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. 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. During the past decade there has been increasing evidence for both integrative and dynamic roles for astrocytes in the central nervous system. Following activation of G protein-coupled receptors, astrocytes exhibit calcium oscillations, leading to the release of the chemical transmitters glutamate and ATP (1Haydon P.G. Nat. Rev. Neurosci. 2001; 2: 185-193Crossref PubMed Scopus (1140) Google Scholar, 2Araque A. Parpura V. Sanzgiri R.P. Haydon P.G. Eur. J. Neurosci. 1998; 10: 2129-2142Crossref PubMed Scopus (420) Google Scholar, 3Fields R.D. Stevens-Graham B. Science. 2002; 298: 556-562Crossref PubMed Scopus (735) Google Scholar). Studies in vitro and in brain slices have led to the hypothesis of tripartite synaptic transmission (4Araque A. Parpura V. Sanzgiri R.P. Haydon P.G. Trends. Neurosci. 1999; 22: 208-215Abstract Full Text Full Text PDF PubMed Scopus (1697) Google Scholar); neuronal activity causes elevations of synaptically associated calcium in astrocytes, which in turn leads to the release of chemical transmitters from these glial cells to locally modulate synaptic transmission (2Araque A. Parpura V. Sanzgiri R.P. Haydon P.G. Eur. J. Neurosci. 1998; 10: 2129-2142Crossref PubMed Scopus (420) Google Scholar, 5Kang J. Jiang L. Goldman S.A. Nedergaard M. Nat. Neurosci. 1998; 1: 683-692Crossref PubMed Scopus (684) Google Scholar, 6Robitaille R. Neuron. 1998; 21: 847-855Abstract Full Text Full Text PDF PubMed Scopus (206) Google Scholar, 7Araque A. Sanzgiri R.P. Parpura V. Haydon P.G. J. Neurosci. 1998; 18: 6822-6829Crossref PubMed Google Scholar). The mechanisms mediating the release of these transmitters from astrocytes are, however, ill-defined and are still the subject of intense debate. At least three distinct release pathways have been proposed as mediating the calcium-dependent release of glutamate from astrocytes: the reversal of plasma membrane glutamate transporters, anion transporter mediate release mechanisms, and calcium-dependent exocytosis (8Attwell D. Nature. 1994; 369: 707-708Crossref PubMed Scopus (58) Google Scholar, 9Nedergaard M. Takano T. Hansen A.J. Nat. Rev. Neurosci. 2002; 3: 748-755Crossref PubMed Scopus (354) Google Scholar, 10Parpura V. Basarsky T.A. Liu F. Jeftinija K. Jeftinija S. Haydon P.G. Nature. 1994; 369: 744-747Crossref PubMed Scopus (1401) Google Scholar). Because the release of glutamate is stimulated by calcium elevations and is not affected by glutamate transport inhibitors and because changes in cell volume have not been detected coincident with release, it has been proposed that this transmitter is released through a vesicle-mediated exocytotic pathway. Several observations made using cultured astrocytes support such a vesicle-mediated exocytotic mechanism of glutamate release, including the calcium dependence of glutamate release (10Parpura V. Basarsky T.A. Liu F. Jeftinija K. Jeftinija S. Haydon P.G. Nature. 1994; 369: 744-747Crossref PubMed Scopus (1401) Google Scholar, 12Parpura V. Haydon P.G. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar, V. Basarsky T. R. Haydon P.G. PubMed Scopus Google the presence of vesicle proteins such as synaptobrevin II and V. Basarsky T. R. Haydon P.G. PubMed Scopus Google Scholar, V. M. 1999; PubMed Scopus Google the release of this transmitter that detected by cells reverse vesicular glutamate transporters and of reverse vesicular glutamate transporters and of L. M. T. S. J. Neurosci. 2001; 21: PubMed Google and the of the release to and A. Haydon P.G. J. Neurosci. PubMed Google Scholar, L. S. D. T. A. Nature. 1998; PubMed Scopus Google Scholar). Although evidence release through such a a has the of a vesicular mechanism of glutamate and release M. Takano T. Hansen A.J. Nat. Rev. Neurosci. 