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Studying vesicle cycling in presynaptic terminals using the genetically encoded probe synaptopHluorin

Nature Protocols volume 1, pages 2970–2978 (2006)Cite this article

Abstract

Genetically encoded fluorescent probes have become indispensable tools in the biological sciences. Studies of synaptic vesicle recycling have been facilitated by a group of GFP-derived probes called pHluorins. These probes exploit changes in pH that accompany exocytosis and recapture of synaptic vesicles. Here we describe how these synaptic tracers can be used in rodent hippocampal neurons to monitor the synaptic vesicle cycle in real time and to obtain mechanistic insights about it. Synapses can be observed in living samples using a wide-field fluorescence microscope and a cooled charge-coupled device camera. A simple specimen chamber allows electrical stimulation of synapses to evoke exocytosis in a precisely controlled manner. We present protocols to measure various parameters of the synaptic vesicle cycle. This technique can be easily adapted to study different classes of synapses from wild-type and mutant mice. Once cultured neurons expressing synaptopHluorin are available, the whole procedure should take about 2 h.

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Figure 1: The principle behind SynaptopHluorin (spH) responses.
Figure 2: The stimulation chamber.
Figure 3: Choosing ROIs and calculating changes in spH fluorescence.
Figure 4: spH responses in hippocampal neurons from Thy1.2-spH transgenic mice.

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References

  1. De Paola, V. et al. Cell type-specific structural plasticity of axonal branches and boutons in the adult neocortex. Neuron 49, 861–875 (2006).

    Article  CAS  Google Scholar 

  2. Feng, G. et al. Imaging neuronal subsets in transgenic mice expressing multiple spectral variants of GFP. Neuron 28, 41–51 (2000).

    Article  CAS  Google Scholar 

  3. Kasthuri, N. & Lichtman, J.W. Structural dynamics of synapses in living animals. Curr. Opin. Neurobiol. 14, 105–111 (2004).

    Article  CAS  Google Scholar 

  4. Keller-Peck, C.R. et al. Asynchronous synapse elimination in neonatal motor units: studies using GFP transgenic nice. Neuron 31, 381–394 (2001).

    Article  CAS  Google Scholar 

  5. Lendvai, B., Stern, E.A., Chen, B. & Svoboda, K. Experience-dependent plasticity of dendritic spines in the developing rat barrel cortex in vivo. Nature 404, 876–881 (2000).

    Article  CAS  Google Scholar 

  6. Miesenbock, G., De Angelis, D.A. & Rothman, J.E. Visualizing secretion and synaptic transmission with pH-sensitive green fluorescence proteins. Nature 394, 192–195 (1998).

    Article  CAS  Google Scholar 

  7. Jahn, R., Lang, T. & Sudhof, T.C. Membrane fusion. Cell 112, 519–533 (2003).

    Article  CAS  Google Scholar 

  8. Murthy, V.N. & De Camilli, P. Cell biology of the presynaptic terminal. Annu. Rev. Neurosci. 26, 701–728 (2003).

    Article  CAS  Google Scholar 

  9. Sudhof, T.C. The synaptic vesicle cycle. Annu. Rev. Neurosci. 27, 509–547 (2004).

    Article  Google Scholar 

  10. Moulder, K.L. & Mennerick, S. Reluctant vesicles contribute to the total readily releasable pool in glutamatergic hippocampal neurons. J. Neurosci. 25, 3842–3850 (2005).

    Article  CAS  Google Scholar 

  11. Rizzoli, S.O. & Betz, W.J. The structural organization of the readily releasable pool of synaptic vesicles. Science 303, 2037–2039 (2004).

    Article  CAS  Google Scholar 

  12. Rosenmund, C. & Stevens, C.F. Definition of the readily releasable pool of vesicles at hippocampal synapses. Neuron 16, 1197–1207 (1996).

    Article  CAS  Google Scholar 

  13. Schikorski, T. & Stevens, C.F. Morphological correlates of functionally defined synaptic vesicle populations. Nat. Neurosci. 4, 391–395 (2001).

    Article  CAS  Google Scholar 

  14. Sankaranarayanan, S., De Angelis, D., Rothman, J.E. & Ryan, T.A. The use of pHluorins for optical measurements of presynaptic activity. Biophys. J. 79, 2199–2208 (2000).

    Article  CAS  Google Scholar 

  15. Granseth, B., Odermatt, B., Royle, S. & Lagnado, L. Clathrin-mediated endocytosis is the dominant mechanism of vesicle retrieval at hippocampal synapses. Neuron 51, 773–786 (2006).

