The synaptic vesicle, a cellular compartment tens to hundreds of nanometres in size, is a main player in the process of exocytosis for neuronal communication. Understanding the regulatory mechanism of neurotransmission and neurological disorders requires the quantification of chemicals transmitted between cells. These challenging single vesicle measurements can be performed using analytical techniques described in this Review. In vivo amperometry at living cells can be used to quantify the amount of neurotransmitter released from a vesicle. By contrast, intracellular vesicle impact electrochemical cytometry allows the amount of molecules to be determined inside single vesicles. Although the dominant mode of exocytosis from vesicles is still under debate, several experiments point to the importance of partial release modes. Making use of fluorescent or isotopically labelled probes enables super-resolution optical and mass spectrometric imaging of molecular composition and activity of single vesicles. Correlating results from these nanoscopic techniques with those from electrochemistry has proved advantageous in understanding the relationship between vesicle structure and function.
Your institute does not have access to this article
Subscribe to Nature+
Get immediate online access to the entire Nature family of 50+ journals
Subscribe to Journal
Get full journal access for 1 year
only $9.92 per issue
All prices are NET prices.
VAT will be added later in the checkout.
Tax calculation will be finalised during checkout.
Get time limited or full article access on ReadCube.
All prices are NET prices.
Takamori, S. et al. Molecular anatomy of a trafficking organelle. Cell 127, 831–846 (2006).
Cox, H. D. & Thompson, C. M. Purification and proteomic analysis of synaptic vesicles. Methods Mol. Biol. 432, 259–274 (2008).
Zito, K., Parnas, D., Fetter, R. D., Isacoff, E. Y. & Goodman, C. S. Watching a synapse grow: noninvasive confocal imaging of synaptic growth in Drosophila. Neuron 22, 719–729 (1999).
Stevens, B. et al. The classical complement cascade mediates CNS synapse elimination. Cell 131, 1164–1178 (2007).
Li, X., Dunevall, J. & Ewing, A. G. Quantitative chemical measurements of vesicular transmitters with electrochemical cytometry. Acc. Chem. Res. 49, 2347–2354 (2016).
Wightman, R. M. et al. Temporally resolved catecholamine spikes correspond to single vesicle release from individual chromaffin cells. Proc. Natl Acad. Sci. USA 88, 10754–10758 (1991). This paper describes one of the earliest amperometric measurements of catecholamines released during exocytosis.
Mellander, L. J. et al. Two modes of exocytosis in an artificial cell. Sci. Rep. 4, 3847–3853 (2014).
Omiatek, D. M., Dong, Y., Heien, M. L. & Ewing, A. G. Only a fraction of quantal content is released during exocytosis as revealed by electrochemical cytometry of secretory vesicles. ACS Chem. Neurosci. 1, 234–245 (2010). The authors of this article demonstrated experimentally that exocytosis is mostly ‘open and closed’ or partial release.
Fernandez-Suarez, M. & Ting, A. Y. Fluorescent probes for super-resolution imaging in living cells. Nat. Rev. Mol. Cell Biol. 9, 929–943 (2008).
Pihel, K., Schroeder, T. J. & Wightman, R. M. Rapid and selective cyclic voltammetric measurements of epinephrine and norepinephrine as a method to measure secretion from single bovine adrenal medullary cells. Anal. Chem. 66, 4532–4537 (1994).
Betz, W. J., Mao, F. & Smith, C. B. Imaging exocytosis and endocytosis. Curr. Opin. Neurobiol. 6, 365–371 (1996).
Cahill, P. S. et al. Microelectrodes for the measurement of catecholamines in biological systems. Anal. Chem. 68, 3180–3186 (1996).
Travis, E. R. & Wightman, R. M. Spatio-temporal resolution of exocytosis from individual cells. Annu. Rev. Biophys. Biomol. Struct. 27, 77–103 (1998).
Aravanis, A. M., Pyle, J. L., Harata, N. C. & Tsien, R. W. Imaging single synaptic vesicles undergoing repeated fusion events: kissing, running, and kissing again. Neuropharmacology 45, 797–813 (2003).
Jin, H., Heller, D. A. & Strano, M. S. Single-particle tracking of endocytosis and exocytosis of single-walled carbon nanotubes in NIH-3T3 cells. Nano Lett. 8, 1577–1585 (2008).