2002; 3: 748-755Crossref PubMed Scopus (354) Google and that release is mediated by a through which transmitter is released from the activation has been to of mediating the release of glutamate through the of the S. J. Neurosci. PubMed Google Scholar). the release of glutamate is mediated by an exocytotic astrocytes express the vesicular glutamate transporters required for the filling of vesicles with of to for exocytosis calcium-dependent glutamate release and membrane that of vesicles with the plasma membrane an increase in membrane coincident with the release of glutamate. In this we have of these Because there is whether cultured astrocytes their in situ we whether proteins to for exocytosis are in situ as as in cell have which vesicle and proteins are expressed in astrocytes in in astrocytes freshly isolated from the nervous and in astrocytes in the presence of SNARE proteins and vesicular glutamate transporters in freshly isolated astrocytes in the of in we the of these proteins for glutamate release by the formation of SNARE we of the of vesicles with the plasma membrane and coincident with vesicle the plasma astrocytes release the chemical transmitter glutamate. Together, these studies support for the hypothesis that calcium-dependent glutamate release from astrocytes is mediated by in from and cell culture from or by the and at the of of murine astrocytes and of astrocytes and as (2Araque A. Parpura V. Sanzgiri R.P. Haydon P.G. Eur. J. Neurosci. 1998; 10: 2129-2142Crossref PubMed Scopus (420) Google Scholar). a cell as a in freshly isolated from using from in a M. J. Neurosci. 2001; 21: PubMed Google Scholar). with and The and with at for with and of through and for immunocytochemistry or to a for from to individual astrocytes and for of and the The an a cell both and a to the cell and The to a and to allow the to the The an to a volume of to cells in the To for by from of or to using an of cell cells at for and The in or are in the with for the and for the performed in a and using and using a system. to have the the to proteins from of or to or cells in and and the through a The at at for The and at by an of from both and whole performed as V. Basarsky T. R. Haydon P.G. PubMed Scopus Google Scholar). and performed as A. Haydon P.G. J. Neurosci. PubMed Google Scholar). and the in these studies in using a The of by at the and as for of and using from synaptobrevin and from and a from of at In performed in which to for of that with a of the and and in astrocytes a of with a for Proc. Natl. Acad. Sci. U. S. A. 1998; PubMed Scopus Google Scholar). express a cell for the of the Because is necessary for to of glial cells to with such as cells with by calcium and in for for of cells and in in cells in and cultured to of for for the for to with The at to the cell The at to within of performed as Proc. Natl. Acad. Sci. U. S. A. 1998; PubMed Scopus Google Scholar). the cells from the or the II for synaptobrevin II from II by and from by and In an of and and synaptobrevin II of The of synaptobrevin II by of astrocytes leads to a in (2Araque A. Parpura V. Sanzgiri R.P. Haydon P.G. Eur. J. Neurosci. 1998; 10: 2129-2142Crossref PubMed Scopus (420) Google Scholar). as of glutamate release from astrocytes the of and the synaptobrevin II SNARE cell as (2Araque A. Parpura V. Sanzgiri R.P. Haydon P.G. Eur. J. Neurosci. 1998; 10: 2129-2142Crossref PubMed Scopus (420) Google Scholar). The identification of by their to and by the presence of synaptic stimulated using with the of an using a a in calcium stimulated as by a calcium During the of a the the calcium to the of with the cell of an calcium the The of this to astrocytes, which we by the of from stimulated to astrocytes stimulated at of at least astrocytes stimulated in and and data from at least three The of as the of to the of astrocytes stimulated in for these to the of for the such as the of the the of cells using the performed at for glutamate to glutamate release from cultured astrocytes B. Parpura V. Haydon P.G. J. Neurosci. PubMed Google Scholar). In the presence of glutamate released from astrocytes, reduces to a that by astrocytes in a and in which of with and by a using a and of the performed by from the cells in the and expressed as the of the and the in using this the of from as a or because of data from at least three using the of by that in to a B. Parpura V. Haydon P.G. J. Neurosci. PubMed Google in which we this glutamate we detected changes in in because in the we in in the we a of in astrocytes using the with for at changes in are as by the and at of and to of for the immunoelectron microscopy with and through the with of and of in the to and