Use of FM1‐43 and Other Derivatives to Investigate Neuronal Function

Michael A. Cousin1

1 University of Edinburgh, Edinburgh, Scotland
Publication Name:  Current Protocols in Neuroscience
Unit Number:  Unit 2.6
DOI:  10.1002/0471142301.ns0206s43
Online Posting Date:  April, 2008
GO TO THE FULL TEXT: PDF or HTML at Wiley Online Library


The fluorescent dye FM1‐43 and its derivatives can be used to monitor the physiology of synaptic vesicle turnover in central nerve terminals. They do so by their ability to reversibly partition into membranes, a process that results in a huge increase in fluorescence in comparison to their quantum yield in solution. This unit provides protocols for quantifying total synaptic vesicle turnover, the kinetics and extent of synaptic vesicle exocytosis, and the kinetics and mode of synaptic vesicle endocytosis. Descriptions of other ways these protocols have been used to derive information about the life cycle of the synaptic vesicle are also provided. Curr. Protoc. Neurosci. 43:2.6.1‐2.6.12. © 2008 by John Wiley & Sons, Inc.

Keywords: FM1‐43; synaptic vesicle; exocytosis; endocytosis; fluorescence; FM2‐10; FM4‐64

PDF or HTML at Wiley Online Library

Table of Contents

  • Introduction
  • Basic Protocol 1: Visualization and Quantification of Synaptic Vesicle Exocytosis
  • Alternate Protocol 1: Visualization and Quantification of Synaptic Vesicle Endocytosis
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
PDF or HTML at Wiley Online Library


Basic Protocol 1: Visualization and Quantification of Synaptic Vesicle Exocytosis

  • Cultured neurons grown on glass coverslips (poly‐lysine‐coated or otherwise; e.g., Chapter 3)
  • Saline+ solution (see recipe), room temperature
  • 10 µM FM1‐43 (Molecular Probes) in saline+ solution (see recipe for saline+ solution)
  • 10 µM FM1‐43 in saline+ solution (see recipe for saline+ solution) supplemented with 50 to 100 mM KCl (reduce NaCl accordingly to maintain osmolarity)
  • Perfusion chamber with parallel platinum electrodes (RC‐21 BRFS, Warner Instrument) and perfusion apparatus (VC‐66CS, Warner Instrument)
  • Inverted epi‐fluorescence microscope with attached imaging system: light source (monochromator or filter wheel), cooled CCD camera, computer, and imaging software (see unit 2.1)
  • Stimulator (D330‐Multistim System, Digitimer Ltd.)
PDF or HTML at Wiley Online Library



