• exocytosis;
  • intracellular Ca2+ dynamics;
  • synaptic transmission;
  • tripartite synapse


  1. Top of page
  2. Abstract
  3. Tripartite synapse: structural association between neuronal and astrocytic elements
  4. Astrocytes respond to glutamatergic synaptic transmission
  5. Exocytotic/vesicular release of glutamate from astrocytes
  6. Neuronal responses to glutamate released from astrocytes
  7. Concluding remarks
  8. Acknowledgements
  9. References

The major excitatory neurotransmitter in the CNS, glutamate, can be released exocytotically by neurons and astrocytes. Glutamate released from neurons can affect adjacent astrocytes by changing their intracellular Ca2+ dynamics and, vice versa, glutamate released from astrocytes can cause a variety of responses in neurons such as: an elevation of [Ca2+]i, a slow inward current, an increase of excitability, modulation of synaptic transmission, synchronization of synaptic events, or some combination of these. This astrocyte-neuron signaling pathway might be a widespread phenomenon throughout the brain with astrocytes possessing the means to be active participants in many functions of the CNS. Thus, it appears that the vesicular release of glutamate can serve as a common denominator for two of the major cellular components of the CNS, astrocytes and neurons, in brain function.

Abbreviations used

intracellular Ca2+ concentration


(S)-α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid




d-2-amino-5-phosponopentanoic acid




ionotropic glutamate receptor


inositol 1, 4, 5-trisphosphate


miniature excitatory PSC


metabotropic glutamate receptor


miniature inhibitory PSC


miniature post-synaptic current


protease-activate receptor 1


prostaglandin E2


slow inward current


soluble N-ethylmaleimide-sensitive fusion protein attachment protein receptor


slow transient current


tetanus neurotoxin




vacuolar type H+-ATPase


vesicular glutamate transporter

Glutamate, traditionally considered as the major excitatory neurotransmitter in the CNS, is used by glutamatergic neurons, and is also released by astrocytes. Glutamate released from astrocytes is able to act on neurons causing a variety of physiological consequences (reviewed in Araque et al. 2001; Volterra and Meldolesi 2005; Haydon and Carmignoto 2006; Halassa et al. 2007a). As a functional component of the tripartite synapse, along with pre- and post-synaptic neuronal elements, astrocytes actively participate in synaptic transmission (Araque et al. 1999b; Halassa et al. 2007a). In this study, the vesicular/exocytotic release mechanism is employed by both neurons and astrocytes to carry out their glutamate-dependent bidirectional signaling. Thus, vesicular release of glutamate can serve as a common denominator for two of the major cellular components of the CNS, astrocytes and neurons, in its function. Although in this review, we focus on glutamate, several other mediators of bidirectional astrocyte-neuron signaling have been reported (reviewed in Parpura 2004; Fields and Burnstock 2006; Haydon and Carmignoto 2006; Halassa et al. 2007a).

Tripartite synapse: structural association between neuronal and astrocytic elements

  1. Top of page
  2. Abstract
  3. Tripartite synapse: structural association between neuronal and astrocytic elements
  4. Astrocytes respond to glutamatergic synaptic transmission
  5. Exocytotic/vesicular release of glutamate from astrocytes
  6. Neuronal responses to glutamate released from astrocytes
  7. Concluding remarks
  8. Acknowledgements
  9. References

Astrocytes can closely associate with synapses (Peters et al. 1991). This relationship is well demonstrated in Fig. 1, where an astrocyte from the cerebellar cortex is in proximity to axonal terminals making synapses onto dendritic spines. Some of these synapses are engulfed by an astrocyte, while others are in partial contact with it. Such microanatomical arrangements are not a phenomenon solely seen in the cerebellum, but are rather ubiquitously present throughout different regions of CNS. For example, in the stratum radiatum of hippocampal CA1 region, 57% of the synapses formed between Schaffer collaterals and CA1 pyramidal neurons are bordered by astrocytes (Ventura and Harris 1999). Here, astrocytic processes surround somewhat less than half (∼0.4) of the synaptic area and occupy part of the extracellular space between neighboring synapses. Intimate morphological associations between neurons and astrocytes are also observed in the magnocellular hypothalamo-neurohypophysial system (reviewed in Hatton 2002). Magnocellular neurons and their dendrites, which receive glutamatergic synaptic input from their afferents, are ensheathed by surrounding astrocytic processes under basal physiological conditions. Under certain physiological challenges, however, such as dehydration and lactation, the system exhibits plasticity, where the retraction of astrocytic processes from the cell bodies and dendrites of magnocellular neurons leads to restructuring of synaptic connections and contributes to the regulation of synaptic efficacy. Here, a consequence of astrocytic processes retrieval is the reduction in coverage of synaptic contacts, which decreases extracellular glutamate clearance and increases glutamate concentration and diffusion in the extracellular space surrounding synapses. This, in turn, affects transmitter release via modulation of pre-synaptic metabotropic glutamate receptors (mGluRs) (reviewed in Oliet et al. 2004). Taken together, these three examples of structural neuron-astrocyte associations at the tripartite synapse support the idea that astrocytes are well positioned to respond to the spillover of transmitter from the synaptic cleft and vice versa as astrocytes can communicate to neuronal synaptic elements because of their ability to release transmitters. In this review, we limit our discussion to glutamate-mediated signaling as a consequence of exocytotic events in both cell types.


Figure 1.  An electron micrograph demonstrates the close association of astrocytes with synapses. The cytoplasm of an astrocyte (blue) contacts axon terminals (At) forming synapses onto spines. Two synapses (sp1 and sp2; arrows) are engulfed by the astrocyte, while two other synapses (at At1 and At2) are in close apposition to the astrocyte; nf, neurofilaments. Modified from (Peters et al. 1991) by (Hatton 2002); reprinted from (Hatton 2002), with permission from American Physiological Society.

