Skip to main content
SpringerLink
Log in
Book cover

Single Molecule Microscopy in Neurobiology pp 81–114Cite as

Single-Molecule Imaging of Recycling Synaptic Vesicles in Live Neurons

  • Protocol
  • First Online:

Part of the book series: Neuromethods ((NM,volume 154))

Abstract

The capacity of neurons to communicate and store information in the brain critically depends on neurotransmission, a process which relies on the release of chemicals called neurotransmitters stored in synaptic vesicles at the presynaptic nerve terminals. Following their fusion with the presynaptic plasma membrane, synaptic vesicles are rapidly reformed via compensatory endocytosis. The investigation of the endocytic pathway dynamics is severely restricted by the diffraction limit of light and, therefore, the recycling of synaptic vesicles, which are roughly 45 nm in diameter, has been primarily studied with electrophysiology, low-resolution fluorescence-based techniques, and electron microscopy. Here, we describe a recently developed technique we named subdiffractional tracking of internalized molecules (sdTIM) that can be used to track and study the mobility of recycling synaptic vesicles in live hippocampal presynapses. The chapter provides detailed guidelines on the application of the sdTIM protocol and highlights controls, adaptations, and limitations of the technique.

This is a preview of subscription content, 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   89.00
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD   119.99
Price excludes VAT (USA)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info
Hardcover Book
USD   169.99
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

Learn about institutional subscriptions

Springer Nature is developing a new tool to find and evaluate Protocols. Learn more

References

  1. Denker A et al (2011) A small pool of vesicles maintains synaptic activity in vivo. Proc Natl Acad Sci U S A 108(41):17177–17182

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  2. Wang T et al (2016) Flux of signalling endosomes undergoing axonal retrograde transport is encoded by presynaptic activity and TrkB. Nat Commun 7:12976

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  3. Wang T et al (2015) Control of autophagosome axonal retrograde flux by presynaptic activity unveiled using botulinum neurotoxin type a. J Neurosci 35(15):6179–6194

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  4. Fowler MW, Staras K (2015) Synaptic vesicle pools: principles, properties and limitations. Exp Cell Res 335(2):150–156

    Article  CAS  PubMed  Google Scholar 

  5. Denker A, Rizzoli SO (2010) Synaptic vesicle pools: an update. Front Synaptic Neurosci 2:135

    PubMed  PubMed Central  Google Scholar 

  6. Rizzoli SO, Betz WJ (2005) Synaptic vesicle pools. Nat Rev Neurosci 6(1):57–69

    Article  CAS  PubMed  Google Scholar 

  7. Chamberland S, Toth K (2016) Functionally heterogeneous synaptic vesicle pools support diverse synaptic signalling. J Physiol 594(4):825–835

    Article  CAS  PubMed  Google Scholar 

  8. Alabi AA, Tsien RW (2012) Synaptic vesicle pools and dynamics. Cold Spring Harb Perspect Biol 4(8):a013680

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  9. Crawford DC, Kavalali ET (2015) Molecular underpinnings of synaptic vesicle pool heterogeneity. Traffic 16(4):338–364

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Kamin D et al (2010) High- and low-mobility stages in the synaptic vesicle cycle. Biophys J 99(2):675–684

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. Gimber N et al (2015) Diffusional spread and confinement of newly exocytosed synaptic vesicle proteins. Nat Commun 6:8392

    Article  CAS  PubMed  Google Scholar 

  12. Hua Y et al (2011) A readily retrievable pool of synaptic vesicles. Nat Neurosci 14(7):833–839

    Article  CAS  PubMed  Google Scholar 

  13. Willig KI et al (2006) STED microscopy reveals that synaptotagmin remains clustered after synaptic vesicle exocytosis. Nature 440(7086):935–939

    Article  CAS  PubMed  Google Scholar 

  14. Hu Y, Qu L, Schikorski T (2008) Mean synaptic vesicle size varies among individual excitatory hippocampal synapses. Synapse 62(12):953–957

    Article  CAS  PubMed  Google Scholar 

  15. Lemke EA, Klingauf J (2005) Single synaptic vesicle tracking in individual hippocampal boutons at rest and during synaptic activity. J Neurosci 25(47):11034–11044

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  16. Westphal V et al (2008) Video-rate far-field optical nanoscopy dissects synaptic vesicle movement. Science 320(5873):246–249

