Caenorhabditis elegans glutamate transporters influence synaptic function and behavior at sites distant from the synapse

To ensure precise neurotransmission and prevent neurotoxic accumulation, l-glutamate (Glu), the major excitatory neurotransmitter in the brain, is cleared from the synapse by glutamate transporters (GluTs). The molecular components of Glu synapses are highly conserved between Caenorhabditis elegans and mammals, yet the absence of synaptic insulation in C. elegans raises fundamental questions about Glu clearance strategies in the nematode nervous system. To gain insight into how Glu clearance is accomplished and how GluTs impact neurotransmission, we probed expression and function of all 6 GluTs found in the C. elegans genome. Disruption of each GluT impacts multiple Glu-dependent behaviors, with GluT combinations commonly increasing the severity of behavioral deficits. Interestingly, the sole GluT that we find expressed in neurons is localized predominantly in presynaptic neurons, in contrast to the postsynaptic concentration of neuronal GluTs typical in mammals. Moreover, 3 of the 6 GluT genes appear strongly expressed on the capillary excretory canal cell, where they affect Glu-dependent behaviors from positions distal to glutamatergic circuits. Indeed, our focused study of GLT-3, one of the distally expressed GluTs, shows that despite this distance, GLT-3 function can balance the activity mediated by synaptic release and synaptic receptors. The effects of distal GluTs on glutamatergic circuits support that Glu diffusion outside the vicinity of the synapse is a critical factor in C. elegans neurotransmission. Together with the presynaptic localization of neuronal GluTs, these observations suggest an unusual strategy for Glu clearance in C. elegans.     

High‐affinity glutamate transporters in chronic pain: an emerging therapeutic target

Neurons responsible for sensing noxious stimuli and conducting pain signals from periphery to the spinal cord are predominantly glutamatergic. Members of the SLC1A family of high‐affinity glutamate transporters (GluTs) are differentially expressed in sensory neurons and surrounding glial cells. These plasma membrane proteins along with glutamate/cystine exchanger, light chain of cystine/glutamate exchanger, are responsible for fine tuning of extracellular glutamate concentrations and, thus, for modulation of excitatory signalling in the spinal cord. Emerging data point at key roles of GluTs in molecular mechanisms of chronic pain and analgesia, incl. development of opioid tolerance. Pharmacological inhibition or antisense down‐regulation of spinal GluTs can induce/aggravate pain behaviours, whereas increasing of expression of GluTs by viral gene transfer or positive pharmacological modulators can mitigate chronic pain. Furthermore, some drugs, originally introduced for targeting different pathological conditions, but in parallel exhibiting analgesic properties (e.g. anti‐convulsants valproate and riluzole, β‐lactam‐ and tetracycline antibiotics, tricyclic anti‐depressants), can enhance glutamate transport in the spinal cord. Thus, molecular modulation of GluTs may turn into prospective therapeutic approach for the management of chronic pain. However, precise pharmacological targeting of this transport system requires in‐depth elucidation of molecular factors and signalling pathways underlying expression and activity of individual GluT subtypes, including their splice variants.

     

Asymmetry of glia near central synapses favors presynaptically directed glutamate escape

Recent findings demonstrate that synaptically released excitatory neurotransmitter glutamate activates receptors outside the immediate synaptic cleft and that the extent of such extrasynaptic actions is regulated by the high affinity glutamate uptake. The bulk of glutamate transporter systems are evenly distributed in the synaptic neuropil, and it is generally assumed that glutamate escaping the cleft affects pre- and postsynaptic receptors to a similar degree. To test whether this is indeed the case, we use quantitative electron microscopy and establish the stochastic pattern of glial occurrence in the three-dimensional (3D) vicinity of two common types of excitatory central synapses, stratum radiatum synapses in hippocampus and parallel fiber synapses in cerebellum. We find that the occurrence of glia postsynaptically is strikingly higher (3-4-fold) than presynaptically, in both types of synapses. To address the functional consequences of this asymmetry, we simulate diffusion and transport of synaptically released glutamate in these two brain areas using a detailed 3D compartmental model of the extracellular space with glutamate transporters arranged unevenly, in accordance with the obtained experimental data. The results predict that glutamate escaping the synaptic cleft is 2-4 times more likely to activate presynaptic compared to postsynaptic receptors. Simulations also show that postsynaptic neuronal transporters (EAAT4 type) at dendritic spines of cerebellar Purkinje cells exaggerate this asymmetry further. Our data suggest that the perisynaptic environment of these common central synapses favors fast presynaptic feedback in the information flow while preserving the specificity of the postsynaptic input.     