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Astrocytes respond to glutamatergic synaptic transmission

  1. Top of page
  2. Abstract
  3. Tripartite synapse: structural association between neuronal and astrocytic elements
  4. Astrocytes respond to glutamatergic synaptic transmission
  5. Exocytotic/vesicular release of glutamate from astrocytes
  6. Neuronal responses to glutamate released from astrocytes
  7. Concluding remarks
  8. Acknowledgements
  9. References

Astrocytes can respond to glutamatergic synaptic transmission via activation of their plasma membrane glutamate receptors and transporters (Fig. 2). Indeed, astrocytes express both ionotropic glutamate receptors (iGluRs) and mGluRs and activation of these receptors can cause intracellular Ca2+ elevation (Verkhratsky and Kettenmann 1996; Porter and McCarthy 1997; Verkhratsky et al. 1998). These cells can express all three types of iGluRs: (S)-α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA), kainate and NMDA receptors. Bergmann glial cells and immature astrocytes can express Ca2+-permeable AMPA receptor subtypes, which allow the influx of extracellular Ca2+ (Seifert and Steinhauser 2001). Also, the expression of Ca2+-permeable NMDA receptors in cortical astrocytes has been reported (Lalo et al. 2006). The predominant mGluR subtype expressed in astrocytes, mGluR5 (Schools and Kimelberg 1999; Cai et al. 2000) is coupled with phospholipase C, which catalyzes the production of inositol 1, 4, 5-trisphosphate (IP3), a second messenger signaling molecule which, after binding to its receptors located on the endoplasmic reticulum, releases Ca2+ from internal stores.


Figure 2.  The tripartite synapse, bidirectional signaling between neurons and astrocytes. After Ca2+ entry into the pre-synaptic terminal, glutamate stored in vesicles is released and signals to the dendritic spine of a post-synaptic neuron. Glutamate can also reach astrocytic glutamate receptors leading to intracellular signaling, while a portion of this transmitter is taken up by surrounding astrocytes and stored, partially, in vesicular compartments. Vesicular glutamate is released by astrocytes upon an increase of [Ca2+]i via regulated exocytosis. The released glutamate can act on adjacent neurons through their pre- and post-synaptic glutamate receptors.

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The astrocytic responsiveness to extracellular glutamate was first demonstrated in cultured astrocytes. Application of glutamate caused various patterns of cytoplasmic Ca2+ responses (Cornell-Bell et al. 1990). Some cells exhibited an initial transient intracellular Ca2+ concentration ([Ca2+]i) increase (spike) followed by different modes of Ca2+ oscillations. These intracellular Ca2+ increases were mainly mediated through non-NMDA iGluRs and mGluRs. The intracellular Ca2+ increases propagated within individual astrocytes when cells were challenged with a low extracellular concentration of glutamate (1 μmol/L), while higher concentrations of extracellular glutamate (1–10 μmol/L) caused the spread of intracellular Ca2+ increases to neighboring cells, forming intercellular Ca2+ waves. Intracellular Ca2+ oscillations were also observed in astrocytes in hippocampal and cortical slices (Pasti et al. 1997). Here, application of (1S, 3R)-1-aminocyclopentane-1, 3-dicarboxylic acid which activates group I mGluRs elicited oscillatory cytoplasmic Ca2+ elevations in astrocytes. Taken together, these studies indicate that astrocytes in culture and slices are not passive cells, but that they possess a form of excitability based on glutamate receptor-mediated intracellular Ca2+ elevations.

Besides the described astrocytic responses to exogenously added glutamate, astrocytes in slices can also respond to synaptically released glutamate, displayed as an increase in [Ca2+]i (Dani et al. 1992; Porter and McCarthy 1996). Dani et al. (1992) monitored the neuronal and astrocytic [Ca2+]i changes in organotypically cultured hippocampal slices (Fig. 3a). They stimulated mossy fibers in the dentate gyrus and measured [Ca2+]i of neurons and astrocytes located in the CA3 region. Following stimulation, in addition to the expected intracellular Ca2+ increases in neurons, they observed that astrocytes exhibited oscillatory Ca2+ elevations. Propagating intra- and intercellular Ca2+ waves were also observed in the astrocytes.