in the for an with a through the for in in to and in the for have been J. J. Neurosci. PubMed Scopus Google Scholar). the at for in a a and a in three in and in the at of VGLUT1 and using In using VGLUT1 detected using this in for by a in in a to for at by of the In VGLUT1 detected using the a Following a in the and a in VGLUT1 using a in of and of in of in and in a of and for in a to for at in the of and as and with in in for by a in and in of using a The for and in and in in for in and in of from the of the with a using a and with and of with of the to in the of not exhibit To of of the by for with of of the from with of and with both for the At least from at least from a of that in as in in the not at least three to The of VGLUT1 in by of of the that both to that not from of in the in by that and by at least to in the and of and with reversal of the for VGLUT1 and Because data of the data The of with to localization in or or glial are as of membrane capacitance measurements made using a at R. J. PubMed Scopus Google Scholar). of the whole cell and by and of The both by a and by the of this from the to to the G of the in we and G and the from a The to and capacitance at and and and at To for in measurements of whole cell capacitance that in we a to the to of The and in made at at a of The cell capacitance made with and in of as in in and as a the for the of a a vesicle-mediated exocytosis mechanism of glutamate release, we whether astrocytes express exocytotic Using reverse transcription-PCR we the presence of transcripts for vesicle proteins in astrocytes is that the formation of a SNARE is for exocytosis R. Rev. 1999; PubMed Scopus Google Scholar). the of culture studies as as studies of cells Liu L. 2002; PubMed Scopus Google Scholar, U. B. S. F. J. M. 2002; PubMed Scopus Google Scholar, R. J. PubMed Scopus Google Scholar, J. J. Full Text Full Text PDF PubMed Scopus Google by the presence of transcripts for the SNARE proteins, synaptobrevin II and V. Basarsky T. R. Haydon P.G. PubMed Scopus Google Scholar, V. M. 1999; PubMed Scopus Google Scholar, R. M. S. T. D. K. 1999; PubMed Scopus Google in astrocytes and that and proteins that with SNARE proteins not of the are in and immunocytochemistry and data not the presence of these proteins in astrocytes. Although these proteins are in both astrocytes and in neuronal astrocytes not express of the proteins in neuronal synaptic for a neuronal synaptic is not in cultured astrocytes. exocytosis mediates the release of astrocytes express the vesicular glutamate transporters required to fill the vesicle with this chemical and immunocytochemistry the presence of vesicular glutamate transporters and Science. PubMed Scopus Google Scholar, S. R. Nature. PubMed Scopus Google in astrocytes with studies of cultured astrocytes is whether such studies processes in to to studies in culture we astrocytes freshly isolated from the M. J. Neurosci. PubMed Scopus Google Scholar). and it is to individual astrocytes from the such that their in a freshly isolated from the of a in which the glial the of L. B. A. A. PubMed Scopus Google Scholar). isolated astrocytes, or in a and to single-cell multiplex to determine whether transcripts for the vesicle proteins in culture demonstrates that astrocytes transcripts for the SNARE proteins synaptobrevin II and as as the proteins and we detected transcripts for VGLUT1 and Because it is that neuronal we that we transcripts from of that isolated with the To for this we whether we for II J. Neuron. Full Text Full Text PDF PubMed Scopus Google Scholar). Although this detected in and it detected in astrocytes, that we transcripts using these freshly isolated detected in freshly isolated astrocytes or of the of cells for freshly isolated freshly isolated and to cells of the of cells for freshly isolated freshly isolated and to cells of the of cells for freshly isolated freshly isolated and to cells with of the of cells for freshly isolated freshly isolated and to cells with of the of cells for freshly isolated freshly isolated and to cells of the of cells for freshly isolated freshly isolated and to cells of the of cells for freshly isolated freshly isolated and to cells cells of the in by three The of that express a vesicle is The