title: Examination of synaptic vesicle recycling using FM dyes during evoked, spontaneous, and miniature synaptic activities.
abstract: Synaptic vesicles in functional nerve terminals undergo exocytosis and endocytosis. This synaptic vesicle recycling can be effectively analyzed using styryl FM dyes, which reveal membrane turnover. Conventional protocols for the use of FM dyes were designed for analyzing neurons following stimulated (evoked) synaptic activity. Recently, protocols have become available for analyzing the FM signals that accompany weaker synaptic activities, such as spontaneous or miniature synaptic events. Analysis of these small changes in FM signals requires that the imaging system is sufficiently sensitive to detect small changes in intensity, yet that artifactual changes of large amplitude are suppressed. Here we describe a protocol that can be applied to evoked, spontaneous, and miniature synaptic activities, and use cultured hippocampal neurons as an example. This protocol also incorporates a means of assessing the rate of photobleaching of FM dyes, as this is a significant source of artifacts when imaging small changes in intensity.
authors.affiliation: Department of Molecular Physiology & Biophysics, University of Iowa Carver College of Medicine.
authors.forename: Sadahiro
authors.initials: S
authors.lastname: Iwabuchi
authors.affiliation: Department of Molecular Physiology & Biophysics, University of Iowa Carver College of Medicine.
authors.forename: Yasuhiro
authors.initials: Y
authors.lastname: Kakazu
authors.affiliation: Department of Molecular Physiology & Biophysics, University of Iowa Carver College of Medicine.
authors.forename: Jin-Young
authors.initials: JY
authors.lastname: Koh
authors.affiliation: Department of Biology & Biochemistry, University of Bath.
authors.forename: Kirsty M
authors.initials: KM
authors.lastname: Goodman
authors.affiliation: Department of Molecular Physiology & Biophysics, University of Iowa Carver College of Medicine; charles-harata@uiowa.edu.
authors.forename: N Charles
authors.initials: NC
authors.lastname: Harata
mesh_terms: D000818:Animals; D023902:Chromosome Pairing / Q000502:physiology*; D005456:Fluorescent Dyes / Q000737:chemistry*; D051379:Mice; D038761:Photobleaching; D017729:Presynaptic Terminals / Q000737:chemistry / Q000502:physiology; D011726:Pyridinium Compounds / Q000737:chemistry*; D000644:Quaternary Ammonium Compounds / Q000737:chemistry*; D051381:Rats; D009435:Synaptic Transmission / Q000502:physiology; D013572:Synaptic Vesicles / Q000737:chemistry / Q000502:physiology*
chemical_list: C113793:FM 1 84; C073804:FM1 43; D005456:Fluorescent Dyes; D011726:Pyridinium Compounds; D000644:Quaternary Ammonium Compounds
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