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

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

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.

Key words

PHluorin Synaptic vesicles Endocytosis Microscopy Live-cell imaging 

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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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