding of this process. The complex, polarized morphology of neurons can restrict protein activity to small cellular subcompartments, while other signals can spread over long distances, which poses unique challenges to monitoring protein dynamics. Fluorescence resonance energy transfer (FRET) is a useful photophysical phenomenon to visualize signaling in space and time within live cells by measuring the efficiency of energy transfer between two fluorescent proteins. Using two-photon fluorescence lifetime imaging microscopy (2pFLIM) to assay FRET-based signaling sensors permits chronic, high-resolution measurements of discrete neuronal signaling events, even in dense, light-scattering brain slices. Here, we describe the imaging setup required to perform 2pFLIM and highlight its application to decipher the orchestrated signaling underlying the structural plasticity of dendritic spines.

Keywords

Fluorescence lifetime imaging microscopy (FLIM) Two-photon microscopy Fluorescence resonance energy transfer (FRET) Sensor biology Dendritic spine Neuronal signal transduction Spine structural plasticity Long-term potentiation (LTP) 

Notes

Acknowledgments

We thank Dr. Lesley Colgan, Dr. Tal Laviv, and other members of the Yasuda lab for thoughtful discussion. We also thank Dr. Nathan Hedrick for contributing to the figures.

Conflict of Interest

Dr. Ryohei Yasuda is the founder of Florida Lifetime Imaging LLC., a company that helps people set up FLIM.