Xia, X. F., Lessmann, V. & Martin, T. F. J. Imaging of evoked dense-core-vesicle exocytosis in hippocampal neurons reveals long latencies and kiss-and-run fusion events. J. Cell Sci. 122, 75–82 (2009).
Leszczyszyn, D. J. et al. Nicotinic receptor-mediated catecholamine secretion from individual chromaffin cells: chemical evidence for exocytosis. J. Biol. Chem. 265, 14736–14737 (1990).
Amatore, C. et al. Relationship between amperometric pre-spike feet and secretion granule composition in chromaffin cells: an overview. Biophys. Chem. 129, 181–189 (2007).
Chow, R. H., von Rüden, L. & Neher, E. Delay in vesicle fusion revealed by electrochemical monitoring of single secretory events in adrenal chromaffin cells. Nature 356, 60–63 (1992).
De Toledo, G. A., Fernández-Chacón, R. & Fernández, J. M. Release of secretory products during transient vesicle fusion. Nature 363, 554–558 (1993).
Ren, L. et al. The evidence for open and closed exocytosis as the primary release mechanism. Q. Rev. Biophys. 49, e12 (2016).
Amatore, C., Oleinick, A. I. & Svir, I. Reconstruction of aperture functions during full fusion in vesicular exocytosis of neurotransmitters. ChemPhysChem 11, 159–174 (2010).
Dunevall, J. et al. Characterizing the catecholamine content of single mammalian vesicles by collision–adsorption events at an electrode. J. Am. Chem. Soc. 137, 4344–4346 (2015). This work demonstrated that it is possible to count molecules in vesicles that undergo stochastic collisions at an electrode, followed by adsorption and opening.
Li, X., Majdi, S., Dunevall, J., Fathali, H. & Ewing, A. G. Quantitative measurement of transmitters in individual vesicles in the cytoplasm of single cells with nanotip electrodes. Angew. Chem. Int. Ed. 54, 11978–11982 (2015). Demonstration of intracellular VIEC for vesicle content in live cells.
Bruns, D. & Jahn, R. Real-time measurement of transmitter release from single synaptic vesicles. Nature 377, 62–65 (1995).
Staal, R. G., Mosharov, E. V. & Sulzer, D. Dopamine neurons release transmitter via a flickering fusion pore. Nat. Neurosci. 7, 341–346 (2004).
Mellander, L. J., Trouillon, R., Svensson, M. I. & Ewing, A. G. Amperometric post spike feet reveal most exocytosis is via extended kiss-and-run fusion. Sci. Rep. 2, 907 (2012).
Chen, Y. A. & Scheller, R. H. SNARE-mediated membrane fusion. Nat. Rev. Mol. Cell Biol. 2, 98–106 (2001).
Feany, M. B. & Bender, W. W. A. Drosophila model of Parkinson's disease. Nature 404, 394–398 (2000).
Finelli, A., Kelkar, A., Song, H. J., Yang, H. & Konsolaki, M. A model for studying Alzheimer's Aβ42-induced toxicity in Drosophila melanogaster. Mol. Cell. Neurosci. 26, 365–375 (2004).
Watson, M. R., Lagow, R. D., Xu, K., Zhang, B. & Bonini, N. M. A. Drosophila model for amyotrophic lateral sclerosis reveals motor neuron damage by human SOD1. J. Biol. Chem. 283, 24972–24981 (2008).
Majdi, S. et al. Electrochemical measurements of optogenetically stimulated quantal amine release from single nerve cell varicosities in Drosophila larvae. Angew. Chem. Int. Ed. 54, 13609–13612 (2015). This article describes amperometric measurement of exocytosis from single neurons in D. melanogaster.
Li, Y.-T. et al. Real-time monitoring of discrete synaptic release events and excitatory potentials within self-reconstructed neuromuscular junctions. Angew. Chem. Int. Ed. 54, 9313–9318 (2015).
Li, Y.-T. et al. Nanoelectrode for amperometric monitoring of individual vesicular exocytosis inside single synapses. Angew. Chem. Int. Ed. 53, 12456–12460 (2014). A report on the use of nanotip electrodes to measure noradrenaline release inside a nanometre synapse.
Strein, T. G. & Ewing, A. G. Characterization of submicron-sized carbon electrodes insulated with a phenol–allylphenol copolymer. Anal. Chem. 64, 1368–1373 (1992).