Figure 3.   Glutamate-mediated astrocyte-neuron bidirectional signaling. (a–c) Effects of glutamate release from neurons on astrocytes. (a) Synaptically released glutamate can cause [Ca2+]i increases in astrocytes. Increases in fluo-3 fluorescence of astrocytes (1–5) and a neuron (6) in hippocampal slice after electrical stimulation of dentate granule cells (modified from Dani et al. 1992). (b) Neuronal transmitter release can synchronize astrocytic network activity. Correlation maps (basal) showing active astrocytes (black squares) and a representative fraction of active neurons (white squares). After tetrodotoxin (TTX) treatment to block action potential discharges, correlated activity decreased among astroglial and neuronal cells (modified from Aguado et al. 2002). (c) Synaptically released glutamate can induce depolarizing currents in astrocytes as a result of electrogenic activity of plasma membrane glutamate transporters. Two superimposed neuronal action potentials caused prolonged neuronal autaptic afterdepolarizations and inward currents in underlying glial cells cultured on microislands; a spontaneous action potential (arrow) caused further glial responses (modified from Mennerick and Zorumski 1994). (d–i) Neuronal responses to glutamate released from astrocytes. (d) Ca2+-dependent release of glutamate from astrocytes can induce [Ca2+]i increases in neurons. Application of bradykinin (BK) to co-cultured neurons and astrocytes caused direct Ca2+ responses in astrocytes leading to glutamate release that stimulates surrounding neurons (modified from Parpura et al. 1994). (e) Glutamate released from astrocytes can evoke a slow inward current in neurons; asterisk indicates stimulus artifacts (modified from Araque et al. 1998b). (f) Astrocytes can induce increased neuronal excitability. Line graphs indicate [Ca2+]i elevations in astrocytes (1–3) and concurrent increased neuronal electrical activity (N) in co-cultured cells. Trace 1 indicates glial [Ca2+]i at the point of stimulus. Elevated [Ca2+]i spreads in a form of intercellular wave through adjacent cells (2 and 3) inducing a response in adjacent neuron (N) as it approaches it (modified from Hassinger et al. 1995). (g) Astrocyte stimulation can reduce the amplitude of evoked EPSCs. Averaged EPSCs in neurons evoked by extracellular stimulation at 1 Hz (1) were reduced when co-cultured astrocytes were mechanically stimulated (2) to release glutamate (modified from Araque et al. 1998b). (h) Glutamate from astrocytes can modulate spontaneous synaptic transmission. A whole-cell recording from a neuron adjacent to an astrocyte that was microinjected with the UV-sensitive Ca2+ cage NP-EGTA. UV photolysis (arrow) increased the Ca2+ level in the astrocyte and caused an increase in the frequency of mEPSCs (modified from Araque et al. 1998a). (i) Astrocytic glutamate release can promote neuronal synchronization. Synchronous slow inward currents in uncoupled neurons, evoked by astrocyte stimulation with (S)-3,5-dihydroxyphenylglycine in TTX (modified from Fellin et al. 2004). Central images of neuron modified from (Hu et al. 2005) and astrocyte from (Hua et al. 2004).

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To investigate whether astrocytes in situ respond to glutamate released from synaptic terminals, Porter and McCarthy (1996) used acutely isolated hippocampal slices. They electrically stimulated Schaffer collaterals and observed an increase of [Ca2+]i in astrocytes from the stratum radiatum of CA1. The astrocytic responses were dependent on neuronal activity and mediated through mGluRs as they were blocked by α-methyl-4-carboxyphenylglycine, a group I mGluR antagonist. Stimulation of Schaffer collaterals in the presence of the voltage-gated K+ channel blocker, 4-aminopyridine, which caused a prolonged release of glutamate from synaptic terminals, resulted in activation of both mGluRs and iGluRs in astrocytes.

The astrocytic response to glutamatergic transmission in acute slices is not limited to the hippocampus. Rather, synaptically released glutamate was shown to induce intracellular Ca2+ oscillations in astrocytes from both visual cortex and CA1 region of the hippocampus (Pasti et al. 1997). The astrocytic Ca2+ responses were blocked by α-methyl-4-carboxyphenylglycine and thus, mediated via mGluRs. Additionally, the frequency of astrocytic Ca2+ oscillations was correlated to the level of neuronal activity. Higher frequency or intensity stimulation of neuronal afferents resulted in an increased frequency of astrocytic Ca2+ oscillations. Thus, the anatomical intimacy with synapses and the expression of various glutamate receptors apparently enable astrocytes to sense the strength of glutamatergic synaptic activity.

Examining astrocytic network activity, Aguado et al. (2002) found that spontaneous astrocytic [Ca2+]i elevations in astrocytes were often synchronized among cells in large groups of astrocytes (dozens) in acute hippocampal slices (Fig. 3b). By applying the voltage-gated Na+ channel blocker, tetrodotoxin (TTX), to block action potentials in the neurons of the slice they found that the spontaneous activity of the astrocytes remained, but the degree of synchronization was reduced. Similar results were obtained when neuronal activity was impaired by applying NMDA and AMPA/kainate receptor antagonists, and increased astrocytic activity and synchronization were observed when slices were treated with bicuculline to induce epileptiform activity in neurons. These results indicate that synchronization of spontaneous astrocytic [Ca2+]i activity is influenced by the activity of neighboring neurons.

As mentioned earlier, astrocytes express high affinity Na+-dependent glutamate transporters (Km∼20 μmol/L) at their plasma membrane which can transport glutamate from the extracellular space into the cytoplasm (reviewed in Danbolt 2001) (Fig. 2). Owing to the activity of these transporters, the resting glutamate concentration in the extracellular space of the CNS is maintained at a low level (∼1–2 μmol/L). Astrocytes mainly express two isoforms of plasma membrane glutamate transporters: l-glutamate/l-aspartate transporter also referred to as excitatory amino acid transporter 1 and glial l-glutamate transporter-1 also called excitatory amino acid transporter 2 (Danbolt 2001). These transporters are non-uniformly distributed in astrocytes, with the cellular processes enwrapping synapses having higher densities of transporters than those processes apposing non-synaptic structures such as capillaries and pia (Chaudhry et al. 1995). The transport stoichiometry of plasma membrane glutamate transporters consists of the uptake of one glutamate molecule, three Na+ and one H+ transported into the astrocytic cytoplasm and one K+ transported out of the astrocyte. Thus, the glutamate transport activity is electrogenic and creates an inward current across the plasma membrane of astrocytes when monitored electrophysiologically (Fig. 3c). Indeed, depolarizing currents induced by synaptically released glutamate were observed in astrocytes associated with single hippocampal neurons forming autaptic synapses in microisland culture (Mennerick and Zorumski 1994). Similarly, glutamate transporter-mediated currents were also recorded in astrocytes in hippocampal slices (Bergles and Jahr 1997, 1998). Here, stimulation of Schaffer collaterals elicited inward currents in astrocytes located in the stratum radiatum of the CA1 region (Bergles and Jahr 1997).