    Article  CAS  PubMed  Google Scholar 

  17. Hoopmann P et al (2010) Endosomal sorting of readily releasable synaptic vesicles. Proc Natl Acad Sci U S A 107(44):19055–19060

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. Lehmann M et al (2015) Multicolor caged dSTORM resolves the ultrastructure of synaptic vesicles in the brain. Angew Chem Int Ed Engl 54(45):13230–13235

    Article  CAS  PubMed  Google Scholar 

  19. Maschi D, Klyachko VA (2017) Spatiotemporal regulation of synaptic vesicle fusion sites in central synapses. Neuron 94(1):65–73.e3

    Article  CAS  PubMed  Google Scholar 

  20. Peng A et al (2012) Differential motion dynamics of synaptic vesicles undergoing spontaneous and activity-evoked endocytosis. Neuron 73(6):1108–1115

    Article  CAS  PubMed  Google Scholar 

  21. Joensuu M et al (2016) Subdiffractional tracking of internalized molecules reveals heterogeneous motion states of synaptic vesicles. J Cell Biol 215(2):277–292

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. Joensuu M et al (2017) Visualizing endocytic recycling and trafficking in live neurons by subdiffractional tracking of internalized molecules. Nat Protoc 12(12):2590–2622

    Article  CAS  PubMed  Google Scholar 

  23. Miesenbock G, De Angelis DA, Rothman JE (1998) Visualizing secretion and synaptic transmission with pH-sensitive green fluorescent proteins. Nature 394(6689):192–195

    Article  CAS  PubMed  Google Scholar 

  24. Royle SJ et al (2008) Imaging phluorin-based probes at hippocampal synapses. Methods Mol Biol 457:293–303

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  25. Villarreal S, Lee SH, Wu LG (2017) Measuring synaptic vesicle endocytosis in cultured hippocampal neurons. J Vis Exp (127)

    Google Scholar 

  26. Fiolka R (2016) Clearer view for TIRF and oblique illumination microscopy. Opt Express 24(26):29556–29567

    Article  CAS  PubMed  Google Scholar 

  27. Giannone G et al (2010) Dynamic superresolution imaging of endogenous proteins on living cells at ultra-high density. Biophys J 99(4):1303–1310

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  28. Rothbauer U et al (2006) Targeting and tracing antigens in live cells with fluorescent nanobodies. Nat Methods 3(11):887–889

    Article  CAS  PubMed  Google Scholar 

  29. Harper CB et al (2016) Botulinum neurotoxin type-A enters a non-recycling pool of synaptic vesicles. Sci Rep 6:19654

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  30. Nair D et al (2013) Super-resolution imaging reveals that AMPA receptors inside synapses are dynamically organized in nanodomains regulated by PSD95. J Neurosci 33(32):13204–13224

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  31. Giannone G et al (2013) High-content super-resolution imaging of live cell by uPAINT. Methods Mol Biol 950:95–110

    Article  CAS  PubMed  Google Scholar 

  32. Kechkar A et al (2013) Real-time analysis and visualization for single-molecule based super-resolution microscopy. PLoS One 8(4):e62918

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  33. Tinevez JY et al (2017) TrackMate: an open and extensible platform for single-particle tracking. Methods 115:80–90

    Article  CAS  PubMed  Google Scholar 

  34. Kubala MH et al (2010) Structural and thermodynamic analysis of the GFP:GFP-nanobody complex. Protein Sci 19(12):2389–2401

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  35. Durisic N et al (2014) Single-molecule evaluation of fluorescent protein photoactivation efficiency using an in vivo nanotemplate. Nat Methods 11(2):156–162

    Article  CAS  PubMed  Google Scholar 

  36. Craig AM, Banker G (1994) Neuronal polarity. Annu Rev Neurosci 17:267–310

    Article  CAS  PubMed  Google Scholar 

  37. Kaech S, Banker G (2006) Culturing hippocampal neurons. Nat Protoc 1(5):2406–2415

    Article  CAS  PubMed  Google Scholar 

  38. Chenouard N et al (2014) Objective comparison of particle tracking methods. Nat Methods 11(3):281–289

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  39. Izeddin I et al (2012) Wavelet analysis for single molecule localization microscopy. Opt Express 20(3):2081–2095

