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.
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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
Wang T et al (2016) Flux of signalling endosomes undergoing axonal retrograde transport is encoded by presynaptic activity and TrkB. Nat Commun 7:12976
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
Fowler MW, Staras K (2015) Synaptic vesicle pools: principles, properties and limitations. Exp Cell Res 335(2):150–156
Denker A, Rizzoli SO (2010) Synaptic vesicle pools: an update. Front Synaptic Neurosci 2:135
Rizzoli SO, Betz WJ (2005) Synaptic vesicle pools. Nat Rev Neurosci 6(1):57–69
Chamberland S, Toth K (2016) Functionally heterogeneous synaptic vesicle pools support diverse synaptic signalling. J Physiol 594(4):825–835
Alabi AA, Tsien RW (2012) Synaptic vesicle pools and dynamics. Cold Spring Harb Perspect Biol 4(8):a013680
Crawford DC, Kavalali ET (2015) Molecular underpinnings of synaptic vesicle pool heterogeneity. Traffic 16(4):338–364
Kamin D et al (2010) High- and low-mobility stages in the synaptic vesicle cycle. Biophys J 99(2):675–684
Gimber N et al (2015) Diffusional spread and confinement of newly exocytosed synaptic vesicle proteins. Nat Commun 6:8392
Hua Y et al (2011) A readily retrievable pool of synaptic vesicles. Nat Neurosci 14(7):833–839
Willig KI et al (2006) STED microscopy reveals that synaptotagmin remains clustered after synaptic vesicle exocytosis. Nature 440(7086):935–939
Hu Y, Qu L, Schikorski T (2008) Mean synaptic vesicle size varies among individual excitatory hippocampal synapses. Synapse 62(12):953–957
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
Westphal V et al (2008) Video-rate far-field optical nanoscopy dissects synaptic vesicle movement. Science 320(5873):246–249
Hoopmann P et al (2010) Endosomal sorting of readily releasable synaptic vesicles. Proc Natl Acad Sci U S A 107(44):19055–19060
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
Maschi D, Klyachko VA (2017) Spatiotemporal regulation of synaptic vesicle fusion sites in central synapses. Neuron 94(1):65–73.e3
Peng A et al (2012) Differential motion dynamics of synaptic vesicles undergoing spontaneous and activity-evoked endocytosis. Neuron 73(6):1108–1115
Joensuu M et al (2016) Subdiffractional tracking of internalized molecules reveals heterogeneous motion states of synaptic vesicles. J Cell Biol 215(2):277–292
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
Miesenbock G, De Angelis DA, Rothman JE (1998) Visualizing secretion and synaptic transmission with pH-sensitive green fluorescent proteins. Nature 394(6689):192–195
Royle SJ et al (2008) Imaging phluorin-based probes at hippocampal synapses. Methods Mol Biol 457:293–303
Villarreal S, Lee SH, Wu LG (2017) Measuring synaptic vesicle endocytosis in cultured hippocampal neurons. J Vis Exp (127)
Fiolka R (2016) Clearer view for TIRF and oblique illumination microscopy. Opt Express 24(26):29556–29567
Giannone G et al (2010) Dynamic superresolution imaging of endogenous proteins on living cells at ultra-high density. Biophys J 99(4):1303–1310
Rothbauer U et al (2006) Targeting and tracing antigens in live cells with fluorescent nanobodies. Nat Methods 3(11):887–889
Harper CB et al (2016) Botulinum neurotoxin type-A enters a non-recycling pool of synaptic vesicles. Sci Rep 6:19654
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
Giannone G et al (2013) High-content super-resolution imaging of live cell by uPAINT. Methods Mol Biol 950:95–110
Kechkar A et al (2013) Real-time analysis and visualization for single-molecule based super-resolution microscopy. PLoS One 8(4):e62918
Tinevez JY et al (2017) TrackMate: an open and extensible platform for single-particle tracking. Methods 115:80–90
Kubala MH et al (2010) Structural and thermodynamic analysis of the GFP:GFP-nanobody complex. Protein Sci 19(12):2389–2401
