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.

The release of transmitters from glia influences synaptic functions. The modalities and physiological functions of glial release are poorly understood. Here we show that glutamate exocytosis from astrocytes of the rat hippocampal dentate molecular layer enhances synaptic strength at excitatory synapses between perforant path afferents and granule cells. The effect is mediated by ifenprodil-sensitive NMDA ionotropic glutamate receptors and involves an increase of transmitter release at the synapse. Correspondingly, we identify NMDA receptor 2B subunits on the extrasynaptic portion of excitatory nerve terminals. The receptor distribution is spatially related to glutamate-containing synaptic-like microvesicles in the apposed astrocytic processes. This glial regulatory pathway is endogenously activated by neuronal activity-dependent stimulation of purinergic P2Y1 receptors on the astrocytes. Thus, we provide the first combined functional and ultrastructural evidence for a physiological control of synaptic activity via exocytosis of glutamate from astrocytes.

The relative distribution of the excitatory amino acid transporter 2 (EAAT2) between synaptic terminals and astroglia, and the importance of EAAT2 for the uptake into terminals is still unresolved. Here we have used antibodies to glutaraldehyde-fixed D-aspartate to identify electron microscopically the sites of D-aspartate accumulation in hippocampal slices. About 3/4 of all terminals in the stratum radiatum CA1 accumulated D-aspartate-immunoreactivity by an active dihydrokainate-sensitive mechanism which was absent in EAAT2 glutamate transporter knockout mice. These terminals were responsible for more than half of all D-aspartate uptake of external substrate in the slices. This is unexpected as EAAT2-immunoreactivity observed in intact brain tissue is mainly associated with astroglia. However, when examining synaptosomes and slice preparations where the extracellular space is larger than in perfusion fixed tissue, it was confirmed that most EAAT2 is in astroglia (about 80%). Neither D-aspartate uptake nor EAAT2 protein was detected in dendritic spines. About 6% of the EAAT2-immunoreactivity was detected in the plasma membrane of synaptic terminals (both within and outside of the synaptic cleft). Most of the remaining immunoreactivity (8%) was found in axons where it was distributed in a plasma membrane surface area several times larger than that of astroglia. This explains why the densities of neuronal EAAT2 are low despite high levels of mRNA in CA3 pyramidal cell bodies, but not why EAAT2 in terminals account for more than half of the uptake of exogenous substrate by hippocampal slice preparations. This and the relative amount of terminal versus glial uptake in the intact brain remain to be discovered. NIH Public Access Author ManuscriptNeuroscience. Author manuscript; available in PMC 2009 November 11. Published in final edited form as:Neuroscience. Glutamate uptake into glia and neurons is essential for controlling the excitatory action of glutamate (for reviews see: Danbolt, 2001;Beart and O'Shea, 2007). It has been much debated whether the uptake activity of glutamatergic nerve terminals represents a major proportion of the total brain tissue uptake activity. The prevailing view is that most brain glutamate uptake is performed by astroglia, because (a) most of the glutamate uptake in forebrain tissue slices and synaptosome preparations is both dihydrokainate sensitive and dependent on the excitatory amino acid transporter (EAAT) 2 (GLT1; slc1a2) gene and protein, and (b) the highest numbers of dihydrokainate sensitive EAAT2 glutamate transporters are found in glial cells (for review see: Danbolt, 2001). This view, however, does not take account of a substantial amount of data showing a significant uptake into glutamatergic nerve terminals (e.g. Beart, 1976; StormMathisen, 1977;Gundersen et al., 1993Gundersen et al., , 1996Suchak et al., 2003;Xu et al., 2003;Waagepetersen et al., 2005; for a detailed discussion about the existence of nerve terminal glutamate up-take, see section 4.2 in Danbolt, 20...