    Article  CAS  PubMed  Google Scholar 

  40. Racine V et al. (2006) Multiple-target tracking of 3D fluorescent objects based on simulated annealing. In: 2006 3rd IEEE international symposium on biomedical imaging: macro to nano, vol 1–3, pp 1020–1023

    Google Scholar 

  41. Kerr RA et al (2008) Fast Monte Carlo simulation methods for biological reaction-diffusion systems in solution and on surfaces. SIAM J Sci Comput 30(6):3126

    Article  PubMed  PubMed Central  Google Scholar 

  42. Wilhelm BG et al (2014) Composition of isolated synaptic boutons reveals the amounts of vesicle trafficking proteins. Science 344(6187):1023–1028

    Article  CAS  PubMed  Google Scholar 

  43. Mutch SA et al (2011) Protein quantification at the single vesicle level reveals that a subset of synaptic vesicle proteins are trafficked with high precision. J Neurosci 31(4):1461–1470

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  44. Takamori S et al (2006) Molecular anatomy of a trafficking organelle. Cell 127(4):831–846

    Article  CAS  PubMed  Google Scholar 

  45. Meunier FA et al (2010) Sustained synaptic-vesicle recycling by bulk endocytosis contributes to the maintenance of high-rate neurotransmitter release stimulated by glycerotoxin. J Cell Sci 123(Pt 7):1131–1140

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  46. Nwabuisi-Heath E, LaDu MJ, Yu C (2012) Simultaneous analysis of dendritic spine density, morphology and excitatory glutamate receptors during neuron maturation in vitro by quantitative immunocytochemistry. J Neurosci Methods 207(2):137–147

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  47. Grillo FW et al (2013) Increased axonal bouton dynamics in the aging mouse cortex. Proc Natl Acad Sci U S A 110(16):E1514–E1523

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  48. Truckenbrodt S, Rizzoli SO (2015) Synaptic vesicle pools: classical and emerging roles. In: Mochida S (ed) Presynaptic terminals. Springer, Tokyo

    Google Scholar 

  49. Iwabuchi S et al (2014) Examination of synaptic vesicle recycling using FM dyes during evoked, spontaneous, and miniature synaptic activities. J Vis Exp (85)

    Google Scholar 

  50. Raingo J et al (2012) VAMP4 directs synaptic vesicles to a pool that selectively maintains asynchronous neurotransmission. Nat Neurosci 15(5):738–745

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  51. Hua Z et al (2011) v-SNARE composition distinguishes synaptic vesicle pools. Neuron 71(3):474–487

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  52. Pan PY, Marrs J, Ryan TA (2015) Vesicular glutamate transporter 1 orchestrates recruitment of other synaptic vesicle cargo proteins during synaptic vesicle recycling. J Biol Chem 290(37):22593–22601

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  53. Kwon SE, Chapman ER (2011) Synaptophysin regulates the kinetics of synaptic vesicle endocytosis in central neurons. Neuron 70(5):847–854

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  54. Granseth B et al (2006) Clathrin-mediated endocytosis is the dominant mechanism of vesicle retrieval at hippocampal synapses. Neuron 51(6):773–786

    Article  CAS  PubMed  Google Scholar 

  55. Voglmaier SM et al (2006) Distinct endocytic pathways control the rate and extent of synaptic vesicle protein recycling. Neuron 51(1):71–84

    Article  CAS  PubMed  Google Scholar 

  56. Ramirez DM et al (2012) Vti1a identifies a vesicle pool that preferentially recycles at rest and maintains spontaneous neurotransmission. Neuron 73(1):121–134

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  57. Harper CB et al (2011) Dynamin inhibition blocks botulinum neurotoxin type A endocytosis in neurons and delays botulism. J Biol Chem 286(41):35966–35976

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  58. Pelkmans L et al (2004) Caveolin-stabilized membrane domains as multifunctional transport and sorting devices in endocytic membrane traffic. Cell 118(6):767–780

    Article  CAS  PubMed  Google Scholar 

  59. Haas BL et al (2015) Single-molecule tracking in live Vibrio cholerae reveals that ToxR recruits the membrane-bound virulence regulator TcpP to the toxT promoter. Mol Microbiol 96(1):4–13

