SDF 1-alpha (CXCL12) triggers glutamate exocytosis from astrocytes on a millisecond time scale: imaging analysis at the single-vesicle level with TIRF microscopy.
Chemokines are small chemotactic molecules widely expressed throughout the central nervous system. A number of papers, during the past few years, have suggested that they have physiological functions in addition to their roles in neuroinflammatory diseases. In this context, the best evidence concerns the CXC-chemokine stromal cell-derived factor (SDF-1alpha or CXCL12) and its receptor CXCR4, whose signalling cascade is also implicated in the glutamate release process from astrocytes. Recently, astrocytic synaptic like microvesicles (SLMVs) that express vesicular glutamate transporters (VGLUTs) and are able to release glutamate by Ca2+-dependent regulated exocytosis, have been described both in tissue and in cultured astrocytes. Here, in order to elucidate whether SDF-1alpha/CXCR4 system can participate to the brain fast communication systems, we investigated whether the activation of CXCR4 receptor triggers glutamate exocytosis in astrocytes. By using total internal reflection (TIRF) microscopy and the membrane-fluorescent styryl dye FM4-64, we adapted an imaging methodology recently developed to measure exocytosis and recycling in synaptic terminals, and monitored the CXCR4-mediated exocytosis of SLMVs in astrocytes. We analyzed the co-localization of VGLUT with the FM dye at single-vesicle level, and observed the kinetics of the FM dye release during single fusion events. We found that the activation of CXCR4 receptors triggered a burst of exocytosis on a millisecond time scale that involved the release of Ca2+ from internal stores. These results support the idea that astrocytes can respond to external stimuli and communicate with the neighboring cells via fast release of glutamate.
Department of Cell Biology and Morphology, FBM, University of Lausanne, rue du Bugnon 9, 1005 Lausanne, Switzerland.
Corrado
C
Calì
Julie
J
Marchaland
Romano
R
Regazzi
Paola
P
Bezzi
D000818:Animals; D000831:Animals, Newborn; D001253:Astrocytes / Q000187:drug effects* / Q000378:metabolism; D002118:Calcium / Q000378:metabolism; D002478:Cells, Cultured; D002614:Chelating Agents / Q000494:pharmacology; D054377:Chemokine CXCL12 / Q000494:pharmacology*; D004305:Dose-Response Relationship, Drug; D004533:Egtazic Acid / Q000031:analogs & derivatives / Q000494:pharmacology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005089:Exocytosis / Q000187:drug effects*; D018698:Glutamic Acid / Q000378:metabolism*; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D007424:Intracellular Fluid / Q000187:drug effects* / Q000378:metabolism; D008734:Methoxyhydroxyphenylglycol / Q000031:analogs & derivatives / Q000494:pharmacology; D008857:Microscopy, Interference / Q000379:methods*; D011726:Pyridinium Compounds / Q000378:metabolism; D000644:Quaternary Ammonium Compounds / Q000378:metabolism; D051381:Rats; D019718:Receptors, CXCR4 / Q000502:physiology; D013997:Time Factors; D014162:Transfection / Q000379:methods; D050599:Vesicular Glutamate Transport Protein 2 / Q000235:genetics / Q000378:metabolism
D002614:Chelating Agents; D054377:Chemokine CXCL12; C486264:Cxcr4 protein, rat; D004791:Enzyme Inhibitors; C092350:FM 4-64; D011726:Pyridinium Compounds; D000644:Quaternary Ammonium Compounds; D019718:Receptors, CXCR4; D050599:Vesicular Glutamate Transport Protein 2; C485184:enhanced green fluorescent protein; C070379:1,2-bis(2-aminophenoxy)ethane N,N,N',N'-tetraacetic acid acetoxymethyl ester; D049452:Green Fluorescent Proteins; D018698:Glutamic Acid; D004533:Egtazic Acid; D008734:Methoxyhydroxyphenylglycol; D002118:Calcium; C010117:3,4-dihydroxyphenylglycol