title: Fusion-related release of glutamate from astrocytes.
abstract: Although cell culture studies have implicated the presence of vesicle proteins in mediating the release of glutamate from astrocytes, definitive proof requires the identification of the glutamate release mechanism and the localization of this mechanism in astrocytes at synaptic locales. In cultured murine astrocytes we show an array of vesicle proteins, including SNARE proteins, and vesicular glutamate transporters that are required to fill vesicles with glutamate. Using immunocytochemistry and single-cell multiplex reverse transcription-PCR we demonstrate the presence of these proteins and their transcripts within astrocytes freshly isolated from the hippocampus. Moreover, immunoelectron microscopy demonstrates the presence of VGLUT1 in processes of astrocytes of the hippocampus. To determine whether calcium-dependent glutamate release is mediated by exocytosis, we expressed the SNARE motif of synaptobrevin II to prevent the formation of SNARE complexes, which reduces glutamate release from astrocytes. To further determine whether vesicular exocytosis mediates calcium-dependent glutamate release from astrocytes, we performed whole cell capacitance measurements from individual astrocytes and demonstrate an increase in whole cell capacitance, coincident with glutamate release. Together, these data allow us to conclude that astrocytes in situ express vesicle proteins necessary for filling vesicles with the chemical transmitter glutamate and that astrocytes release glutamate through a vesicle- or fusion-related mechanism.
authors.affiliation: Department of Neuroscience, University of Pennsylvania School of Medicine, Philadelphia, PA 19104, USA.
authors.forename: Qi
authors.initials: Q
authors.lastname: Zhang
authors.forename: Tina
authors.initials: T
authors.lastname: Pangrsic
authors.forename: Marko
authors.initials: M
authors.lastname: Kreft
authors.forename: Mojca
authors.initials: M
authors.lastname: Krzan
authors.forename: Nianzhen
authors.initials: N
authors.lastname: Li
authors.forename: Jai-Yoon
authors.initials: JY
authors.lastname: Sul
authors.forename: Michael
authors.initials: M
authors.lastname: Halassa
authors.forename: Elisabeth
authors.initials: E
authors.lastname: Van Bockstaele
authors.forename: Robert
authors.initials: R
authors.lastname: Zorec
authors.forename: Philip G
authors.initials: PG
authors.lastname: Haydon
mesh_terms: D020816:Amino Acid Motifs; D000818:Animals; D001253:Astrocytes / Q000378:metabolism*; D015153:Blotting, Western; D001921:Brain / Q000378:metabolism; D002118:Calcium / Q000378:metabolism; D002478:Cells, Cultured; D004594:Electrophysiology; D005089:Exocytosis; D018698:Glutamic Acid / Q000378:metabolism*; D006624:Hippocampus / Q000378:metabolism; D007150:Immunohistochemistry; D008565:Membrane Proteins / Q000737:chemistry / Q000378:metabolism; D051379:Mice; D008822:Mice, Transgenic; D008854:Microscopy, Electron; D008856:Microscopy, Fluorescence; D016253:Microscopy, Immunoelectron; D017434:Protein Structure, Tertiary; D050683:R-SNARE Proteins; D012333:RNA, Messenger / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D050600:SNARE Proteins; D013569:Synapses / Q000378:metabolism*; D013997:Time Factors; D033921:Vesicular Transport Proteins
chemical_list: D008565:Membrane Proteins; D050683:R-SNARE Proteins; D012333:RNA, Messenger; D050600:SNARE Proteins; D033921:Vesicular Transport Proteins; D018698:Glutamic Acid; D002118:Calcium