As the discharge of glutamate from synaptic vesicles dramatically elevates the glutamate concentration (∼1 mmol/L) in the synaptic cleft, one role of astrocytes in glutamatergic synaptic transmission is to remove excess glutamate diffusing out of the synaptic cleft, ensuring the specificity of synaptic transmission. Hence, inhibition of astrocytic glutamate uptake prolonged non-NMDA post-synaptic currents when receptor desensitization was blocked (Mennerick and Zorumski 1994), indicating that astrocytes can modulate post-synaptic responses by controlling the glutamate concentration in the synaptic cleft.

These results, taken together, indicate that astrocytes can respond to glutamatergic transmission by activation of plasma membrane glutamate transporters and receptors. As briefly outlined, the activation of transporters can also affect synaptic transmission. Activation of glutamatergic receptors on astrocytes can cause an increase in [Ca2+]i leading to exocytotic/vesicular release of glutamate from astrocytes which, in turn, can influence neurons (Fig. 2).

Exocytotic/vesicular release of glutamate from astrocytes

  1. Top of page
  2. Abstract
  3. Tripartite synapse: structural association between neuronal and astrocytic elements
  4. Astrocytes respond to glutamatergic synaptic transmission
  5. Exocytotic/vesicular release of glutamate from astrocytes
  6. Neuronal responses to glutamate released from astrocytes
  7. Concluding remarks
  8. Acknowledgements
  9. References

It should be noted that although in this review we focus on exocytotic glutamate release from astrocytes, this transmitter can also be released from astrocytes by various additional mechanisms: (i) reversal of uptake by glutamate transporters (Szatkowski et al. 1990), (ii) anion channel opening induced by cell swelling (Kimelberg et al. 1990), (iii) exchange via the cystine–glutamate antiporter (Warr et al. 1999), (iv) diffusional release through ionotropic purinergic receptors (Duan et al. 2003), and (v) functional ‘hemichannels’ or unpaired connexons on the cell surface (Ye et al. 2003) . For further discussion on various mechanisms of glutamate release form astrocytes, we refer to recent reviews (Parpura et al. 2004; Malarkey and Parpura 2007). Additionally, as a detailed review of astrocytic exocytosis has been covered in a recent article (Montana et al. 2006), we only briefly describe this mechanism of glutamate release here.

Calcium-dependent glutamate release from astrocytes was initially described in culture (Parpura et al. 1994) and followed by similar observations in brain slices (e.g. Bezzi et al. 1998). Different types of stimuli, including receptor activation, mechanical stimulation, photostimulation, electrical stimulation, all of which can induce intracellular Ca2+ increases, result in glutamate release from astrocytes (Parpura et al. 1994; Araque et al. 1998b; Bezzi et al. 1998). Intracellular Ca2+ is both sufficient and necessary for astrocytic exocytosis of glutamate. Treatment of astrocytes with a Ca2+ ionophore in the presence of extracellular Ca2+ or the photolysis of caged Ca2+ induces glutamate release from astrocytes (Parpura et al. 1994; Parpura and Haydon 2000; Fellin et al. 2004; Liu et al. 2004). Depletion of internal Ca2+ stores in astrocytes or pre-loading them with a Ca2+ chelator significantly inhibits glutamate release (Araque et al. 1998a,b, 2000; Bezzi et al. 1998; Hua et al. 2004). The level of Ca2+ increase necessary for astrocytic exocytosis is also within the physiological range (Parpura and Haydon 2000; Pasti et al. 2001; Kreft et al. 2004).

The source of Ca2+ for exocytotic glutamate release is dual. Although external Ca2+ plays a role, the predominant source is from internal stores. This is substantiated by the reduction of mechanically induced glutamate release from astrocytes in the presence of thapsigargin, a blocker of store specific Ca2+-ATPase and Cd2+, a blocker of Ca2+ entry from the extracellular space (Hua et al. 2004). Additional data suggest that mechanically induced glutamate release requires the co-activation of both IP3- and ryanodine/caffeine-sensitive internal Ca2+ stores, which operate jointly (Hua et al. 2004). Ca2+ entry across the plasma membrane that supports mechanically induced glutamate release in astrocytes involves store-operated channels that are activated by the depletion of internal Ca2+ stores (Malarkey, Ni, and Parpura, unpublished observation). Here, transient receptor potential C1 protein, which plays a role in the regulation of Ca2+ homeostasis (Golovina 2005; Malarkey and Parpura 2005), mediates the Ca2+ entry across the plasma membrane.

The Ca2+-dependency of glutamate release from astrocytes suggests regulated exocytosis as a mechanism underlying this release. Indeed, similar to neurons, astrocytes express a plethora of exocytotic proteins which are functionally involved in the Ca2+-dependent glutamate release pathway (reviewed in Montana et al. 2006) (Fig. 4). Vesicle-associated membrane protein 2 also referred to as synaptobrevin 2, synaptosome-associated protein of 23 kDa, a homolog of neuronal synaptosome-associated protein of 25 kDa, and syntaxin, which are the core components of the soluble N-ethylmaleimide-sensitive fusion protein attachment protein receptor (SNARE) complex mediating the fusion of vesicles with the plasma membrane, have all been found in astrocytes (Parpura et al. 1995; Jeftinija et al. 1997; Hepp et al. 1999; Maienschein et al. 1999).


