Glutamate-induced Exocytosis of Glutamate from Astrocytes (original) (raw)
Related papers
Fusion-related Release of Glutamate from Astrocytes
Journal of Biological Chemistry, 2004
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.
Astrocytes contain a vesicular compartment that is competent for regulated exocytosis of glutamate
Nature Neuroscience, 2004
Astrocytes establish rapid cell-to-cell communication through the release of chemical transmitters. The underlying mechanisms and functional significance of this release are, however, not well understood. Here we identify an astrocytic vesicular compartment that is competent for glutamate exocytosis. Using postembedding immunogold labeling of the rat hippocampus, we show that vesicular glutamate transporters (VGLUT1/2) and the vesicular SNARE protein, cellubrevin, are both expressed in small vesicular organelles that resemble synaptic vesicles of glutamatergic terminals. Astrocytic vesicles, which are not as densely packed as their neuronal counterparts, can be observed in small groups at sites adjacent to neuronal structures bearing glutamate receptors. Fluorescently tagged VGLUT-containing vesicles were studied dynamically in living astrocytes by total internal reflection fluorescence (TIRF) microscopy. After activation of metabotropic glutamate receptors, astrocytic vesicles underwent rapid (milliseconds) Ca 2+ -and SNARE-dependent exocytic fusion that was accompanied by glutamate release. These data document the existence of a Ca 2+ -dependent quantal glutamate release activity in glia that was previously considered to be specific to synapses.
Morphological evidence for vesicular glutamate release from astrocytes
Neuroscience, 2009
There is now growing evidence that astrocytes, like neurons, can release transmitters. One transmitter that in a vast number of studies has been shown to be released from astrocytes is glutamate. Although asytrocytic glutamate may be released by several mechanisms, the evidence in favor of exocytosis is most compelling. Astrocytes may respond to neuronal activity by such exocytotic release of glutamate. The astrocyte derived glutamate can in turn activate neuronal glutamate receptors, in particular N-methyl-D-aspartate (NMDA) receptors. Here we review the morphological data supporting that astrocytes possess the machinery for exocytosis of glutamate. We describe the presence of small synaptic-like microvesicles, SNARE proteins and vesicular glutamate transporters in astrocytes, as well as NMDA receptors situated in vicinity of the astrocytic vesicles.
Vesicular Glutamate Transporter-Dependent Glutamate Release from Astrocytes
Journal of Neuroscience, 2004
Astrocytes exhibit excitability based on variations of their intracellular Ca 2ϩ concentrations, which leads to glutamate release, that in turn can signal to adjacent neurons. This glutamate-mediated astrocyte-neuron signaling occurs at physiological intracellular Ca 2ϩ levels in astrocytes and includes modulation of synaptic transmission. The mechanism underlying Ca 2ϩ -dependent glutamate release from astrocytes is most likely exocytosis, because astrocytes express the protein components of the soluble N-ethyl maleimide-sensitive fusion protein attachment protein receptors complex, including synaptobrevin 2, syntaxin, and synaptosome-associated protein of 23 kDa. Although these proteins mediate Ca 2ϩ -dependent glutamate release from astrocytes, it is not well understood whether astrocytes express functional vesicular glutamate transporters (VGLUTs) that are critical for vesicle refilling. Here, we find in cultured and freshly isolated astrocytes the presence of brain-specific Na ϩ -dependent inorganic phosphate cotransporter and differentiation-associated Na ϩ -dependent inorganic phosphate cotransporter that have recently been identified as VGLUTs 1 and 2. Indirect immunocytochemistry showed a punctate pattern of VGLUT immunoreactivity throughout the entire cell body and processes, whereas pharmacological inhibition of VGLUTs abolished mechanically and agonist-evoked Ca 2ϩ -dependent glutamate release from astrocytes. Taken together, these data indicate that VGLUTs play a functional role in exocytotic glutamate release from astrocytes.
SNARE protein-dependent glutamate release from astrocytes
The Journal of neuroscience : the official journal of the Society for Neuroscience, 2000
We investigated the cellular mechanisms underlying the Ca(2+)-dependent release of glutamate from cultured astrocytes isolated from rat hippocampus. Using Ca(2+) imaging and electrophysiological techniques, we analyzed the effects of disrupting astrocytic vesicle proteins on the ability of astrocytes to release glutamate and to cause neuronal electrophysiological responses, i.e., a slow inward current (SIC) and/or an increase in the frequency of miniature synaptic currents. We found that the Ca(2+)-dependent glutamate release from astrocytes is not caused by the reverse operation of glutamate transporters, because the astrocyte-induced glutamate-mediated responses in neurons were affected neither by inhibitors of glutamate transporters (beta-threo-hydroxyaspartate, dihydrokainate, and L-trans-pyrrolidine-2,4-dicarboxylate) nor by replacement of extracellular sodium with lithium. We show that Ca(2+)-dependent glutamate release from astrocytes requires an electrochemical gradient nece...