Durisic N et al (2014) Single-molecule evaluation of fluorescent protein photoactivation efficiency using an in vivo nanotemplate. Nat Methods 11(2):156–162
Craig AM, Banker G (1994) Neuronal polarity. Annu Rev Neurosci 17:267–310
Kaech S, Banker G (2006) Culturing hippocampal neurons. Nat Protoc 1(5):2406–2415
Chenouard N et al (2014) Objective comparison of particle tracking methods. Nat Methods 11(3):281–289
Izeddin I et al (2012) Wavelet analysis for single molecule localization microscopy. Opt Express 20(3):2081–2095
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
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
Wilhelm BG et al (2014) Composition of isolated synaptic boutons reveals the amounts of vesicle trafficking proteins. Science 344(6187):1023–1028
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
Takamori S et al (2006) Molecular anatomy of a trafficking organelle. Cell 127(4):831–846
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
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
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
Truckenbrodt S, Rizzoli SO (2015) Synaptic vesicle pools: classical and emerging roles. In: Mochida S (ed) Presynaptic terminals. Springer, Tokyo
Iwabuchi S et al (2014) Examination of synaptic vesicle recycling using FM dyes during evoked, spontaneous, and miniature synaptic activities. J Vis Exp (85)
Raingo J et al (2012) VAMP4 directs synaptic vesicles to a pool that selectively maintains asynchronous neurotransmission. Nat Neurosci 15(5):738–745
Hua Z et al (2011) v-SNARE composition distinguishes synaptic vesicle pools. Neuron 71(3):474–487
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
Kwon SE, Chapman ER (2011) Synaptophysin regulates the kinetics of synaptic vesicle endocytosis in central neurons. Neuron 70(5):847–854
Granseth B et al (2006) Clathrin-mediated endocytosis is the dominant mechanism of vesicle retrieval at hippocampal synapses. Neuron 51(6):773–786
Voglmaier SM et al (2006) Distinct endocytic pathways control the rate and extent of synaptic vesicle protein recycling. Neuron 51(1):71–84
Ramirez DM et al (2012) Vti1a identifies a vesicle pool that preferentially recycles at rest and maintains spontaneous neurotransmission. Neuron 73(1):121–134
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
Pelkmans L et al (2004) Caveolin-stabilized membrane domains as multifunctional transport and sorting devices in endocytic membrane traffic. Cell 118(6):767–780
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
Jiang M, Chen G (2006) High Ca2+-phosphate transfection efficiency in low-density neuronal cultures. Nat Protoc 1(2):695–700
Zeitelhofer M et al (2007) High-efficiency transfection of mammalian neurons via nucleofection. Nat Protoc 2(7):1692–1704
Belevich I et al (2016) Microscopy image browser: a platform for segmentation and analysis of multidimensional datasets. PLoS Biol 14(1):e1002340
Sudhof TC, Rothman JE (2009) Membrane fusion: grappling with SNARE and SM proteins. Science 323(5913):474–477
Chanaday NL, Kavalali ET (2018) Presynaptic origins of distinct modes of neurotransmitter release. Curr Opin Neurobiol 51:119–126
Maritzen T, Haucke V (2018) Coupling of exocytosis and endocytosis at the presynaptic active zone. Neurosci Res 127:45–52
Heller JP, Rusakov DA (2017) The nanoworld of the tripartite synapse: insights from super-resolution microscopy. Front Cell Neurosci 11:374
Monnier N et al (2015) Inferring transient particle transport dynamics in live cells. Nat Methods 12(9):838–840
Persson F et al (2013) Extracting intracellular diffusive states and transition rates from single-molecule tracking data. Nat Methods 10(3):265–269
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).
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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
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DOI: https://doi.org/10.1007/978-1-0716-0532-5_5
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