References

  1. 1.
    Murakoshi H, Yasuda R (2012) Postsynaptic signaling during plasticity of dendritic spines. Trends Neurosci 35:135–143PubMedPubMedCentralCrossRefGoogle Scholar
  2. 2.
    Nishiyama J, Yasuda R (2015) Biochemical computation for spine structural plasticity. Neuron 87:63–75PubMedPubMedCentralCrossRefGoogle Scholar
  3. 3.
    Tsay D, Yuste R (2004) On the electrical function of dendritic spines. Trends Neurosci 27:77–83PubMedCrossRefPubMedCentralGoogle Scholar
  4. 4.
    Bloodgood BL, Sabatini BL (2005) Neuronal activity regulates diffusion across the neck of dendritic spines. Science 310:866–869PubMedCrossRefPubMedCentralGoogle Scholar
  5. 5.
    Svoboda K, Tank DW, Denk W (1996) Direct measurement of coupling between dendritic spines and shafts. Science 272:716–719PubMedCrossRefPubMedCentralGoogle Scholar
  6. 6.
    Matsuzaki M, Honkura N, Ellis-Davies GCR et al (2004) Structural basis of long-term potentiation in single dendritic spines. Nature 429:761–766PubMedPubMedCentralCrossRefGoogle Scholar
  7. 7.
    Hedrick NG, Harward SC, Hall CE et al (2016) Rho GTPase complementation underlies BDNF-dependent homo- and heterosynaptic plasticity. Nature 538:104–108PubMedPubMedCentralCrossRefGoogle Scholar
  8. 8.
    Harvey CD, Yasuda R, Zhong H et al (2008) The spread of Ras activity triggered by activation of a single dendritic spine. Science 321:136–140PubMedPubMedCentralCrossRefGoogle Scholar
  9. 9.
    Zhai S, Ark ED, Parra-Bueno P et al (2013) Long-distance integration of nuclear ERK signaling triggered by activation of a few dendritic spines. Science 342:1107–1111PubMedPubMedCentralCrossRefGoogle Scholar
  10. 10.
    Impey S, Obrietan K, Wong ST et al (1998) Cross talk between ERK and PKA is required for Ca2+ stimulation of CREB-dependent transcription and ERK nuclear translocation. Neuron 21:869–883PubMedCrossRefPubMedCentralGoogle Scholar
  11. 11.
    Harvey CD, Ehrhardt AG, Cellurale C et al (2008) A genetically encoded fluorescent sensor of ERK activity. Proc Natl Acad Sci U S A 105:19264–19269PubMedPubMedCentralCrossRefGoogle Scholar
  12. 12.
    Suzuki K, Tsunekawa Y, Hernandez-Benitez R et al (2016) In vivo genome editing via CRISPR/Cas9 mediated homology-independent targeted integration. Nature 540:144–149PubMedPubMedCentralCrossRefGoogle Scholar
  13. 13.
    Mikuni T, Nishiyama J, Sun Y et al (2016) High- throughput, high-resolution mapping of protein localization in mammalian brain by in vivo genome editing. Cell 165:1803–1817PubMedPubMedCentralCrossRefGoogle Scholar
  14. 14.
    Miyawaki A (2003) Visualization of the spatial and temporal dynamics of intracellular signaling. Dev Cell 4:295–305PubMedCrossRefPubMedCentralGoogle Scholar
  15. 15.
    Förster T (1948) Zwischenmolekulare Energiewanderung und Fluoreszenz. Ann Phys 437:55–75CrossRefGoogle Scholar
  16. 16.
    Lakowicz JR (1999) Principles of fluorescence spectroscopy. Kluwer Academic/Plenum, New YorkCrossRefGoogle Scholar
  17. 17.
    Ueda Y, Kwok S, Hayashi Y (2013) Application of FRET probes in the analysis of neuronal plasticity. Front Neural Circuits 7:163PubMedPubMedCentralCrossRefGoogle Scholar
  18. 18.
    Takao K, Okamoto K-I, Nakagawa T et al (2005) Visualization of synaptic Ca2+/calmodulin-dependent protein kinase II activity in living neurons. J Neurosci 25:3107–3112Google Scholar
  19. 19.
    Lee S-JR, Escobedo-Lozoya Y, Szatmari EM et al (2009) Activation of CaMKII in single dendritic spines during long-term potentiation. Nature 458:299–304PubMedPubMedCentralCrossRefGoogle Scholar
  20. 20.
    Chang J-Y, Parra-Bueno P, Laviv T et al (2017) CaMKII autophosphorylation is necessary for optimal integration of Ca2+ signals during LTP induction, but not maintenance. Neuron 94:800–808PubMedPubMedCentralCrossRefGoogle Scholar
  21. 21.