Ca2+ sources for the exocytotic release of glutamate from astrocytes
Biochimica et Biophysica Acta (BBA) - Molecular Cell Research, 2011
Astrocytes can exocytotically release the gliotransmitter glutamate from vesicular compartments. Increased cytosolic Ca 2+ concentration is necessary and sufficient for this process. The predominant source of Ca 2+ for exocytosis in astrocytes resides within the endoplasmic reticulum (ER). Inositol 1,4,5-trisphosphate and ryanodine receptors of the ER provide a conduit for the release of Ca 2+ to the cytosol. The ER store is (re)filled by the store-specific Ca 2+ -ATPase. Ultimately, the depleted ER is replenished by Ca 2+ which enters from the extracellular space to the cytosol via store-operated Ca 2+ entry; the TRPC1 protein has been implicated in this part of the astrocytic exocytotic process. Voltage-gated Ca 2+ channels and plasma membrane Na + /Ca 2+ exchangers are additional means for cytosolic Ca 2+ entry. Cytosolic Ca 2+ levels can be modulated by mitochondria, which can take up cytosolic Ca 2+ via the Ca 2+ uniporter and release Ca 2+ into cytosol via the mitochondrial Na + /Ca 2+ exchanger, as well as by the formation of the mitochondrial permeability transition pore. The interplay between various Ca 2+ sources generates cytosolic Ca 2+ dynamics that can drive Ca 2+ -dependent exocytotic release of glutamate from astrocytes. An understanding of this process in vivo will reveal some of the astrocytic functions in health and disease of the brain. This article is part of a Special Issue entitled: 11th European Symposium on Calcium. j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / b b a m c r 985 V. Parpura et al. / Biochimica et Biophysica Acta 1813 (2011) 984-991
Vesicular release of glutamate utilizes the proton gradient between the vesicle and synaptic cleft
Frontiers in Synaptic …, 2010
Glutamate is released from synaptic vesicles following formation of a fusion pore, connecting the vesicle interior with the synaptic cleft. Release is proposed to result from either full fusion of the vesicle with the terminal membrane or by ‘kiss-and-run,’ where release occurs through the fusion pore. ‘Kiss-and-run’ seems implausible as passive diffusion of glutamate through the pore is too slow to account for the rapidity of release. Vesicular accumulation of glutamate is driven by a proton gradient, resulting in the co-release of protons during exocytosis. We tested whether the proton gradient between the vesicle and cleft contributes to glutamate exocytosis. Collapse of the gradient reduced hippocampal glutamatergic transmission, an effect that was not associated with presynaptic changes in excitability, transmitter release probability, or postsynaptic sensitivity. These data indicate that approximately half of glutamate release utilizes the proton gradient between vesicle and cleft, suggesting a significant proportion of release by ‘kiss-and-run.’
Animal Cells and Systems
Bafilomycin A1, a vacuolar H +-ATPase inhibitor, and botulinum toxin B and tetanus toxin, both vesicle fusion inhibitors, are widely known exocytosis blockers that have been used to inhibit the presynaptic release of neurotransmitters. However, protein trafficking mechanisms, such as the insertion of postsynaptic receptors and astrocytic glutamatereleasing channels into the plasma membrane, also require exocytosis. In our previous study, exocytosis inhibitors reduced the surface expression of astrocytic glutamate-releasing channels. Here, we further investigated whether exocytosis inhibitors influence the surface expression of postsynaptic receptors. Using pH-sensitive superecliptic pHluorin (SEP)-tagged postsynaptic glutamate receptors, including GluA1, GluA2, GluN1, and GluN2A, we found that bafilomycin A1, botulinum toxin B, and/or tetanus toxin reduce the SEP fluorescence of SEP-GluA1, SEP-GluA2, SEP-GluN1, and SEP-GluN2A. These findings indicate that presynaptic vesicle exocytosis inhibitors also affect the postsynaptic trafficking machinery for surface expression. Finally, this study provides profound insights assembling presynaptic, postsynaptic and astrocytic viewpoints into the interpretation of the data obtained using these synaptic vesicle exocytosis inhibitors.
Characterization of Glutamate Uptake into Synaptic Vesicles
Journal of Neurochemistry, 1985
Recent evidence indicates that L-glutamate is taken up into synaptic vesicles in an ATP-dependent manner, supporting the notion that synaptic vesicles may be involved in glutamate synaptic transmission. In this study, we further characterized the ATP-dependent vesicular uptake of glutamate. Evidence is provided that a Mg-ATPase, not Ca-ATPase, is responsible for the ATP hydrolysis coupled to the glutamate uptake. The ATPdependent glutamate uptake was inhibited by agents known to dissipate the electrochemical proton gradient across the membrane of chromaffin granules. Hence, it is suggested that the vesicular uptake of glutamate is driven by electrochemical proton gradients generated by the Mg-ATPase. Of particular interest is the finding that the ATP-dependent glutamate uptake is markedly stimulated by chloride over a physiologically relevant, millimolar concentration range, suggesting an important role of intranerve terminal chloride in the accumulation of glutamate in synaptic vesicles. The vesicular glutamate translocator is highly specific for L-glutamate, and failed to interact with aspartate, its related agents, and most of the glutamate analogs tested. It is proposed that this vesicular translocator plays a crucial role in determining the fate of glutamate as a neurotransmitter. Key Words: Glutamate-Synaptic vesicle-Uptake-Mg-ATPase-Excitatory neurotransmitter. Naito S. and Ueda T. Characterization of glutamate uptake into synaptic vesicles.