    Article  CAS  PubMed  Google Scholar 

  60. Jiang M, Chen G (2006) High Ca2+-phosphate transfection efficiency in low-density neuronal cultures. Nat Protoc 1(2):695–700

    Article  CAS  PubMed  Google Scholar 

  61. Zeitelhofer M et al (2007) High-efficiency transfection of mammalian neurons via nucleofection. Nat Protoc 2(7):1692–1704

    Article  CAS  PubMed  Google Scholar 

  62. Belevich I et al (2016) Microscopy image browser: a platform for segmentation and analysis of multidimensional datasets. PLoS Biol 14(1):e1002340

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  63. Sudhof TC, Rothman JE (2009) Membrane fusion: grappling with SNARE and SM proteins. Science 323(5913):474–477

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  64. Chanaday NL, Kavalali ET (2018) Presynaptic origins of distinct modes of neurotransmitter release. Curr Opin Neurobiol 51:119–126

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  65. Maritzen T, Haucke V (2018) Coupling of exocytosis and endocytosis at the presynaptic active zone. Neurosci Res 127:45–52

    Article  CAS  PubMed  Google Scholar 

  66. Heller JP, Rusakov DA (2017) The nanoworld of the tripartite synapse: insights from super-resolution microscopy. Front Cell Neurosci 11:374

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  67. Monnier N et al (2015) Inferring transient particle transport dynamics in live cells. Nat Methods 12(9):838–840

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  68. Persson F et al (2013) Extracting intracellular diffusive states and transition rates from single-molecule tracking data. Nat Methods 10(3):265–269

    Article  PubMed  Google Scholar 

Download references

Acknowledgments

The super-resolution imaging was carried out at the Queensland Brain Institute’s (QBI) Advanced Microimaging and Analysis Facility. We thank all the authors of the original studies for their contribution [21, 22], and our collaborators for helpful discussions, and we would like to further extend our gratitude to N. Valmas for the schematic illustrations presented here, R. Amor for technical support on imaging, I. Morrow for support on EM, and R. Tweedale (QBI) for critical appraisal of the chapter. This work was supported by an Australian Research Council Discovery Project grant (DP150100539), an Australian Research Council Linkage Infrastructure, Equipment, and Facilities grant (LE130100078), and a National Health and Medical Research Council (NHMRC) grant (1120381) to F.A.M. M.J. is supported by an Academy of Finland Postdoctoral Research Fellowship (298124). F.A.M. is a NHMRC Senior Research Fellow (1060075).

Author information

Authors and Affiliations

  1. Clem Jones Centre for Ageing Dementia Research, Queensland Brain Institute, The University of Queensland, Brisbane, QLD, Australia

    Merja Joensuu, Ramon Martínez-Mármol, Mahdie Mollazade, Pranesh Padmanabhan & Frédéric A. Meunier

  2. Minerva Foundation Institute for Medical Research, Helsinki, Finland

    Merja Joensuu

Authors
  1. Merja Joensuu
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Ramon Martínez-Mármol
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Mahdie Mollazade
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Pranesh Padmanabhan
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Frédéric A. Meunier
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding authors

Correspondence to Merja Joensuu or Frédéric A. Meunier .

Editor information

Editors and Affiliations

  1. Laboratory of Cellular and Molecular Neurobiology, Graduate School of Frontier Biosciences, Osaka University, Suita, Osaka, Japan

    Nobuhiko Yamamoto

  2. Laboratory for Cell Polarity Regulation, RIKEN Center for Biosystems Dynamics Research, Suita, Osaka, Japan

    Yasushi Okada

Rights and permissions

Reprints and permissions

Copyright information

© 2020 Springer Science+Business Media, LLC, part of Springer Nature

About this protocol

Check for updates. Verify currency and authenticity via CrossMark

Cite this protocol

Joensuu, M., Martínez-Mármol, R., Mollazade, M., Padmanabhan, P., Meunier, F.A. (2020). Single-Molecule Imaging of Recycling Synaptic Vesicles in Live Neurons. In: Yamamoto, N., Okada, Y. (eds) Single Molecule Microscopy in Neurobiology . Neuromethods, vol 154. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-0532-5_5

Download citation

  • DOI: https://doi.org/10.1007/978-1-0716-0532-5_5

  • Published:

  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-0716-0531-8

  • Online ISBN: 978-1-0716-0532-5

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics

Search

Navigation