title: Imaging Synaptic Vesicle Exocytosis-Endocytosis with pH-Sensitive Fluorescent Proteins.
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.
authors: Department of Neuroscience, UT Southwestern Medical Center, 5323 Harry Hines Blvd., Dallas, TX, 75390-9111, USA.
authors: Olusoji A T
authors: OA
authors: Afuwape
authors: Department of Neuroscience, UT Southwestern Medical Center, 5323 Harry Hines Blvd., Dallas, TX, 75390-9111, USA. Ege.Kavalali@UTsouthwestern.edu.|Department of Physiology, UT Southwestern Medical Center, Dallas, TX, 75390-9111, USA. Ege.Kavalali@UTsouthwestern.edu.
authors: Ege T
authors: ET
authors: Kavalali
mesh_terms: D000818:Animals; D000831:Animals, Newborn; D004705:Endocytosis; D005089:Exocytosis / Q000502:physiology; D005456:Fluorescent Dyes / Q000737:chemistry; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism*; D057809:HEK293 Cells; D006624:Hippocampus / Q000378:metabolism / Q000648:ultrastructure; D006801:Humans; D006863:Hydrogen-Ion Concentration; D009474:Neurons / Q000378:metabolism / Q000648:ultrastructure*; D061848:Optical Imaging / Q000379:methods*; D061251:Primary Cell Culture; D021381:Protein Transport; D050683:R-SNARE Proteins / Q000235:genetics / Q000378:metabolism*; D051381:Rats; D013194:Staining and Labeling / Q000379:methods*; D009435:Synaptic Transmission / Q000502:physiology; D013572:Synaptic Vesicles / Q000378:metabolism / Q000648:ultrastructure*; D050496:Vesicular Glutamate Transport Proteins / Q000235:genetics / Q000378:metabolism
chemical_list: D005456:Fluorescent Dyes; C485187:PHluorin; D050683:R-SNARE Proteins; D050496:Vesicular Glutamate Transport Proteins; D049452:Green Fluorescent Proteins
keywords: Endocytosis; Live-cell imaging; Microscopy; PHluorin; Synaptic vesicles