    Article  CAS  Google Scholar 

  16. 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).

    Article  CAS  Google Scholar 

  17. Wienisch, M. & Klingauf, J. Vesicular proteins exocytosed and subsequently retrieved by compensatory endocytosis are nonidentical. Nat. Neurosci. 9, 1019–1027 (2006).

    Article  CAS  Google Scholar 

  18. Voglmaier, S.M. et al. Distinct endocytic pathways control the rate and extent of synaptic vesicle protein recycling. Neuron 51, 71–84 (2006).

    Article  CAS  Google Scholar 

  19. Ashby, M.C., Maier, S.R., Nishimune, A. & Henley, J.M. Lateral diffusion drives constitutive exchange of AMPA receptor at dendritic spines and is regulated by spine morphology. J. Neurosci. 26, 7046–7055 (2006).

    Article  CAS  Google Scholar 

  20. Jacob, T.C. et al. Gephyrin regulates the cell surface dynamics of synaptic GABAA receptors. J. Neurosci. 25, 10469–10478 (2005).

    Article  CAS  Google Scholar 

  21. Pelkey, K.A., Yuan, X., Lavezzari, G., Roche, K.W. & McBain, C.J. mGluR7 undergoes rapid internalization in response to activation by the allosteric agonist AMN082. Neuropharmacology published online 14 August 2006 (doi: 10.1016/j.neuropharm.2006.07.020).

  22. Ng, M. et al. Transmission of olfactory information between three populations of neurons in the antennal lobe of the fly. Neuron 36, 463–474 (2002).

    Article  CAS  Google Scholar 

  23. Bozza, T., McGann, J.P., Mombaerts, P. & Wachowiak, M. In vivo imaging of neuronal activity by targeted expression of a genetically encoded probe in the mouse. Neuron 42, 9–21 (2004).

    Article  CAS  Google Scholar 

  24. Samuel, A.D.T., Silva, R. & Murthy, V.N. Synaptic activity of the AFD neurons in Caenorhabditis elegans correlates with thermotactic memory. J. Neurosci. 23, 373–376 (2003).

    Article  CAS  Google Scholar 

  25. Kim, J. et al. Presynaptic activation of silent synapses and growth of new synapses contribute to intermediate and long-term facilitation in Aplysia. Neuron 40, 151–165 (2003).

    Article  CAS  Google Scholar 

  26. Li, Z. et al. Synaptic vesicle recycling studied in transgenic mice expressing synaptopHluorin. Proc. Natl. Acad. Sci. 102, 6131–6136 (2005).

    Article  CAS  Google Scholar 

  27. Araki, R. et al. Transgenic mouse lines expressing synaptopHluorin in hippocampus and cerebellar cortex. Genesis 42, 53–60 (2005).

    Article  CAS  Google Scholar 

  28. Caroni, P. Overexpression of growth-associated proteins in the neurons of adult transgenic mice. J. Neurosci. Methods 71, 3–9 (1997).

    Article  CAS  Google Scholar 

  29. Jiang, M. & Chen, G. High Ca2+-phosphate transfection efficiency in low-density neuronal cultures. Nat. Protocols 1, 695–700 (2006).

    Article  CAS  Google Scholar 

  30. Xia, Z., Dudek, H., Miranti, C.K. & Greenberg, M.E. Calcium influx via the NMDA receptor induces immediate early gene transcription by a MAP kinase/ERK-dependent mechanism. J. Neurosci. 16, 5425–5436 (1996).

    Article  CAS  Google Scholar 

  31. 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).

    Article  CAS  Google Scholar 

  32. Li, Z. & Murthy, V.N. Visualizing postendocytic traffic of synaptic vesicles at hippocampal synapses. Neuron 31, 593–605 (2001).

    Article  CAS  Google Scholar 

  33. Schikorski, T. & Stevens, C.F. Quantitative ultrastructural analysis of hippocampal excitatory synapses. J. Neurosci. 17, 5858–5867 (1997).

    Article  CAS  Google Scholar 

  34. 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).

    Article  CAS  Google Scholar 

  35. Newton, A.J., Kirchhausen, T. & Murthy, V.N. Inhibition of dynamin completely blocks compensatory synaptic vesicle endocytosis. Proc. Natl. Acad. Sci. U S A. 103, 17955–17960 (2006).

