Abstract
FM dyes have been used to label and then monitor synaptic vesicles, secretory granules and other endocytic structures in a variety of preparations. Here, we describe the general procedure for using FM dyes to study endosomal trafficking in general, and synaptic vesicle recycling in particular. The dye, dissolved in normal saline solution, is added to a chamber containing the preparation to be labeled. Stimulation evokes exocytosis, and compensatory endocytosis that follows traps FM dye inside the retrieved vesicles. The extracellular dye is then washed from the chamber, and labeled endocytic structures are examined with a fluorescence microscope. Fluorescence intensity provides a direct measure of the labeled vesicle number, a good measure of the amount of exocytosis. If the preparation is stimulated again, without dye in the chamber, dimming of the preparation provides a measure of exocytosis of labeled vesicles. With a synaptic preparation on hand, this protocol requires 1 day.
This is a preview of subscription content, access via your institution
Access options
Subscribe to this journal
Receive 12 print issues and online access
$259.00 per year
only $21.58 per issue
Buy this article
- Purchase on Springer Link
- Instant access to full article PDF
Prices may be subject to local taxes which are calculated during checkout
Similar content being viewed by others
References
Betz, W.J., Mao, F. & Bewick, G.S. Activity-dependent fluorescent staining and destaining of living vertebrate motor nerve terminals. J. Neurosci. 12, 363–375 (1992).
Betz, W.J. & Bewick, G.S. Optical analysis of synaptic vesicle recycling at the frog neuromuscular junction. Science 255, 200–203 (1992).
Lichtman, J.W., Wilkinson, R.S. & Rich, M.M. Multiple innervation of tonic endplates revealed by activity-dependent uptake of fluorescent probes. Nature 314, 357–359 (1985).
Grinvald, A., Frostig, R.D., Lieke, E. & Hildesheim, R. Optical imaging of neuronal activity. Physiol. Rev. 68, 1285–1366 (1988).
Ryan, T.A. et al. The kinetics of synaptic vesicle recycling measured at single presynaptic boutons. Neuron 11, 713–724 (1993).
Ramaswami, M., Krishnan, K.S. & Kelly, R.B. Intermediates in synaptic vesicle recycling revealed by optical imaging of Drosophila neuromuscular junctions. Neuron 13, 363–375 (1994).
Pocock, J.M., Cousin, M.A., Parkin, J. & Nicholls, D.G. Glutamate exocytosis from cerebellar granule cells: the mechanism of a transition to an L-type Ca2+ channel coupling. Neuroscience 67, 595–607 (1995).
Lagnado, L., Gomis, A. & Job, C. Continuous vesicle cycling in the synaptic terminal of retinal bipolar cells. Neuron 17, 957–967 (1996).
Wang, C. & Zucker, R.S. Regulation of synaptic vesicle recycling by calcium and serotonin. Neuron 21, 155–167 (1998).
Teng, H., Cole, J.C., Roberts, R.L. & Wilkinson, R.S. Endocytic active zones: hot spots for endocytosis in vertebrate neuromuscular terminals. J. Neurosci. 19, 4855–4866 (1999).
Kay, A.R. et al. Imaging synaptic activity in intact brain and slices with FM1-43 in C. elegans, lamprey, and rat. Neuron 24, 809–817 (1999).
Pyle, J.L., Kavalali, E.T., Choi, S. & Tsien, R.W. Visualization of synaptic activity in hippocampal slices with FM1-43 enabled by fluorescence quenching. Neuron 24, 803–808 (1999).
Smith, C.B. & Betz, W.J. Simultaneous independent measurement of endocytosis and exocytosis. Nature 380, 531–534 (1996).
Stafford, S.J., Shorte, S.L. & Schofield, J.G. Use of a fluorescent dye to measure secretion from intact bovine anterior pituitary cells. Biosci. Rep. 13, 9–17 (1993).
Brumback, A.C., Lieber, J.L., Angleson, J.K. & Betz, W.J. Using FM1-43 to study neuropeptide granule dynamics and exocytosis. Methods 33, 287–294 (2004).
