title: Lack of evidence for vesicular glutamate transporter expression in mouse astrocytes.
abstract: The concept of a tripartite synapse including a presynaptic terminal, a postsynaptic spine, and an astrocytic process that responds to neuronal activity by fast gliotransmitter release, confers to the electrically silent astrocytes an active role in information processing. However, the mechanisms of gliotransmitter release are still highly controversial. The reported expression of all three vesicular glutamate transporters (VGLUT1-3) by astrocytes suggests that astrocytes, like neurons, may release glutamate by exocytosis. However, the demonstration of astrocytic VGLUT expression is largely based on immunostaining, and the possibility of nonspecific labeling needs to be systematically addressed. We therefore examined the expression of VGLUT1-3 in astrocytes, both in culture and in situ. We used Western blots and single-vesicle imaging by total internal reflection fluorescence microscopy in live cultured astrocytes, and confocal microscopy, at the cellular level in cortical, hippocampal, and cerebellar brain slices, combined with quantitative image analysis. Control experiments were systematically performed in cultured astrocytes using wild-type, VGLUT1-3 knock-out, VGLUT1(Venus) knock-in, and VGLUT2-EGFP transgenic mice. In fixed brain slices, we quantified the degree of overlap between VGLUT1-3 and neuronal or astrocytic markers, both in an object-based manner using fluorescence line profiles, and in a pixel-based manner using dual-color scatter plots followed by the calculation of Pearson's correlation coefficient over all pixels with intensities significantly different from background. Our data provide no evidence in favor of the expression of any of the three VGLUTs by gray matter protoplasmic astrocytes of the primary somatosensory cortex, the thalamic ventrobasal nucleus, the hippocampus, and the cerebellum.
authors: INSERM U603, CNRS UMR8154, Laboratoire de Neurophysiologie et Nouvelles Microscopies, PRES Sorbonne Paris Cité, Université Paris Descartes, Paris, F-75006 France.
authors: Dongdong
authors: D
authors: Li
authors: Karine
authors: K
authors: Hérault
authors: Kätlin
authors: K
authors: Silm
authors: Alexis
authors: A
authors: Evrard
authors: Sonja
authors: S
authors: Wojcik
authors: Martin
authors: M
authors: Oheim
authors: Etienne
authors: E
authors: Herzog
authors: Nicole
authors: N
authors: Ropert
mesh_terms: D026922:Amino Acid Transport Systems, Acidic / Q000378:metabolism; D000818:Animals; D000831:Animals, Newborn; D001253:Astrocytes / Q000378:metabolism*; D002478:Cells, Cultured; D002540:Cerebral Cortex / Q000166:cytology; D000076122:Disks Large Homolog 4 Protein; D027342:Excitatory Amino Acid Transporter 2 / Q000378:metabolism; D005260:Female; D005786:Gene Expression Regulation / Q000235:genetics; D049452:Green Fluorescent Proteins / Q000235:genetics; D051528:Guanylate Kinases / Q000378:metabolism; D006624:Hippocampus / Q000166:cytology; D007091:Image Processing, Computer-Assisted; D066298:In Vitro Techniques; D008297:Male; D008565:Membrane Proteins / Q000378:metabolism; D051379:Mice; D008822:Mice, Transgenic; D018613:Microscopy, Confocal; D009419:Nerve Tissue Proteins / Q000378:metabolism; D050598:Vesicular Glutamate Transport Protein 1 / Q000378:metabolism; D050496:Vesicular Glutamate Transport Proteins / Q000145:classification / Q000235:genetics / Q000378:metabolism*
chemical_list: D026922:Amino Acid Transport Systems, Acidic; D000076122:Disks Large Homolog 4 Protein; D027342:Excitatory Amino Acid Transporter 2; D049452:Green Fluorescent Proteins; D051528:Guanylate Kinases; D008565:Membrane Proteins; D009419:Nerve Tissue Proteins; D050598:Vesicular Glutamate Transport Protein 1; D050496:Vesicular Glutamate Transport Proteins; C501447:Dlg4 protein, mouse; C504941:Slc17a8 protein, mouse