    Article  CAS  Google Scholar 

  36. Atluri, P.P. & Ryan, T.A. The kinetics of synaptic vesicle reacidification at hippocampal nerve terminals. J. Neurosci. 26, 2313–2320 (2006).

    Article  CAS  Google Scholar 

  37. Yudowski, G.A., Puthenveedu, M.A. & von Zastrow, M. Distinct modes of regulated receptor insertion to the somatodendritic plasma membrane. Nat. Neurosci. 9, 622–627 (2006).

    Article  CAS  Google Scholar 

  38. Dittman, J.S. & Kaplan, J.M. Factors regulating the abundance and localization of synaptobrevin in the plasma membrane. Proc. Natl. Acad. Sci. 103, 11399–11404 (2006).

    Article  CAS  Google Scholar 

  39. Yu, D., Ponomarev, A. & Davis, R.L. Altered representation of the spatial code for odors after olfactory classical conditioning: memory trace formation by synaptic recruitment. Neuron 42, 437–449 (2004).

    Article  CAS  Google Scholar 

  40. Poskanzer, K.E., Marek, K.W., Sweeney, S.T. & Davis, G.W. Synaptotagmin I is necessary for compensatory synaptic vesicle endocytosis in vivo. Nature 426, 559–563 (2003).

    Article  CAS  Google Scholar 

  41. Reiff, D.F. et al. In vivo performance of genetically encoded indicators of neural activity in flies. J. Neurosci. 25, 4766–4778 (2005).

    Article  CAS  Google Scholar 

  42. Sturman, D.A., Shakiryanova, D., Hewes, R.S., Deitcher, D.L. & Levitan, E.S. Nearly neutral secretory vesicles in Drosophila nerve terminals. Biophys. J. 90, L45–L47 (2006).

    Article  CAS  Google Scholar 

  43. McGann, J.P. et al. Odorant representations are modulated by intra- but not interglomerular presynaptic inhibition of olfactory sensory neurons. Neuron 48, 1039–1053 (2005).

    Article  CAS  Google Scholar 

  44. Wachowiak, M. et al. Inhibition of olfactory receptor neuron input to olfactory bulb glomeruli mediated by suppression of presynaptic calcium influx. J. Neurophysiol. 94, 2700–2712 (2005).

    Article  CAS  Google Scholar 

  45. Gandhi, S.P. & Stevens, C.F. Three modes of synaptic vesicular recycling revealed by single-vesicle imaging. Nature 423, 607–613 (2003).

    Article  CAS  Google Scholar 

  46. Sankaranarayanan, S. & Ryan, T.A. Calcium accelerates endocytosis of vSNAREs at hippocampal synapses. Nat. Neurosci. 4, 129–136 (2001).

    Article  CAS  Google Scholar 

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Author information

Author notes

  1. Juan Burrone and Zhiying Li: These authors contributed equally to this work.

Authors and Affiliations

  1. MRC Center for Developmental Neurobiology, King's College London, London, SE1 1UL, UK

    Juan Burrone

  2. Rockefeller University, New York, 10021, New York, USA

    Zhiying Li

  3. Department of Molecular and Cellular Biology Harvard University, 16 Divinity Avenue, Cambridge, 02138, Massachusetts, USA

    Venkatesh N Murthy

Authors
  1. Juan Burrone
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  2. Zhiying Li
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  3. Venkatesh N Murthy
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Corresponding author

Correspondence to Venkatesh N Murthy.

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Supplementary information

Supplementary Note

Protocol for preparing dissociated rodent hippocampal neuron cultures. (PDF 100 kb)

Supplementary Video 1

A typical experiment with neurons obtained from a transgenic mouse line in which a subset of neurons express synaptopHluorin. Fluorescence increases as neurotransmitter is released and spH is exposed to the extracellular space during neuronal stimulation. When stimulation ceases, the fluorescence decays back to baseline due to endocytosis and reacidification of synaptic vesicles. (AVI 3729 kb)

Supplementary Video 2

A typical experiment with neurons obtained from rat hippocampus, where synaptopHluorin expression is achieved by transient transfection. Fluorescence increases as neurotransmitter is released and spH is exposed to the extracellular space during neuronal stimulation. When stimulation ceases, the fluorescence decays back to baseline due to endocytosis and reacidification of synaptic vesicles. (AVI 3112 kb)

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Burrone, J., Li, Z. & Murthy, V. Studying vesicle cycling in presynaptic terminals using the genetically encoded probe synaptopHluorin. Nat Protoc 1, 2970–2978 (2006). https://doi.org/10.1038/nprot.2006.449

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