Borges, R., Travis, E. R., Hochstetler, S. E. & Wightman, R. M. Effects of external osmotic pressure on vesicular secretion from bovine adrenal medullary cells. J. Biol. Chem. 272, 8325–8331 (1997).
Camacho, M., Machado, J. D., Montesinos, M. S., Criado, M. & Borges, R. Intragranular pH rapidly modulates exocytosis in adrenal chromaffin cells. J. Neurochem. 96, 324–334 (2006).
Haynes, C. L., Siff, L. N. & Wightman, R. M. Temperature-dependent differences between readily releasable and reserve pool vesicles in chromaffin cells. Biochim. Biophys. Acta 1773, 728–735 (2007).
Machado, J. D., Morales, A., Gomez, J. F. & Borges, R. cAMP modulates exocytotic kinetics and increases quantal size in chromaffin cells. Mol. Pharmacol. 60, 514–520 (2001).
Calvo-Gallardo, E. et al. Faster kinetics of quantal catecholamine release in mouse chromaffin cells stimulated with acetylcholine, compared with other secretagogues. J. Neurochem. 139, 722–736 (2016).
Pothos, E. N. et al. Stimulation-dependent regulation of the pH, volume and quantal size of bovine and rodent secretory vesicles. J. Physiol. 542, 453–476 (2002).
Elhamdani, A., Palfrey, H. C. & Artalejo, C. R. Quantal size is dependent on stimulation frequency and calcium entry in calf chromaffin cells. Neuron 31, 819–830 (2001).
Sombers, L. A., Maxson, M. M. & Ewing, A. G. Loaded dopamine is preferentially stored in the halo portion of PC12 cell dense core vesicles. J. Neurochem. 93, 1122–1131 (2005).
Omiatek, D. M., Santillo, M. F., Heien, M. L. & Ewing, A. G. Hybrid capillary-microfluidic device for the separation, lysis, and electrochemical detection of vesicles. Anal. Chem. 81, 2294–2302 (2009).
Omiatek, D. M. et al. The real catecholamine content of secretory vesicles in the CNS revealed by electrochemical cytometry. Sci. Rep. 3, 1447–1452 (2013).
Cheng, W. & Compton, R. G. Investigation of single-drug-encapsulating liposomes using the nano-impact method. Angew. Chem. Int. Ed. 53, 13928–13930 (2014).
Lovric´, J. et al. On the mechanism of electrochemical vesicle cytometry: chromaffin cell vesicles and liposomes. Faraday Discuss. 193, 65–79 (2016).
Trouillon, R. & Ewing, A. G. Actin controls the vesicular fraction of dopamine released during extended kiss and run exocytosis. ACS Chem. Biol. 9, 812–820 (2014).
Najafinobar, N. et al. Excited fluorophores enhance the opening of vesicles at electrode surfaces in vesicle electrochemical cytometry. Angew. Chem. Int. Ed. 55, 15081–15085 (2016).
Lebègue, E., Anderson, C. M., Dick, J. E., Webb, L. J. & Bard, A. J. Electrochemical detection of single phospholipid vesicle collisions at a Pt ultramicroelectrode. Langmuir 31, 11734–11739 (2015).
Cheng, W. & Compton, R. G. Measuring the content of a single liposome through electrocatalytic nanoimpact “titrations”. ChemElectroChem 3, 2017–2020 (2016).
Li, X., Dunevall, J. & Ewing, A. G. Using single-cell amperometry to reveal how cisplatin treatment modulates the release of catecholamine transmitters during exocytosis. Angew. Chem. Int. Ed. 55, 9041–9044 (2016).
Ren, L. et al. Zinc regulates chemical-transmitter storage in nanometer vesicles and exocytosis dynamics as measured by amperometry. Angew. Chem. Int. Ed. 56, 4970–4975 (2017).
Colliver, T. L., Pyott, S. J., Achalabun, M. & Ewing, A. G. VMAT-mediated changes in quantal size and vesicular volume. J. Neurosci. 20, 5276–5282 (2000).
Fathali, H., Dunevall, J., Majdi, S. & Cans, A.-S. Extracellular osmotic stress reduces the vesicle size while keeping a constant neurotransmitter concentration. ACS Chem. Neurosci. 8, 368–375 (2017).
Huang, B., Bates, M. & Zhuang, X. Super-resolution fluorescence microscopy. Annu. Rev. Biochem. 78, 993–1016 (2009).