    Yasuda R, Harvey CD, Zhong H et al (2006) Supersensitive Ras activation in dendrites and spines revealed by two-photon fluorescence lifetime imaging. Nat Neurosci 9:283–291PubMedCrossRefPubMedCentralGoogle Scholar
  22. 22.
    Oliveira AF, Yasuda R (2013) An improved ras sensor for highly sensitive and quantitative FRET-FLIM imaging. PLoS One 8:e52874PubMedPubMedCentralCrossRefGoogle Scholar
  23. 23.
    Ni Q, Titov D, Zhang J (2006) Analyzing protein kinase dynamics in living cells with FRET reporters. Methods 40:279–286PubMedCrossRefPubMedCentralGoogle Scholar
  24. 24.
    Yasuda R (2006) Imaging spatiotemporal dynamics of neuronal signaling using fluorescence resonance energy transfer and fluorescence lifetime imaging microscopy. Curr Opin Neurobiol 16:551–561PubMedCrossRefPubMedCentralGoogle Scholar
  25. 25.
    Yasuda R (2012) Studying signal transduction in single dendritic spines. Cold Spring Harb Perspect Biol.  https://doi.org/10.1101/cshperspect.a005611
  26. 26.
    Gratton E, Breusegem S, Sutin J et al (2003) Fluorescence lifetime imaging for the two-photon microscope: time-domain and frequency-domain methods. J Biomed Opt 8:381–390PubMedCrossRefPubMedCentralGoogle Scholar
  27. 27.
    Murakoshi H, Lee S-J, Yasuda R (2008) Highly sensitive and quantitative FRET–FLIM imaging in single dendritic spines using improved non-radiative YFP. Brain Cell Biol 36:31–42PubMedPubMedCentralCrossRefGoogle Scholar
  28. 28.
    Tramier M, Gautier I, Piolot T et al (2002) Picosecond-hetero-FRET microscopy to probe protein-protein interactions in live cells. Biophys J 83:3570–3577PubMedPubMedCentralCrossRefGoogle Scholar
  29. 29.
    Peter M, Ameer-Beg SM, Hughes MKY et al (2005) Multiphoton-FLIM quantification of the EGFP-mRFP1 FRET pair for localization of membrane receptor-kinase interactions. Biophys J 88:1224–1237PubMedCrossRefPubMedCentralGoogle Scholar
  30. 30.
    Ganesan S, Ameer-beg SM, Ng TTC et al (2006) A dark yellow fluorescent protein (YFP)-based Resonance Energy-Accepting Chromoprotein (REACh) for Forster resonance energy transfer with GFP. Proc Natl Acad Sci USA 103:4089–4094PubMedCrossRefPubMedCentralGoogle Scholar
  31. 31.
    Laviv T, Kim BB, Chu J et al (2016) Simultaneous dual-color fluorescence lifetime imaging with novel red-shifted fluorescent proteins. Nat Methods 13:989–992PubMedPubMedCentralCrossRefGoogle Scholar
  32. 32.
    Murakoshi H, Wang H, Yasuda R (2011) Local, persistent activation of Rho GTPases during plasticity of single dendritic spines. Nature 472:100–104PubMedPubMedCentralCrossRefGoogle Scholar
  33. 33.
    Harward SC, Hedrick NG, Hall CE et al (2016) Autocrine BDNF–TrkB signalling within a single dendritic spine. Nature 538:99–103PubMedPubMedCentralCrossRefGoogle Scholar
  34. 34.
    Sato M, Ueda Y, Takagi T et al (2003) Production of PtdInsP3 at endomembranes is triggered by receptor endocytosis. Nat Cell Biol 5:1016–1022PubMedCrossRefPubMedCentralGoogle Scholar
  35. 35.
    Ueda Y, Hayashi Y (2013) PIP3 regulates spinule formation in dendritic spines during structural long-term potentiation. J Neurosci 33:11040–11047PubMedPubMedCentralCrossRefGoogle Scholar
  36. 36.
    Tang S, Yasuda R (2017) Imaging ERK and PKA Activation in single dendritic spines during structural plasticity. Neuron 93:1315–1324PubMedPubMedCentralCrossRefGoogle Scholar
  37. 37.
    Chen Y, Saulnier JL, Yellen G et al (2014) A PKA activity sensor for quantitative analysis of endogenous GPCR signaling via 2-photon FRET-FLIM imaging. Front Pharmacol 5:56PubMedPubMedCentralCrossRefGoogle Scholar
  38. 38.
    Bonnot A, Guiot E, Hepp R et al (2014) Single-fluorophore biosensors based on conformation-sensitive GFP variants. FASEB J 28:1375–1385PubMedPubMedCentralCrossRefGoogle Scholar
  39. 39.
    Tillo SE, Xiong W-H, Takahashi M et al (2017) Liberated PKA catalytic subunits associate with the membrane via myristoylation to preferentially phosphorylate membrane substrates. Cell Rep 19:617–629PubMedPubMedCentralCrossRefGoogle Scholar
