Abstract
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.
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References
Kavalali ET, Jorgensen EM (2014) Visualizing presynaptic function. Nat Neurosci 17:10–16
Miesenbock G, De Angelis DA, Rothman JE (1998) Visualizing secretion and synaptic transmission with pH-sensitive green fluorescent proteins. Nature 394:192–195
Zhu Y, Xu J, Heinemann SF (2009) Two pathways of synaptic vesicle retrieval revealed by single-vesicle imaging. Neuron 61:397–411
Granseth B, Odermatt B, Royle SJ, Lagnado L (2006) Clathrin-mediated endocytosis is the dominant mechanism of vesicle retrieval at hippocampal synapses. Neuron 51:773–786
Voglmaier SM et al (2006) Distinct endocytic pathways control the rate and extent of synaptic vesicle protein recycling. Neuron 51:71–84
Li H et al (2011) Concurrent imaging of synaptic vesicle recycling and calcium dynamics. Front Mol Neurosci 4:34
Ramirez DM, Khvotchev M, Trauterman B, Kavalali ET (2012) Vti1a identifies a vesicle pool that preferentially recycles at rest and maintains spontaneous neurotransmission. Neuron 73:121–134
Raingo J et al (2012) VAMP4 directs synaptic vesicles to a pool that selectively maintains asynchronous neurotransmission. Nat Neurosci 15:738–745
Sankaranarayanan S, De Angelis D, Rothman JE, Ryan TA (2000) The use of pHluorins for optical measurements of presynaptic activity. Biophys J 79:2199–2208
Shaner NC et al (2004) Improved monomeric red, orange and yellow fluorescent proteins derived from Discosoma sp. red fluorescent protein. Nat Biotechnol 22:1567–1572
Li Y, Tsien RW (2012) pHTomato, a red, genetically encoded indicator that enables multiplex interrogation of synaptic activity. Nat Neurosci 15:1047–1053
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 207:419–432
Gandhi SP, Stevens CF (2003) Three modes of synaptic vesicular recycling revealed by single-vesicle imaging. Nature 423:607–613
Takamori S et al (2006) Molecular anatomy of a trafficking organelle. Cell 127:831–846
Leitz J, Kavalali ET (2014) Fast retrieval and autonomous regulation of single spontaneously recycling synaptic vesicles. Elife 3:e03658
Leitz J, Kavalali ET (2011) Ca(2)(+) influx slows single synaptic vesicle endocytosis. J Neurosci 31:16318–16326
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Afuwape, O.A.T., Kavalali, E.T. (2016). Imaging Synaptic Vesicle Exocytosis-Endocytosis with pH-Sensitive Fluorescent Proteins. In: Schwartzbach, S., Skalli, O., Schikorski, T. (eds) High-Resolution Imaging of Cellular Proteins. Methods in Molecular Biology, vol 1474. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-6352-2_11
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DOI: https://doi.org/10.1007/978-1-4939-6352-2_11
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