of cells is in that these the by three not in this a of a from the in as as and to for for the vesicle that within the by not by within In and the presence of within the not of the of cells for freshly isolated freshly isolated and to cells of the in by three The of that express a vesicle is The of cells is in that these the by three not in this a of a from the in as as and to for for the vesicle that within the by not by within In and the presence of within the not in a performed freshly isolated astrocytes the presence of the proteins by the transcripts for the exocytotic proteins a from an isolated which we performed for the neuronal synaptic and for Because are associated with the processes of astrocytes, we for a vesicle that is not expressed by astrocytes, to the of synaptic demonstrates in to the as by to freshly isolated astrocytes, is not detected within the processes of the Although VGLUT1 is expressed in not it within processes Because of that this VGLUT1 within astrocytes within we these and the from a The in show that VGLUT1 is within the volume of the the presence of this within astrocytes. In to VGLUT1 we the presence of synaptobrevin of of of and of within processes and demonstrate that both the transcripts and for a of vesicle proteins are within isolated astrocytes. VGLUT1 in in whether vesicular glutamate transporter is within astrocytes in situ we performed immunoelectron microscopy from astrocytes with that by an and VGLUT1 using demonstrates the presence of VGLUT1 in as as within the processes of astrocytes that with In these it that VGLUT1 is within processes to for an a to an and processes by the of of of processes for VGLUT1 through the of not detected in the of To that we detected using and VGLUT1 with In with these we detected within processes in to these processes by the presence of and by with a Together, this demonstrates the presence of VGLUT1 within processes in situ in to synaptic SNARE and that cultured astrocytes, freshly isolated astrocytes, and astrocytes in situ express the exocytotic proteins, we to cell culture to whether this exocytotic is necessary for the release of glutamate from astrocytes. a to glutamate released from astrocytes B. Parpura V. Haydon P.G. J. Neurosci. PubMed Google and that the of a of ATP causes a increase in which in turn the release of glutamate release of glutamate is because it is by the calcium and the T. T. S. T. K. J. PubMed Scopus Google and The calcium-dependent release of glutamate is by the with vesicular of released glutamate A. Haydon P.G. J. Neurosci. PubMed Google Scholar). The formation of the the SNARE is to for the of vesicles with the plasma studies using have implicated a SNARE as for the calcium-dependent release of glutamate from astrocytes A. Haydon P.G. J. Neurosci. PubMed Google Scholar, L. S. D. T. A. Nature. 1998; PubMed Scopus Google Scholar, Jeftinija PubMed Scopus Google Scholar). In these the of the SNARE and as a the SNARE not Because it is to the of an of transmitter release the is using we have an to SNARE formation and whether it glutamate release. studies have that the of an SNARE is to prevent the formation of the SNARE vesicle-mediated exocytosis of D. D. PubMed Scopus Google Scholar, Proc. Natl. Acad. Sci. U. S. A. 2001; PubMed Scopus Google Scholar, Neuron. Full Text Full Text PDF PubMed Scopus Google Scholar). the calcium-dependent release of chemical transmitter from astrocytes is mediated by exocytosis, it that the of the synaptobrevin II SNARE motif the release of glutamate calcium within astrocytes. To and of the SNARE motif in astrocytes we a of with a for Proc. Natl. Acad. Sci. U. S. A. 1998; PubMed Scopus Google Scholar). express a cell for the of the Because is required for to of glial cells to with such as Using this we within astrocytes. In we that cells that expressed to this and the of astrocytes in cell In it that we in isolated because is it is that this a the presence of within The of the SNARE not the of astrocytes for at least in processes such as calcium and to SNARE To whether the of the SNARE motif calcium-dependent release of we performed we the glutamate for glutamate release and that glutamate release is by the of the SNARE motif we as for released glutamate and that the in by of astrocytes (2Araque A. Parpura V. Sanzgiri R.P. Haydon P.G. Eur. J. Neurosci. 