Figure 4.  Glutamate release from astrocytes utilizes a vesicular, exocytotic pathway. Astrocytes express the SNARE proteins: synaptobrevin 2, syntaxin 1, and synaptosome-associated protein of 23 kDa (SNAP-23), a homolog of neuronal SNAP-25. Synaptotagmin 4 appears to be a Ca2+ sensor for glutamate (Glu) release from astrocytes, a function carried out by synaptotagmin 1 in neurons. Packaging of glutamate into vesicles by vesicular glutamate transporters (VGLUTs) requires a proton gradient created by vacuolar type H+-ATPase (V-ATPase).

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Synaptotagmin 4, a homolog of the Ca2+ sensor protein synaptotagmin 1, expressed by neurons (reviewed in Koh and Bellen 2003), was found in astrocytes and governs their Ca2+-dependent glutamate release process (Zhang et al. 2004a). In neuronal exocytosis, the uptake of glutamate into vesicles is carried out by vesicle membrane bound proteins, the vesicular glutamate transporters (VGLUTs), which transport glutamate into vesicles using the proton concentration gradient established across the vesicular membrane by another vesicular membrane protein, vacuolar type H+-ATPase (V-ATPase). Astrocytes express all the three isoforms of VGLUTs (1, 2, and 3) as well as V-ATPase (Bezzi et al. 2004; Montana et al. 2004; Wilhelm et al. 2004; Zhang et al. 2004b). The subcellular expression of VGLUTs co-localized with that of synaptobrevin 2 (Montana et al. 2004) or cellubrevin (Bezzi et al. 2004). Inhibition of the activity of either VGLUTs or V-ATPase attenuated glutamate release from astrocytes (Araque et al. 2000; Pasti et al. 2001; Montana et al. 2004). In support of these functional data, clear vesicle-like organelles were observed in cultured astrocytes and astrocytes in vivo providing microanatomical evidence for astrocytic exocytosis (Maienschein et al. 1999; Bezzi et al. 2004). Furthermore, astrocytes display two modes of exocytotic events: (i) the formation of a transient fusion pore between vesicles and the plasma membrane, commonly referred to as ‘kiss-and-run’ events and (ii) full fusion events, where the vesicle collapses into the plasma membrane (Bezzi et al. 2004; Kreft et al. 2004; Zhang et al. 2004b; Chen et al. 2005; Crippa et al. 2006; Bowser and Khakh 2007), as recently reviewed in (Montana et al. 2006).

Neuronal responses to glutamate released from astrocytes

  1. Top of page
  2. Abstract
  3. Tripartite synapse: structural association between neuronal and astrocytic elements
  4. Astrocytes respond to glutamatergic synaptic transmission
  5. Exocytotic/vesicular release of glutamate from astrocytes
  6. Neuronal responses to glutamate released from astrocytes
  7. Concluding remarks
  8. Acknowledgements
  9. References

The consequences of Ca2+-dependent exocytotic glutamate release from astrocytes onto neurons present themselves as: an elevation of [Ca2+]i, a slow inward current (SIC), an increase of excitability, modulation of synaptic transmission, synchronization of synaptic events, or some combination of these (Fig. 3d–i). Such effects of the glutamate-mediated astrocyte-neuron signaling pathway have been observed in neurons of different brain regions, including visual cortex, hippocampus, thalamus, and nucleus accumbens. Thus, this signaling pathway might be a widespread phenomenon throughout the brain with astrocytes having the means to be active in many functions of the CNS.

Intracellular Ca2+ elevation

Exocytotic release of glutamate from astrocytes was initially found to induce intracellular Ca2+ increases in neurons in culture (Parpura et al. 1994). Application of bradykinin to a mixed culture of astrocytes and neurons, which elicited Ca2+-dependent glutamate release from astrocytes but had no effect on neurons, resulted in an increase in cytoplasmic Ca2+ concentration in neurons (Fig. 3d). This Ca2+ increase in neurons was mediated through NMDA receptors as it was blocked by the antagonist d-2-amino-5-phosponopentanoic acid (d-AP5) without affecting astrocytic responses.

Glutamate release from astrocytes induced using various stimuli can also cause intracellular Ca2+ elevations in neurons in slice preparations. Application of an mGluR agonist caused [Ca2+]i elevation in astrocytes, presumably from intracellular stores, followed by [Ca2+]i elevations in adjacent neurons that were attenuated by application of iGluR antagonists (Pasti et al. 1997; Fellin et al. 2004), pointing to glutamate release from an astrocytic source causing the response. It should be noted that these experiments were carried out after a short pre-incubation (40 min–2 h) with tetanus neurotoxin (TeNT), to specifically block synaptic transmission in neurons; astrocytes require longer incubation times (commonly 24 h) with TeNT to show an effect on its substrate, synaptobrevin 2. In acute hippocampal slices, again after short pre-incubation with TeNT (40 min), application of prostaglandin E2 (PGE2) caused astrocytic intracellular Ca2+ increases followed by Ca2+ increases in neighboring neurons (Bezzi et al. 1998). As they were abolished in the presence of iGluR antagonists, neuronal Ca2+ responses, apparently, resulted from astrocytic Ca2+-dependent glutamate release.

Slow inward and similar currents

Another effect that glutamate released from astrocytes has on neurons is to evoke a SIC, seen when using electrophysiological methods to monitor neuronal activity (Fig. 3e). Initial studies of astrocyte-neuron signaling using electrical or mechanical stimulation to selectively elevate [Ca2+]i in astrocytes showed that this caused SIC in adjacent neurons of hippocampal cultures (Araque et al. 1998b). Application of thapsigargin or microinjection of a Ca2+ chelator revealed that [Ca2+]i elevation in astrocytes was necessary to evoke SIC in neurons, pointing towards exocytosis as a mechanism underlying glutamate release from astrocytes. This current appeared to be mediated by iGluRs in the neurons, as application of the NMDA and AMPA receptor antagonists, d-AP5 and 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX), respectively, abolished the SIC.