Takahashi, N. et al. Two-photon excitation imaging of pancreatic islets with various fluorescent probes. Diabetes 51 (Suppl 1): S25–S28 (2002).
Giovannucci, D.R., Yule, D.I. & Stuenkel, E.L. Optical measurement of stimulus-evoked membrane dynamics in single pancreatic acinar cells. Am. J. Physiol. 275, C732–C739 (1998).
Vida, T.A. & Emr, S.D. A new vital stain for visualizing vacuolar membrane dynamics and endocytosis in yeast. J. Cell Biol. 128, 779–792 (1995).
Vlahakis, N.E., Schroeder, M.A., Pagano, R.E. & Hubmayr, R.D. Deformation-induced lipid trafficking in alveolar epithelial cells. Am. J. Physiol. Lung Cell Mol. Physiol. 280, L938–L946 (2001).
Sterling, T.M. & Nemere, I. 1,25-dihydroxyvitamin D3 stimulates vesicular transport within 5 s in polarized intestinal epithelial cells. J. Endocrinol. 185, 81–91 (2005).
Lee, S.C., Vielhauer, N.S., Leaver, E.V. & Pappone, P.A. Differential regulation of Ca(2+) signaling and membrane trafficking by multiple p2 receptors in brown adipocytes. J. Membr. Biol. 207, 131–142 (2005).
Bolte, S. et al. FM-dyes as experimental probes for dissecting vesicle trafficking in living plant cells. J. Microsc. 214, 159–173 (2004).
Angleson, J.K., Cochilla, A.J., Kilic, G., Nussinovitch, I. & Betz, W.J. Regulation of dense core release from neuroendocrine cells revealed by imaging single exocytic events. Nat. Neurosci. 2, 440–446 (1999).
Henkel, A.W., Lubke, J. & Betz, W.J. FM1-43 dye ultrastructural localization in and release from frog motor nerve terminals. Proc. Natl. Acad. Sci. USA 93, 1918–1923 (1996).
Harata, N., Ryan, T.A., Smith, S.J., Buchanan, J. & Tsien, R.W. Visualizing recycling synaptic vesicles in hippocampal neurons by FM 1-43 photoconversion. Proc. Natl. Acad. Sci. USA 98, 12748–12753 (2001).
Rizzoli, S.O. & Betz, W.J. The structural organization of the readily releasable pool of synaptic vesicles. Science 303, 2037–2039 (2004).
Richards, D.A., Bai, J. & Chapman, E.R. Two modes of exocytosis at hippocampal synapses revealed by rate of FM1-43 efflux from individual vesicles. J. Cell Biol. 168, 929–939 (2005).
Zenisek, D., Steyer, J.A., Feldman, M.E. & Almers, W. A membrane marker leaves synaptic vesicles in milliseconds after exocytosis in retinal bipolar cells. Neuron 35, 1085–1097 (2002).
Richards, D.A., Guatimosim, C. & Betz, W.J. Two endocytic recycling routes selectively fill two vesicle pools in frog motor nerve terminals. Neuron 27, 551–559 (2000).
Bewick, G.S. & Betz, W.J. Illumination partly reverses the postsynaptic blockade of the frog neuromuscular junction by the styryl pyridinium dye RH414. Proc. Biol. Sci. 258, 201–207 (1994).
Mazzone, S.B. et al. Fluorescent styryl dyes FM1-43 and FM2-10 are muscarinic receptor antagonists: intravital visualization of receptor occupancy. J. Physiol 575, 23–35 (2006).
Nishikawa, S. & Sasaki, F. Internalization of styryl dye FM1-43 in the hair cells of lateral line organs in Xenopus larvae. J. Histochem. Cytochem. 44, 733–741 (1996).
Gale, J.E., Marcotti, W., Kennedy, H.J., Kros, C.J. & Richardson, G.P. FM1-43 dye behaves as a permeant blocker of the hair-cell mechanotransducer channel. J. Neurosci. 21, 7013–7025 (2001).