  40. 40.
    Takahashi N, Sawada W, Noguchi J et al (2015) Two-photon fluorescence lifetime imaging of primed SNARE complexes in presynaptic terminals and β cells. Nat Commun 6:8531PubMedPubMedCentralCrossRefGoogle Scholar
  41. 41.
    Bosch M, Castro J, Saneyoshi T et al (2014) Structural and molecular remodeling of dendritic spine substructures during long-term potentiation. Neuron 82:444–459PubMedPubMedCentralCrossRefGoogle Scholar
  42. 42.
    Mongeon R, Venkatachalam V, Yellen G (2016) Cytosolic NADH-NAD + redox visualized in brain slices by two-photon fluorescence lifetime biosensor imaging. Antioxid Redox Signal 25:553–563PubMedPubMedCentralCrossRefGoogle Scholar
  43. 43.
    Díaz-García CM, Mongeon R, Lahmann C et al (2017) Neuronal stimulation triggers neuronal glycolysis and not lactate uptake. Cell Metab 26:361–374PubMedPubMedCentralCrossRefGoogle Scholar
  44. 44.
    Stoppini L, Buchs PA, Muller D (1991) A simple method for organotypic cultures of nervous tissue. J Neurosci Meth 37:173–182Google Scholar
  45. 45.
    McAllister AK (2000) Biolistic transfection of neurons. Sci STKE 2000:pl1PubMedCrossRefPubMedCentralGoogle Scholar
  46. 46.
    Woods G, Zito K (2008) Preparation of gene gun bullets and biolistic transfection of neurons in slice culture. J Vis Exp (12):675Google Scholar
  47. 47.
    Malenka RC, Kauer JA, Zucker RS et al (1988) Postsynaptic calcium is sufficient for potentiation of hippocampal synaptic transmission. Science 242:81–84CrossRefGoogle Scholar
  48. 48.
    Malenka RC, Nicoll RA (1999) Long-term potentiation–a decade of progress? Science 285:1870–1874PubMedCrossRefPubMedCentralGoogle Scholar
  49. 49.
    Lynch G, Larson J, Kelso S et al (1983) Intracellular injections of EGTA block induction of hippocampal long-term potentiation. Nature 305:719–721PubMedCrossRefPubMedCentralGoogle Scholar
  50. 50.
    Matsuzaki M, Ellis-Davies GC, Nemoto T et al (2001) Dendritic spine geometry is critical for AMPA receptor expression in hippocampal CA1 pyramidal neurons. Nat Neurosci 4:1086–1092PubMedPubMedCentralCrossRefGoogle Scholar
  51. 51.
    Canepari M, Nelson L, Papageorgiou G et al (2001) Photochemical and pharmacological evaluation of 7-nitroindolinyl-and 4-methoxy-7-nitroindolinyl-amino acids as novel, fast caged neurotransmitters. J Neurosci Meth 112:29–42Google Scholar
  52. 52.
    Ellis-Davies GCR, Matsuzaki M, Paukert M et al (2007) 4-Carboxymethoxy-5,7-dinitroindolinyl-Glu: an improved caged glutamate for expeditious ultraviolet and two-photon photolysis in brain slices. J Neurosci 27:6601–6604PubMedPubMedCentralCrossRefGoogle Scholar
  53. 53.
    Fino E, Araya R, Peterka DS et al (2009) RuBi-Glutamate: two-photon and visible-light photoactivation of neurons and dendritic spines. Front Neural Circuits 3:2PubMedPubMedCentralCrossRefGoogle Scholar
  54. 54.
    Patterson MA, Szatmari EM, Yasuda R (2010) AMPA receptors are exocytosed in stimulated spines and adjacent dendrites in a Ras-ERK-dependent manner during long-term potentiation. Proc Natl Acad Sci USA 107:15951–15956PubMedCrossRefPubMedCentralGoogle Scholar
  55. 55.
    Gähwiler BH (1988) Organotypic cultures of neural tissue. Trends Neurosci 11:484–489PubMedCrossRefPubMedCentralGoogle Scholar
  56. 56.
    Hedrick N, Yasuda R (2014) Imaging signaling transduction in single dendritic spines. In: Nägerl U, Triller A (eds) Nanoscale imaging of synapses. Neuromethods, vol 84. Humana Press, New YorkGoogle Scholar
  57. 57.
    Lisman J, Yasuda R, Raghavachari S (2012) Mechanisms of CaMKII action in long-term potentiation. Nat Rev Neurosci 13:169–182PubMedPubMedCentralCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2019

Authors and Affiliations

  1. 1.Max Planck Florida Institute for NeuroscienceJupiterUSA

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