Several subsequent studies in culture provided additional information about the mechanisms underlying SIC evoked by astrocytic glutamate release. Stimulation of astrocytes by PGE2 can elicit iGluR-mediated SIC, blocked by CNQX and d-AP5, in adjacent neurons by a mechanism that required elevation of astrocytic [Ca2+]i (Sanzgiri et al. 1999). Parpura and Haydon (2000) demonstrated that [Ca2+]i fluctuations readily attainable in astrocytes under normal physiological conditions could evoke SIC in neurons. Here, they used photolysis of a caged Ca2+ compound to directly increase the [Ca2+]i in astrocytes, the resulting SICs could be blocked by the presence of d-AP5 and CNQX. Additional evidence for involvement of an exocytotic mechanism was provided from the injection of the light chain of botulinum toxin B, which cleaves synaptobrevin 2, into astrocytes or the application of bafilomycin A1, which blocks V-ATPase; both interventions prevented SIC in the adjacent neurons (Araque et al. 2000). Furthermore, expression of the cytoplasmic SNARE domain of synaptobrevin 2 in astrocytes, which prevents assembly of the SNARE complex and vesicular fusion, resulted in similar disruption of neuronal SIC (Zhang et al. 2004b).

Fellin et al. (2004) examined astrocyte-mediated neuronal SIC in hippocampal slices. They found that stimulation of Schaffer collaterals caused SIC in CA1 pyramidal neurons. These currents could also occur spontaneously and were NMDA receptor-mediated, as they were blocked by d-AP5. Using TTX, to block action potential-evoked synaptic transmission, similar NMDA receptor-mediated SIC could be generated by the group I mGluR agonist (S)-3,5-dihydroxyphenylglycine (DHPG), presumably via stimulation of astrocytes. To further investigate this, they used photolysis of caged Ca2+ to stimulate astrocytes directly, which caused SIC in adjacent neurons. Similarly, in acute slices from the nucleus accumbens, activation of mGluR5 receptors on astrocytes which causes [Ca2+]i oscillations leads to SIC in medium spiny neurons, an effect mediated by NR2B subunit containing NMDA receptors on the neurons (D’Ascenzo et al. 2007). Again, direct stimulation of astrocytes using photolysis of caged Ca2+ also caused SIC. These findings in acute slice preparations are consistent with other SIC studies performed in cultured astrocytes.

Two additional currents, similar to SIC, recorded in neurons as a consequence of glial glutamate release were also described: slowly decaying NMDA receptor-mediated inward currents in neurons of the ventrobasal thalamus (Parri et al. 2001) and slow transient currents (STCs) mediated by NMDA receptor activation in hippocampal pyramidal cells (Angulo et al. 2004).

Parri et al. (2001) showed that astrocytes display intrinsic [Ca2+]i oscillations, using acutely prepared slices from the ventrobasal thalamus. As these spontaneous astrocytic [Ca2+]i oscillations could not be blocked by TTX, they were not caused by neuronal activity. However, the presence of blockers of internal store-specific Ca2+-ATPase and of Ca2+ entry from the extracellular space reduced the number of spontaneously active astrocytes. These spontaneous [Ca2+]i oscillations in individual astrocytes could initiate [Ca2+]i waves in adjacent astrocytes, and also cause NMDA receptor-mediated neuronal excitability, electrophysiologically recorded as inward currents in neurons. Findings are consistent with those from previous studies of the Ca2+-dependent mechanism of glutamate release from astrocytes mediating glutamatergic currents in adjacent neurons. Thus, astrocytes are able to initiate astrocyte-neuron signaling based on their intrinsic activity.

Angulo et al. (2004) using hippocampal slices recorded STCs in pyramidal neurons that were the result of spontaneous glial activity, as they occurred in the presence of TTX. Also, stimulation with DHPG, PGE2 or mechanical stimulation which causes [Ca2+]i elevation in astrocytes, was followed by STCs in neurons of hippocampal slices, implicating a glial source of glutamate release. Again, these currents were blocked by d-AP5, indicating the activation of neuronal NMDA receptors. The mechanism underlying this glutamate release was unclear, as the stimuli used implicate a Ca2+-dependent mechanism, while the lack of action of V-ATPase blockers argues for an alternative mechanism. It is possible, however, that emptying of the astrocytic vesicular pool may require longer incubation times with inhibitors of V-ATPase in slice preparation than times reported in culture (Montana et al. 2004).

At the present time, these different currents appear to have astrocytic release of glutamate as a common theme. Whether the differences may reflect a functional property of astrocyte-neuron signaling or are simply a result of variations in materials and methods used in the experimental approaches are important issues remaining in the field.

Increase of excitability

As the release of glutamate from astrocytes can result in inward currents in neurons, as described above, then astrocytes should be able to influence the electrical excitability of the surrounding neurons by this same mechanism. Hassinger et al. (1995) noticed increased neuronal excitability in hippocampal neurons cultured onto a monolayer of astrocytes (Fig. 3f). When astrocytes were stimulated, mechanically or electrically, a [Ca2+]i wave propagated through the culture. As waves in astrocytes passed the nearby neurons, they caused increases of [Ca2+]i in neurons and also an increase in the frequency of action potential discharge that was reduced by application of GluR antagonists. Similarly, Araque et al. (1999a) reported that astrocytes induced slow depolarization in adjacent, current-clamped, hippocampal neurons. Subthreshold depolarization in neurons became suprathreshold by summation with the astrocyte-induced slow depolarization, resulting in discharges of action potentials. The conditions of astrocytic stimulation here correspond to those of voltage-clamped neurons exhibiting SIC as a result of astrocytic release of glutamate. Consistent with this finding, using acute slices of nucleus accumbens, D’Ascenzo et al. (2007) demonstrated that glutamate release from astrocytes, leading to SICs in medium spiny neurons, can cause an increase in the frequency of action potential discharge in these neurons when they reside at their up-state membrane potentials (−65 mV).