Griesinger, C.B., Richards, C.D. & Ashmore, J.F. Fm1-43 reveals membrane recycling in adult inner hair cells of the mammalian cochlea. J. Neurosci. 22, 3939–3952 (2002).
Chi, P., Greengard, P. & Ryan, T.A. Synapsin dispersion and reclustering during synaptic activity. Nat. Neurosci. 4, 1187–1193 (2001).
Renger, J.J., Egles, C. & Liu, G. A developmental switch in neurotransmitter flux enhances synaptic efficacy by affecting AMPA receptor activation. Neuron 29, 469–484 (2001).
Winterer, J., Stanton, P.K. & Muller, W. Direct monitoring of vesicular release and uptake in brain slices by multiphoton excitation of the styryl FM 1-43. Biotechniques 40, 343–351 (2006).
Kraszewski, K. et al. Synaptic vesicle dynamics in living cultured hippocampal neurons visualized with CY3-conjugated antibodies directed against the lumenal domain of synaptotagmin. J. Neurosci. 15, 4328–4342 (1995).
Miesenbock, G., De Angelis, D.A. & Rothman, J.E. Visualizing secretion and synaptic transmission with pH-sensitive green fluorescent proteins. Nature 394, 192–195 (1998).
Sankaranarayanan, S. & Ryan, T.A. Real-time measurements of vesicle-SNARE recycling in synapses of the central nervous system. Nat. Cell Biol. 2, 197–204 (2000).
Fernandez-Alfonso, T., Kwan, R. & Ryan, T.A. Synaptic vesicles interchange their membrane proteins with a large surface reservoir during recycling. Neuron 51, 179–186 (2006).
Wienisch, M. & Klingauf, J. Vesicular proteins exocytosed and subsequently retrieved by compensatory endocytosis are nonidentical. Nat. Neurosci. 9, 1019–1027 (2006).
Granseth, B., Odermatt, B., Royle, S.J. & Lagnado, L. Clathrin-mediated endocytosis is the dominant mechanism of vesicle retrieval at hippocampal synapses. Neuron 51, 773–786 (2006).
Waters, J. & Smith, S.J. Phorbol esters potentiate evoked and spontaneous release by different presynaptic mechanisms. J. Neurosci. 20, 7863–7870 (2000).
Virmani, T., Ertunc, M., Sara, Y., Mozhayeva, M. & Kavalali, E.T. Phorbol esters target the activity-dependent recycling pool and spare spontaneous vesicle recycling. J. Neurosci. 25, 10922–10929 (2005).
Sara, Y., Virmani, T., Deak, F., Liu, X. & Kavalali, E.T. An isolated pool of vesicles recycles at rest and drives spontaneous neurotransmission. Neuron 45, 563–573 (2005).
Henkel, A.W., Simpson, L.L., Ridge, R.M. & Betz, W.J. Synaptic vesicle movements monitored by fluorescence recovery after photobleaching in nerve terminals stained with FM1-43. J. Neurosci. 16, 3960–3967 (1996).
Darcy, K.J., Staras, K., Collinson, L.M. & Goda, Y. Constitutive sharing of recycling synaptic vesicles between presynaptic boutons. Nat. Neurosci. 9, 315–321 (2006).
Gaffield, M.A., Rizzoli, S.O. & Betz, W.J. Mobility of synaptic vesicles in different pools in resting and stimulated frog motor nerve terminals. Neuron 51, 317–325 (2006).
Shtrahman, M., Yeung, C., Nauen, D.W., Bi, G.Q. & Wu, X.L. Probing vesicle dynamics in single hippocampal synapses. Biophys. J. 89, 3615–3627 (2005).
Jordan, R., Lemke, E.A. & Klingauf, J. Visualization of synaptic vesicle movement in intact synaptic boutons using fluorescence fluctuation spectroscopy. Biophys. J. 89, 2091–2102 (2005).
