Imaging Synaptic Vesicle Exocytosis-Endocytosis with pH-Sensitive Fluorescent Proteins

  • Olusoji A. T. Afuwape
  • Ege T. Kavalali
Part of the Methods in Molecular Biology book series (MIMB, volume 1474)


The introduction of pHluorin, a pH-sensitive GFP, by Miesenbock and colleagues provided a versatile tool to studies of vesicle trafficking, in particular synaptic vesicle exocytosis and endocytosis. By tagging pHluorin to the luminal region of the synaptic vesicular protein synaptobrevin (also called VAMP, vesicle-associated membrane protein) or other synaptic vesicle-specific proteins such as the vesicular glutamate transporter-1, we are able to directly track synaptic vesicle endocytosis in response to stimuli in a molecularly specific manner. Here, we describe the process of imaging synaptic vesicle endocytosis in response to extracellular stimulation in dissociated neuronal cultures of hippocampal neurons obtained from rats—also applicable to mice—using pHluorin-tagged vesicular glutamate transporter-1 as a reporter.

Key words

PHluorin Synaptic vesicles Endocytosis Microscopy Live-cell imaging 


  1. 1.
    Kavalali ET, Jorgensen EM (2014) Visualizing presynaptic function. Nat Neurosci 17:10–16CrossRefGoogle Scholar
  2. 2.
    Miesenbock G, De Angelis DA, Rothman JE (1998) Visualizing secretion and synaptic transmission with pH-sensitive green fluorescent proteins. Nature 394:192–195CrossRefGoogle Scholar
  3. 3.
    Zhu Y, Xu J, Heinemann SF (2009) Two pathways of synaptic vesicle retrieval revealed by single-vesicle imaging. Neuron 61:397–411CrossRefPubMedCentralGoogle Scholar
  4. 4.
    Granseth B, Odermatt B, Royle SJ, Lagnado L (2006) Clathrin-mediated endocytosis is the dominant mechanism of vesicle retrieval at hippocampal synapses. Neuron 51:773–786CrossRefGoogle Scholar
  5. 5.
    Voglmaier SM et al (2006) Distinct endocytic pathways control the rate and extent of synaptic vesicle protein recycling. Neuron 51:71–84CrossRefGoogle Scholar
  6. 6.
    Li H et al (2011) Concurrent imaging of synaptic vesicle recycling and calcium dynamics. Front Mol Neurosci 4:34CrossRefPubMedCentralGoogle Scholar
  7. 7.
    Ramirez DM, Khvotchev M, Trauterman B, Kavalali ET (2012) Vti1a identifies a vesicle pool that preferentially recycles at rest and maintains spontaneous neurotransmission. Neuron 73:121–134CrossRefPubMedCentralGoogle Scholar
  8. 8.
    Raingo J et al (2012) VAMP4 directs synaptic vesicles to a pool that selectively maintains asynchronous neurotransmission. Nat Neurosci 15:738–745CrossRefPubMedCentralGoogle Scholar
  9. 9.
    Sankaranarayanan S, De Angelis D, Rothman JE, Ryan TA (2000) The use of pHluorins for optical measurements of presynaptic activity. Biophys J 79:2199–2208CrossRefPubMedCentralGoogle Scholar
  10. 10.
    Shaner NC et al (2004) Improved monomeric red, orange and yellow fluorescent proteins derived from Discosoma sp. red fluorescent protein. Nat Biotechnol 22:1567–1572CrossRefGoogle Scholar
  11. 11.
    Li Y, Tsien RW (2012) pHTomato, a red, genetically encoded indicator that enables multiplex interrogation of synaptic activity. Nat Neurosci 15:1047–1053CrossRefPubMedCentralGoogle Scholar
  12. 12.
    Shen Y, Rosendale M, Campbell RE, Perrais D (2014) pHuji, a pH-sensitive red fluorescent protein for imaging of exo- and endocytosis. J Cell Biol 207:419–432CrossRefPubMedCentralGoogle Scholar
  13. 13.
    Gandhi SP, Stevens CF (2003) Three modes of synaptic vesicular recycling revealed by single-vesicle imaging. Nature 423:607–613CrossRefGoogle Scholar
  14. 14.
    Takamori S et al (2006) Molecular anatomy of a trafficking organelle. Cell 127:831–846CrossRefGoogle Scholar
  15. 15.
    Leitz J, Kavalali ET (2014) Fast retrieval and autonomous regulation of single spontaneously recycling synaptic vesicles. Elife 3:e03658CrossRefPubMedCentralGoogle Scholar
  16. 16.
    Leitz J, Kavalali ET (2011) Ca(2)(+) influx slows single synaptic vesicle endocytosis. J Neurosci 31:16318–16326CrossRefPubMedCentralGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2016

Authors and Affiliations

  1. 1.Department of NeuroscienceUT Southwestern Medical CenterDallasUSA
  2. 2.Department of PhysiologyUT Southwestern Medical CenterDallasUSA

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