Astrocytic modulation of neuronal excitability was also demonstrated in rat eyecup preparations (Newman and Zahs 1998). Light pulses were used to stimulate electrical activity in neurons of the retinal ganglion cell layer, recorded extracellularly as action potential discharges (spikes). Intercellular [Ca2+]i waves were mechanically induced in the eyecup astrocytes and Müller glia. When this wave propagated near neurons in the ganglion cell layer, a change in frequency of the light-evoked spike activity of the neurons was observed, either increasing or decreasing in frequency. This modulation was dependent on glial [Ca2+]i elevation as it was proportional to the magnitude of [Ca2+]i elevation in the glia and failed to occur if the [Ca2+]i wave subsided before reaching the neuron. Modulation of spike frequency was also retarded by application of thapsigargin. Inhibitory modulation of the frequency of spike activity was thought to occur through glutamate released from glial cells exciting inhibitory interneurons which, in turn, inhibited the retinal ganglion neurons.

Modulation of synaptic transmission

The initial investigations into astrocytic modulation of action potential-evoked synaptic transmission used mixed cultures of hippocampal neurons and astrocytes to reveal that mechanically or electrically induced [Ca2+]i elevations in astrocytes caused a reduction in the amplitude of recorded post-synaptic currents evoked by action potentials which were induced by electrical stimulation of pre-synaptic neurons (Araque et al. 1998b) (Fig. 3g). This current depression affected both excitatory and inhibitory synaptic currents and was determined to be mediated through pre-synaptic mGluRs as the effect was reduced in the presence of mGluR antagonists.

Ca2+-dependent glutamate release from astrocytes is also implicated in affecting spontaneous synaptic events (Fig. 3h). Araque et al. (1998a) showed that glutamate released from astrocytes could increase the frequency of miniature post-synaptic currents (mPSCs), both excitatory and inhibitory (miniature excitatory PSC; mEPSCs and miniature inhibitory PSC; mIPSCs). The effect on mPSCs was mediated by extrasynaptic NMDA receptors. Using a Ca2+ chelator, thapsigargin or a Ca2+ cage indicated that the glutamate mediating the effect on mPSCs was released from astrocytes by a Ca2+-dependent mechanism that, as revealed by subsequent work using bafilomycin A1 and botulinum toxin B, involved exocytosis (Araque et al. 2000).

In hippocampal slice preparations, astrocytes were shown to be able to influence spontaneous synaptic transmission in a variety of situations. Electrically stimulated astrocytes in the vicinity of inhibitory interneurons caused an increase of the frequency of mIPSCs in CA1 pyramidal neurons (Kang et al. 1998). This effect was determined to occur via iGluRs on the interneuron and was dependent on [Ca2+]i elevation in the astrocytes. Further studies using AMPA and kainate receptor antagonists showed that glutamate released from astrocytes could act on kainate receptors of interneurons to cause the increase in the frequency of the spontaneous IPSCs (Liu et al. 2004). Another group found that stimulating astrocytes, by intracellular photorelease of IP3 from its cage to raise their [Ca2+]i, caused an increase in the frequency of spontaneous AMPA EPSCs in pyramidal neurons that was inhibited by group I mGluR antagonists (Fiacco and McCarthy 2004). Interestingly, the same group demonstrated a lack of evidence for Ca2+-dependent glutamate-mediated astrocyte-neuron signaling when using a transgenic mouse that expresses an astrocyte-specific Gq-coupled receptor of which the stimulation leads to astrocytic [Ca2+]i elevations (Fiacco et al. 2007).

Using a G protein coupled protease-activate receptor 1 (PAR1) stimulation of astrocytes to induce glutamate release was shown to enhance NMDA-mediated EPSCs, mEPSCs and excitatory post-synaptic potentials in neurons of hippocampal slices (Lee et al. 2007). Ca2+-dependent glutamate release from astrocytes through PAR1 activation by thrombin or a selective PAR1 activating peptide was confirmed using GluR1-expressing ‘sniffer cells.’ This release was able to induce NMDA-mediated [Ca2+]i elevations and inward currents in co-cultured neurons, which were determined not to respond to PAR1 activation. PAR1 activation of astrocytes in slices resulted in depolarization and inward currents in neurons and reduced the Mg2+ block of NMDA receptors in these neurons, effects that were attenuated by NMDA receptor antagonist. PAR1 activation also prolonged the NMDA-sensitive decay component of mEPSCs and excitatory post-synaptic potentials.