Betz, W.J., Mao, F. & Smith, C.B. Imaging exocytosis and endocytosis. Curr. Opin. Neurobiol. 6, 365–371 (1996).
Ryan, T.A. Presynaptic imaging techniques. Curr. Opin. Neurobiol. 11, 544–549 (2001).
Cousin, M.A. & Robinson, P.J. Mechanisms of synaptic vesicle recycling illuminated by fluorescent dyes. J. Neurochem. 73, 2227–2239 (1999).
Royle, S.J. & Lagnado, L. Endocytosis at the synaptic terminal. J. Physiol 553, 345–355 (2003).
de Lange, R.P., de Roos, A.D. & Borst, J.G. Two modes of vesicle recycling in the rat calyx of Held. J. Neurosci. 23, 10164–10173 (2003).
Richards, D.A., Guatimosim, C., Rizzoli, S.O. & Betz, W.J. Synaptic vesicle pools at the frog neuromuscular junction. Neuron 39, 529–541 (2003).
Kuromi, H. & Kidokoro, Y. Two distinct pools of synaptic vesicles in single presynaptic boutons in a temperature-sensitive Drosophila mutant, shibire. Neuron 20, 917–925 (1998).
Rizzoli, S.O. & Betz, W.J. Synaptic vesicle pools. Nat. Rev. Neurosci. 6, 57–69 (2005).
Neves, G. & Lagnado, L. The kinetics of exocytosis and endocytosis in the synaptic terminal of goldfish retinal bipolar cells. J. Physiol 515, 181–202 (1999).
Kuromi, H. & Kidokoro, Y. Tetanic stimulation recruits vesicles from reserve pool via a cAMP-mediated process in Drosophila synapses. Neuron 27, 133–143 (2000).
Ryan, T.A. & Smith, S.J. Vesicle pool mobilization during action potential firing at hippocampal synapses. Neuron 14, 983–989 (1995).
Cousin, M.A., Held, B. & Nicholls, D.G. Exocytosis and selective neurite calcium responses in rat cerebellar granule cells during field stimulation. Eur. J. Neurosci. 7, 2379–2388 (1995).
Acknowledgements
This work is supported by grants from MDA and NIH to W.J.B. We thank Steve Fadul for technical assistance, Michael Grybko for discussions on brain slices, Dr Silvio Rizzoli for discussions on phototoxicity, and Dr Joe Johnson and Dr Leah Sheridan for discussions on the text.
Author information
Authors and Affiliations
Corresponding author
Ethics declarations
Competing interests
The authors declare no competing financial interests.
Supplementary information
Supplementary Video 1
FM 1-43 destain in frog motor nerve terminal. The nerve was stimulated at 30 Hz. One revolution of the clock represents one minute. (AVI 1363 kb)
Rights and permissions
About this article
Cite this article
Gaffield, M., Betz, W. Imaging synaptic vesicle exocytosis and endocytosis with FM dyes. Nat Protoc 1, 2916–2921 (2006). https://doi.org/10.1038/nprot.2006.476
Published:
Issue Date:
DOI: https://doi.org/10.1038/nprot.2006.476
This article is cited by
-
Drosophila Atlastin regulates synaptic vesicle mobilization independent of bone morphogenetic protein signaling
Biological Research (2023)
-
Pneumolysin boosts the neuroinflammatory response to Streptococcus pneumoniae through enhanced endocytosis
Nature Communications (2022)
-
CDH2 mutation affecting N-cadherin function causes attention-deficit hyperactivity disorder in humans and mice
Nature Communications (2021)
-
Interactions of Antibodies to the Gram-Negative Gastric Bacterium Helicobacter pylori with the Synaptic Calcium Sensor Synaptotagmin 5, Correlate to Impaired Vesicle Recycling in SiMa Human Neuroblastoma Cells
Journal of Molecular Neuroscience (2021)
-
MicroRNA-153 impairs presynaptic plasticity by blocking vesicle release following chronic brain hypoperfusion
Cell Communication and Signaling (2020)
Comments
By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.