Hell, S. W. & Wichmann, J. Breaking the diffraction resolution limit by stimulated emission: stimulated-emission-depletion fluorescence microscopy. Opt. Lett. 19, 780–782 (1994).
Hein, B., Willig, K. I. & Hell, S. W. Stimulated emission depletion (STED) nanoscopy of a fluorescent protein-labeled organelle inside a living cell. Proc. Natl Acad. Sci. USA 105, 14271–14276 (2008).
Moneron, G. & Hell, S. W. Two-photon excitation STED microscopy. Opt. Express 17, 14567–14583 (2009).
Neupane, B., Ligler, F. S. & Wang, G. Review of recent developments in stimulated emission depletion microscopy: applications on cell imaging. J. Biomed. Opt. 19, 080901 (2014).
Török, P. & Munro, P. R. T. The use of Gauss–Laguerre vector beams in STED microscopy. Opt. Express 12, 3605–3617 (2004).
Kotlyar, V. V., Almazov, A. A., Khonina, S. N. & Soifer, V. A. Generation of phase singularity through diffracting a plane or Gaussian beam by a spiral phase plate. J. Opt. Soc. Am. A 22, 849–861 (2005).
Schmidt, R. et al. Spherical nanosized focal spot unravels the interior of cells. Nat. Methods 5, 539–544 (2008).
Curdt, F. et al. isoSTED nanoscopy with intrinsic beam alignment. Opt. Express 23, 30891–30903 (2015).
Rust, M. J., Bates, M. & Zhuang, X. Sub-diffraction-limit imaging by stochastic optical reconstruction microscopy (STORM). Nat. Methods 3, 793–795 (2006).
Betzig, E. et al. Imaging intracellular fluorescent proteins at nanometer resolution. Science 313, 1642–1645 (2006).
Xu, K., Babcock, H. P. & Zhuang, X. Dual-objective STORM reveals three-dimensional filament organization in the actin cytoskeleton. Nat. Methods 9, 185–188 (2012).
Dani, A., Huang, B., Bergan, J., Dulac, C. & Zhuang, X. Superresolution imaging of chemical synapses in thebrain. Neuron 68, 843–856 (2010).
Wilhelm, B. G., Groemer, T. W. & Rizzoli, S. O. The same synaptic vesicles drive active and spontaneous release. Nat. Neurosci. 13, 1454–1456 (2010).
Wilhelm, B. G. et al. Composition of isolated synaptic boutons reveals the amounts of vesicle trafficking proteins. Science 344, 1023–1028 (2014). This report describes protein organization of the synaptic bouton and vesicle, including a 3D model of synaptic structure.
Westphal, V. et al. Video-rate far-field optical nanoscopy dissects synaptic vesicle movement. Science 320, 246–249 (2008).
Huang, B., Wang, W., Bates, M. & Zhuang, X. Three-dimensional super-resolution imaging by stochastic optical reconstruction microscopy. Science 319, 810–813 (2008). The first demonstration of 3D imaging using STORM, applied here to observe the structure of protein microtubules and clathrin-coated pits in cells.
Balzarotti, F. et al. Nanometer resolution imaging and tracking of fluorescent molecules with minimal photon fluxes. Science 355, 606–612 (2017). This paper describes MINFLUX, a STED variant that minimizes the emitter photons needed for locating single molecules to 1 nm precision. This may allow the study of dynamics, distribution and structure of single molecules in living cells.
Opazo, F. et al. Aptamers as potential tools for super-resolution microscopy. Nat. Methods 9, 938–939 (2012).
Tanaka, K. A. K. et al. Membrane molecules mobile even after chemical fixation. Nat. Methods 7, 865–866 (2010).
Ries, J., Kaplan, C., Platonova, E., Eghlidi, H. & Ewers, H. A simple, versatile method for GFP-based super-resolution microscopy via nanobodies. Nat. Methods 9, 582–584 (2012).
Pleiner, T. et al. Nanobodies: site-specific labeling for super-resolution imaging, rapid epitope-mapping and native protein complex isolation. eLife 4, e11349 (2015).
Szymborska, A. et al. Nuclear pore scaffold structure analyzed by super-resolution microscopy and particle averaging. Science 341, 655–658 (2015).
Vreja, I. C. et al. Super-resolution microscopy of clickable amino acids reveals the effects of fluorescent protein tagging on protein assemblies. ACS Nano 9, 11034–11041 (2015).