1998; 10: 2129-2142Crossref PubMed Scopus (420) Google in and by the of the SNARE motif and that the of glutamate release by SNARE in this with that in from a in with that from the to cell in Although the of the SNARE motif and the release of glutamate in of these the of and the of the SNARE motif at a of the calcium because the ATP the calcium of a vesicle-mediated mechanism of transmitter release is to determine whether the that transmitter release causes the of vesicle membrane to the plasma To this we made whole cell capacitance measurements to the of the plasma membrane A. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar). and the increase in membrane are detected as an increase in whole cell have that of the calcium a increase in calcium that in turn stimulated the exocytotic of vesicular membrane with the plasma membrane as by an increase in whole cell capacitance J. A. A. J. Neurosci. PubMed Google Scholar). Because calcium-dependent exocytosis is in astrocytes, we whether activation of by which the calcium-dependent release of glutamate L. S. D. T. A. Nature. 1998; PubMed Scopus Google a increase in membrane capacitance and release of glutamate. is an because it requires that the whole cell not the proteins required to activation to calcium and exocytosis A. B. Google Scholar). To of we as for a cell in that the glutamate release, whether there a in membrane of a increase in in In of these we detected a increase in whole cell capacitance In of a a in capacitance glutamate release. Because vesicles are to capacitance with the cell membrane A. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google and to it is not that we not individual Although their M. R. PubMed Scopus Google Scholar, Nature. 2002; PubMed Scopus Google in which we not have the to glutamate release, or requires the of of of Nature. 2002; PubMed Scopus Google which is not in this has a of the mechanism of calcium-dependent glutamate release from astrocytes. demonstrate an array of vesicle proteins in astrocytes freshly isolated from the nervous including the SNARE proteins synaptobrevin proteins and as as VGLUT1 and The presence of the SNARE proteins is not that cells are to express synaptobrevin II and Liu L. 2002; PubMed Scopus Google Scholar, U. B. S. F. J. M. 2002; PubMed Scopus Google Scholar, R. J. PubMed Scopus Google Scholar, J. J. Full Text Full Text PDF PubMed Scopus Google Scholar). we to release of chemical transmitter from astrocytes. we demonstrate the of VGLUT1 in processes to in which that this exocytotic is as a of it synaptic we demonstrate that the activation of glutamate to a calcium causes an increase in cell which we as to the of vesicles with the plasma membrane to an of the plasma membrane B. J. 1998; Full Text Full Text PDF PubMed Scopus Google and the release of glutamate. these data us to that glutamate is released from astrocytes by an exocytotic mechanism. it is still necessary to studies in which the presence of glutamate within vesicles is with including to and A. Haydon P.G. J. Neurosci. PubMed Google Scholar, L. S. D. T. A. Nature. 1998; PubMed Scopus Google and the presence of release of glutamate L. M. T. S. J. Neurosci. 2001; 21: PubMed Google it is that astrocytes release glutamate in an exocytotic at with the for astrocytes in released glutamate by plasma membrane transporters it is to within the and to as a of glutamate. Although glutamate are in to neuronal astrocytes glutamate at J. F. R. PubMed Scopus Google Scholar, R. J. PubMed Scopus Google Scholar, D. B. M. Neuron. Full Text PDF PubMed Scopus Google a for the of vesicular glutamate transporters that have of Science. PubMed Scopus Google Scholar, J. T. J. J. D. J. Proc. Natl. Acad. Sci. U. S. A. 2002; PubMed Scopus Google Scholar, J. 2002; Google Scholar). Moreover, a performed at the of in the that glutamate is to support synaptic transmission T. T. Neuron. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar). glutamate synaptic of the of synaptic in astrocytes have a glutamate these and to support the filling of glial vesicles with glutamate. studies to their of of required the presence of the and that cells for the Together, these data support the that astrocytes express and that these proteins are in processes to with immunoelectron microscopy is whether from the of and in processes in The of the is that it a that not from the J. J. Neurosci. PubMed Google Scholar, A. J. Neurosci. PubMed Google Scholar, J. A. A. J. Neurosci. PubMed Google Scholar). a with of is that to in the The in the is by in Because the of of is and to prevent of that not to glial In further support of the of to we not in we performed measurements of whole cell capacitance glutamate release through the of we detected the of membrane and the release of transmitter in of cells by evidence to support an exocytotic mechanism of glutamate release. is not in three of these cells we to a in whole cell however, is that the of transmitter release with exocytosis as has been in cells of and J. Nature. PubMed Scopus Google Scholar). Because exocytosis not detected as an increase in membrane and because astrocytes express and this the to release glutamate a in cell in this of the cells we Although there are a synaptic vesicle release and the of glutamate release from astrocytes, we that are not the Although proteins are astrocytes not express the synaptic vesicle Moreover, the of release from astrocytes is the release from of the of exocytosis that astrocytes are by at least of neuronal M. M. M. S. M. M. T. Haydon P.G. R. Google Scholar). we detected changes in whole cell capacitance of in cell a vesicle to capacitance with the cell the increase in cell capacitance of that we in cell culture to the of vesicles with the plasma Although this a it is that in situ with in J. Neurosci. 2002; 22: PubMed Google Scholar). it is not to that an release glutamate through the of of of vesicles immunoelectron microscopy that vesicular glutamate transporters are in processes to In to the of this is of because this in astrocytes in the of vesicle proteins in astrocytes is not a culture is it a associated with is not from this is glutamate released from astrocytes in neuronal in the of the plasma membrane transporter studies have that the of calcium within astrocytes is to the release of as detected by a glutamate L. S. D. T. A. Nature. 1998; PubMed Scopus Google Scholar). that this transmitter released and the transporter it is that released glutamate has at from of transporters and have a in the studies performed using to an of synaptic have that glutamate released from astrocytes to modulate synaptic transmission A. Sanzgiri R.P. Parpura V. Haydon P.G. J. Neurosci. 1998; 18: 6822-6829Crossref PubMed Google Scholar). is that astrocytes not vesicles within their processes in there are within an it is to conclude that with the of vesicles in that processes in situ vesicle proteins a immunoelectron microscopy is immunoelectron microscopy studies performed cultured astrocytes have a of vesicles associated with the vesicle synaptobrevin II V. M. 1999; PubMed Scopus Google Scholar). an in situ determine the of associated with these proteins, as as their and with to the the of a and an it is not that such a is The has to of transmitter in to that at of to Because the for of vesicles in is to of the of of it is not that the is with of In the a in which calcium are at of to not such a of because vesicle and with transmitter is of a to the calcium Because there are vesicles within the processes in to it is not that studies have to VGLUT1 in which immunoelectron microscopy performed to the of of a J. T. J. J. D. J. Proc. Natl. Acad. Sci. U. S. A. 2002; PubMed Scopus Google Scholar). is that astrocytes express three of the vesicular glutamate and astrocytes express or whether a to that exocytosis mediates the release of glutamate from astrocytes and that the mechanism for this release is in it is necessary to the in which we the to In to transmitters released by the to modulate synaptic it is to that transmitters released from synaptically associated astrocytes at the tripartite are in synaptic synaptically released exocytotic release of the and astrocytes are in an the of the Because has the to from associated J. Neurosci. 2002; 22: PubMed Google these glial cells both the synaptic and the of synaptic for for for for and for synaptobrevin II for VGLUT1 and and and for the of with