Literature Cited

Literature Cited
   Angleson, J.K. and Betz, W.J. 1997. Monitoring secretion in real time: Capacitance, amperometry and fluorescence compared. Trends Neurosci. 20: 281‐287.
   Betz, W.J. and Henkel, A.W. 1994. Okadaic acid disrupts clusters of synaptic vesicles in frog motor nerve terminals. J. Cell. Biol. 124: 843‐854.
   Betz, W.J., Mao, F., and Bewick, G.S. 1992. Activity‐dependent fluorescent staining and destaining of living vertebrate motor nerve terminals. J. Neurosci. 12: 363‐375.
   Betz, W.J., Mao F., and Smith, C.B. 1996. Imaging exocytosis and endocytosis. Curr. Opin. Neurobiol. 6: 365‐371.
   Chi, P., Greengard, P., and Ryan, T.A. 2001. Synapsin dispersion and reclustering during synaptic activity. Nat. Neurosci. 4: 1187‐1193.
   Cochilla, A.J., Angleson, J.K., and Betz, W.J. 1999. Monitoring secretory membrane with FM1‐43 fluorescence. Annu. Rev. Neurosci. 22: 1‐10.
   Cousin, M.A. 2000. Synaptic vesicle endocytosis: Calcium works overtime in the nerve terminal. Mol. Neurobiol. 22: 115‐128.
   Cousin, M.A. and Nicholls, D.G. 1997. Synaptic vesicle recycling in cultured cerebellar granule cells: Role of vesicular acidification and refilling. J. Neurochem. 69: 1927‐1935.
   Cousin, M.A. and Robinson, P.J. 1999. Mechanisms of synaptic vesicle recycling illuminated by fluorescent dyes. J. Neurochem. 73: 2227‐2239.
   Evans, G.J.O. and Cousin, M.A. 2007. Activity‐dependent control of slow synaptic vesicle endocytosis by cyclin‐dependent kinase 5. J. Neurosci. 27: 401‐411.
   Feng, J., Chi, P., Blanpied, T.A., Xu, Y., Magarinos, A.M., Ferreira, A., Takahashi, R.H., Kao, H.T., McEwen, B.S., Ryan, T.A., Augustine, G.J., and Greengard, P. 2002. Regulation of neurotransmitter release by synapsin III. J. Neurosci. 22: 4372‐4380.
   Fernandez‐Alfonso, T., Kwan, R., and Ryan, T.A. 2006. Synaptic vesicles interchange their membrane proteins with a large surface reservoir during recycling Neuron 51: 179‐186.
   Harata, N., Pyle, J.L., Aravanis, A.M., Mozhayeva, M., Kavalali, E.T., and Tsien, R.W. 2001. Limited numbers of recycling vesicles in small CNS nerve terminals: Implications for neural signaling and vesicular cycling. Trends Neurosci. 24: 637‐643.
   Henkel, A.W., Lubke, J., and Betz, W.J. 1996a. FM1‐43 dye ultrastructural localization in and release from frog motor nerve terminals. Proc. Natl. Acad. Sci. U.S.A. 93: 1918‐1923.
   Henkel, A.W., Simpson, L.L., Ridge, R.M.A.P., and Betz, W.J. 1996b. Synaptic vesicle movements monitored by fluorescence recovery after photobleaching in nerve terminals stained with FM1‐43. J. Neurosci. 16: 3960‐3967.
   Klingauf, J., Kavalali, E.T., and Tsien, R.W. 1998. Kinetics and regulation of fast endocytosis at hippocampal synapses. Nature 394: 581‐585.
   Li, Z. and Murthy, V.N. 2001. Visualizing postendocytic traffic of synaptic vesicles at hippocampal synapses. Neuron 31: 593‐605.
   Ma, L., Zablow, L., Kandel, E.R., and Siegelbaum, S.A. 1999. Cyclic AMP induces functional presynaptic boutons in hippocampal CA3‐CA1 neuronal cultures. Nat. Neurosci. 2: 24‐30.
   Micheva, K.D. and Smith, S.J. 2005. Strong effects of subphysiological temperature on the function and plasticity of mammalian presynaptic terminals. J. Neurosci. 25: 7481‐7488.
   Murthy, V.N. and Stevens, C.F. 1998. Synaptic vesicles retain their identity through the endocytic cycle. Nature 392: 497‐501.
   Neale, E.A., Bowers, L.M., Jia, M., Bateman, K.E., and Williamson, L.C. 1999. Botulinum neurotoxin A blocks synaptic vesicle exocytosis but not endocytosis at the nerve terminal. J. Cell Biol. 147: 1249‐1260.
   Neves, G. and Lagnado, L. 1999. The kinetics of exocytosis and endocytosis in the synaptic terminal of goldfish retinal bipolar cells. J. Physiol. 515: 181‐202.
   Parsons, R.L., Calupca, M.A., Merriam, L.A., and Prior, C. 1999. Empty synaptic vesicles recycle and undergo exocytosis at vesamicol‐treated motor nerve terminals. J. Neurophysiol. 81: 2696‐2700.
   Pocock, J.M., Cousin, M.A., Parkin J., and Nicholls, D.G. 1995. Glutamate exocytosis from cerebellar granule cells: The mechanism of a transition to an L‐type Ca2+ channel coupling. J. Neurosci. 67: 595‐607.
   Pyle, J.L., Kavalali, E.T., Piedras‐Renteria, E.S., and Tsien, R.W. 2000. Rapid reuse of readily releasable pool vesicles at hippocampal synapses. Neuron 28: 221‐231.
   Richards, D.A., Guatimosim, C., and Betz, W.J. 2000. Two endocytic recycling routes selectively fill two vesicle pools in frog motor nerve terminals. Neuron 27: 551‐559.
   Rouze, N.C. and Schwartz, E.A. 1998. Continuous and transient vesicle cycling at a ribbon synapse. J. Neurosci. 18: 8614‐8624.
   Ryan, T.A. and Reuter, H. 2001. Measurements of vesicle recycling in central neurons. News Physiol. Sci. 16: 10‐14.
   Ryan, T.A. and Smith, S.J. 1995. Vesicle pool mobilization during action potential firing at hippocampal synapses. Neuron 14: 983‐989.
   Ryan, T.A., Li, L., Chin, L.S., Greengard, P., and Smith, S.J. 1996a. Synaptic vesicle recycling in synapsin I knock‐out mice. J. Cell Biol. 134: 1219‐1227.
   Ryan, T.A., Smith, S.J., and Reuter, H. 1996b. The timing of synaptic vesicle endocytosis. Proc. Natl. Acad. Sci. U.S.A. 93: 5567‐5571.
   Ryan, T.A., Ziv, N.E., and Smith, S.J. 1996c. Potentiation of evoked vesicle turnover at individually resolved synaptic boutons. Neuron 17: 125‐134.
   Sara, Y., Mozhayeva, M.G., Liu, X., and Kavalali, E.T. 2002. Fast vesicle recycling supports neurotransmission during sustained stimulation at hippocampal synapses. J. Neurosci. 22: 1608‐1617.
   Schikorski, T. and Stevens, C.F. 2001. Morphological correlates of functionally defined synaptic vesicle populations. Nat. Neurosci. 4: 391‐395.
   Smith, C.B. and Betz, W.J. 1996. Simultaneous independent measurement of endocytosis and exocytosis. Nature 380: 531‐534.
   Vanden Berghe, P. and Klingauf, J. 2006. Synaptic vesicles in rat hippocampal boutons recycle to different pools in a use‐dependent fashion. J. Physiol. 572: 707‐720.
   Virmani, T., Han, W., Liu, X., Sudhof, T.C., and Kavalali, E.T. 2003. Synaptotagmin 7 splice variants differentially regulate synaptic vesicle recycling. EMBO J. 22: 5347‐5357.
   Wienisch, M. and Klingauf, J. 2006. Vesicular proteins exocytosed and subsequently retrieved by compensatory endocytosis are nonidentical. Nat. Neurosci. 9: 1019‐1027.
PDF or HTML at Wiley Online Library