Plass, T., Milles, S., Koehler, C., Schultz, C. & Lemke, E. A. Genetically encoded copper-free click chemistry. Angew. Chem. Int. Ed. 50, 3878–3881 (2011).
de Castro, M. A., Rammner, B. & Opazo, F. Aptamer stainings for super-resolution microscopy. Methods Mol. Biol. 1380, 197–210 (2016).
Revelo, N. H. et al. A new probe for super-resolution imaging of membranes elucidates trafficking pathways. J. Cell Biol. 205, 591–606 (2014).
Jung, S. et al. Disruption of adaptor protein 2μ (AP-2μ) in cochlear hair cells impairs vesicle reloading of synaptic release sites and hearing. EMBO J. 34, 2686–2702 (2015).
Opazo, F. et al. Limited intermixing of synaptic vesicle components upon vesicle recycling. Traffic 11, 800–812 (2010).
Spuhler, I. A., Conley, G. M., Scheffold, F. & Sprecher, S. G. Super resolution imaging of genetically labeled synapses in Drosophila brain tissue. Front. Cell. Neurosci. 10, 142 (2016).
Pech, U., Revelo, N. H., Seitz, K. J., Rizzoli, S. O. & Fiala, A. Optical dissection of experience-dependent pre- and postsynaptic plasticity in the Drosophila brain. Cell Rep. 10, 2083–2095 (2015).
Willig, K. I., Rizzoli, S. O., Westphal, V., Jahn, R. & Hell, S. W. STED microscopy reveals that synaptotagmin remains clustered after synaptic vesicle exocytosis. Nature 440, 935–939 (2006). This article considers the nature of vesicular membrane protein synaptotagmin I during vesicle recycling using STED.
Hoopmann, P. et al. Endosomal sorting of readily releasable synaptic vesicles. Proc. Natl Acad. Sci. USA 107, 19055–19060 (2010).
Yeung, C., Shtrahman, M. & Wu, X.-L. Stick-and-diffuse and caged diffusion: a comparison of two models of synaptic vesicle dynamics. Biophys. J. 92, 2271–2280 (2007).
Saka, S. K. et al. Correlated optical and isotopic nanoscopy. Nat. Commun. 5, 3664 (2014).
Vreja, I. C. et al. Secondary-ion mass spectrometry of genetically encoded targets. Angew. Chem. Int. Ed. 54, 5784–5788 (2015).
Kabatas, S. et al. A contamination-insensitive probe for imaging specific biomolecules by secondary ion mass spectrometry. Chem. Commun. 51, 13221–13224 (2015).
Watanabe, S. et al. Protein localization in electron micrographs using fluorescence nanoscopy. Nat. Methods 8, 80–84 (2011).
Fouquet, W. et al. Maturation of active zone assembly by Drosophila Bruchpilot. J. Cell Biol. 186, 129–145 (2009).
Kim, D. et al. Correlative stochastic optical reconstruction microscopy and electron microscopy. PLoS ONE 10, e0124581 (2015).
Lovric´, J. et al. Nano secondary ion mass spectrometry imaging of dopamine distribution across nanometer vesicles. ACS Nano 11, 3446–3455 (2017). The paper describes applications of multimodal nanoSIMS, TEM and single-cell electrochemistry to image single vesicles and quantify neurotransmitter content and release. The results implicate a role for vesicle inner structure on regulating release kinetics.
Dean, K. M. & Palmer, A. E. Advances in fluorescence labeling strategies for dynamic cellular imaging. Nat. Chem. Biol. 10, 512–523 (2014).
The authors thank S. O. Rizzoli for helpful comments, and the many colleagues and collaborators that have contributed to work cited in this Review. This work was supported by the Knut and Alice Wallenberg Foundation, the Swedish Research Council, the European Research Council, and the US National Institutes of Health.
The authors declare no competing interests.
About this article
Cite this article
Phan, N., Li, X. & Ewing, A. Measuring synaptic vesicles using cellular electrochemistry and nanoscale molecular imaging. Nat Rev Chem 1, 0048 (2017). https://doi.org/10.1038/s41570-017-0048
Simulations of amperometric monitoring of exocytosis: moderate pH variations within the cell-electrode cleft with the buffer diffusion
Analytical and Bioanalytical Chemistry (2021)