In hippocampal slices, astrocytes modulate the perforant path-granule cell synapses in the hippocampal dentate molecular layer. Using this system, Jourdain et al. (2007) provided physiological and anatomical evidence for the role of glutamate exocytosis from astrocytes in controlling the strength of synaptic transmission. Here, stimulation of astrocytes increased the frequency but not the amplitude of spontaneous EPSCs, and the amplitude of action potential-evoked EPSCs on granule cells, implying there is a pre-synaptic mechanism of action. Pharmacology suggested that the NR2B subunit-containing NMDA receptors were responsible for this enhancement of synaptic transmission. The effect was abolished after injecting the light chain of TeNT into astrocytes to block astrocytic exocytosis. Consistent with the electrophysiology pointing to vesicular/exocytotic release of glutamate from astrocytes as the underlying mechanism for synaptic potentiation, electron microscopic evidence revealed the existence of synaptic-like vesicles in astrocytic processes; such vesicles were shown previously to contain VGLUTs (Bezzi et al. 2004). Analysis of the ultrastructural distribution of vesicles and NR2B subunits indicated that vesicles were preferentially located in the astrocytic processes in close apposition to the extrasynaptic area of synaptic terminals decorated with NR2B subunits. Interestingly, ATP was the endogenous signaling molecule that mediated astrocytic [Ca2+]i increases through the activation of their purinergic P2Y1 receptors which then lead to exocytotic glutamate release from these cells and signaling to neurons. This P2Y1 receptor-dependent pathway was engaged constitutively or by neuronal activity-dependent stimulation of the perforant path. The effects that astrocytes have on synaptic transmission in these various situations all stem from Ca2+-mediated glutamate release from astrocytes.


Injection of fluorescent dyes into the cytoplasm of individual hippocampal astrocytes that were fixed so that gap junctions were blocked and the dye was retained in the injected cell reveals that each astrocyte occupies an exclusive domain in the brain, only at the cell peripheries do their processes overlap (Bushong et al. 2002; Ogata and Kosaka 2002). Similarly, cortical astrocytes also occupy discrete territories as revealed by labeling individual astrocytes with enhanced green fluorescent protein using a transgenic and viral approach (Halassa et al. 2007b). Hippocampal astrocytes in situ display extensive processes which enable a single astrocyte to make contact with more than a hundred thousand synapses (Bushong et al. 2002). Single cortical astrocytes from tissue sections and animals in vivo enwrap up to eight neuronal bodies and make contact with 300–600 neuronal dendrites (Halassa et al. 2007b). Thus, with their ability to increase neuronal excitability, astrocytes may be able to simultaneously affect several neurons within their domain, or area of influence, resulting in a synchronization of neuronal activity. Two recent studies have provided evidence in support of this idea.

While examining NMDA receptor-mediated STCs induced in neurons via glutamate released from spontaneously active glial cells in hippocampal slices, Angulo et al. (2004) observed that some neurons adjacent to each other would display STCs that occurred almost simultaneously. This synchronization was not because of synaptic transmission as experiments were conducted in the presence of TTX and it did not seem to be the result of electrical coupling between neurons, based on the time courses of these events. The synchronized STCs occurred in neurons that had bodies within 100 μm of each other and appeared to happen too quickly (< 100 ms) to be the result of [Ca2+]i wave propagation among astrocytes, indicating that, most likely, the release of glutamate from a single astrocyte was synchronizing these events.

Examining synchronization, Fellin et al. (2004) used Schaffer collateral stimulation, DHPG, PGE2, or low extracellular Ca2+ to stimulate astrocytes and cause NMDA receptor-mediated SICs in adjacent pyramidal neurons in hippocampal slices. They were able to detect synchronized currents in groups of neurons, which were not because of synaptic transmission as application of TTX or TeNT did not abolish them (Fig. 3i). By applying depolarizing current to each neuron, they confirmed that electrical coupling was not occurring. They also concluded that synchronization was the result of glutamate release from a single astrocyte as induced SICs started in each neuron faster than a [Ca2+]i wave could propagate among astrocytes.

Concluding remarks

  1. Top of page
  2. Abstract
  3. Tripartite synapse: structural association between neuronal and astrocytic elements
  4. Astrocytes respond to glutamatergic synaptic transmission
  5. Exocytotic/vesicular release of glutamate from astrocytes
  6. Neuronal responses to glutamate released from astrocytes
  7. Concluding remarks
  8. Acknowledgements
  9. References

Experimental evidence from the last decade reveals many previously unknown functions of astrocytes and expands our understanding of astrocytes beyond being supportive and nutritional elements of the brain. Anatomically and functionally, these cells are an active participant in glutamatergic synaptic transmission as illustrated in Fig. 2. Expression of various glutamate receptors confers on them the ability to respond to synaptically released glutamate and monitor the changes of synaptic activity via an intracellular Ca2+-based mechanism. Astrocytes exhibit neuronal activity-dependent and intrinsic intracellular Ca2+ oscillations. The propagation of intracellular and intercellular Ca2+ waves in astrocytes may represent a type of signaling pathway to correlate astrocytic activity as well as neuronal activity in the brain. Astrocytic plasma membrane glutamate transporters function to clear the excess glutamate that escapes from the synaptic cleft and contribute to the maintenance of functional glutamatergic synapses. The exocytotic glutamate release from astrocytes following intracellular Ca2+ elevations provides astrocytes with a mechanism to actively take part in synaptic activity and integrate astrocytes into the information processing of the CNS. Astrocytic glutamate modulates the intracellular Ca2+ concentration in neurons, neuronal excitability, miniature and action potential-evoked PSCs, and synchonization of activity among the neuronal network. Taken together, it appears that the vesicular glutamate release mechanism is a common denominator for neurons and astrocytes in their bidirectional communication. As several other transmitters can mediate the crosstalk between astrocytes and neurons, most notably purines (i.e. ATP and adenosine), it will be necessary to determine how these various signaling pathways (co)operate under physiological or pathophysiological conditions.


  1. Top of page
  2. Abstract
  3. Tripartite synapse: structural association between neuronal and astrocytic elements
  4. Astrocytes respond to glutamatergic synaptic transmission
  5. Exocytotic/vesicular release of glutamate from astrocytes
  6. Neuronal responses to glutamate released from astrocytes
  7. Concluding remarks
  8. Acknowledgements
  9. References
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