Dynamics of the extracellular gate and ion-substrate coupling in the glutamate transporter

Glutamate transporters (GluTs) are the primary regulators of extracellular concentration of the neurotransmitter glutamate in the central nervous system. In this study, we have investigated the dynamics and coupling of the substrate and Na⁺ binding sites, and the mechanism of cotransport of Na⁺ ions, using molecular dynamics simulations of a membrane-embedded model of GluT in its apo (empty form) and various Na⁺- and/or substrate-bound states. The results shed light on the mechanism of the extracellular gate and on the sequence of binding of the substrate and Na⁺ ions to GluT during the transport cycle. The results suggest that the helical hairpin HP2 plays the key role of the extracellular gate for the substrate binding site, and that the opening and closure of the gate is controlled by substrate binding. GluT adopts an open conformation in the absence of the substrate exposing the binding sites of the substrate and Na⁺ ions to the extracellular solution. Based on the calculated trajectories, we propose that Na1 is the first element to bind GluT, as it is found to be important for the completion of the substrate binding site. The subsequent binding of the substrate, in turn, is shown to result in an almost complete closure of the extracellular gate and the formation of the Na2 binding site. Finally, binding of Na2 locks the extracellular gate and completes the formation of the occluded state of GluT.     

Neuronal glutamate transporters control activation of postsynaptic metabotropic glutamate receptors and influence cerebellar long-term depression.

Neuronal and glial isoforms of glutamate transporters show distinct distributions on membranes surrounding excitatory synapses, but specific roles for transporter subtypes remain unidentified. At parallel fiber (PF) synapses in cerebellum, neuronal glutamate transporters and metabotropic glutamate receptors (mGluRs) have overlapping postsynaptic distributions suggesting that postsynaptic transporters selectively regulate mGluR activation. We examined interactions between transporters and mGluRs by evoking mGluR-mediated excitatory postsynaptic currents (mGluR EPSCs) in slices of rat cerebellum. Selective inhibition of postsynaptic transporters enhanced mGluR EPSCs greater than 3-fold. Moreover, impairing glutamate uptake facilitated mGluR-dependent long-term depression at PF synapses. Our results demonstrate that uniquely positioned glutamate transporters strongly influence mGluR activation at cerebellar PF synapses. Postsynaptic glutamate uptake may serve as a general mechanism for regulating mGluR-initiated synaptic depression.     

Long-term potentiation of neuronal glutamate transporters

Persistent, use-dependent modulation of synaptic strength has been demonstrated for fast synaptic transmission mediated by glutamate and has been hypothesized to underlie persistent behavioral changes ranging from memory to addiction. Glutamate released at synapses is sequestered by the action of excitatory amino acid transporters (EAATs) in glia and postsynaptic neurons. So, the efficacy of glutamate transporter function is crucial for regulating glutamate spillover to adjacent presynaptic and postsynaptic receptors and the consequent induction of plastic or excitotoxic processes. Here, we report that tetanic stimulation of cerebellar climbing fiber-Purkinje cell synapses results in long-term potentiation (LTP) of a climbing fiber-evoked glutamate transporter current recorded in Purkinje cells. This LTP is postsynaptically expressed and requires activation of an mGluR1/PKC cascade. Together with a simultaneously induced long-term depression (LTD) of postsynaptic AMPA receptors, this might reflect an integrated antiexcitotoxic cellular response to strong climbing fiber synaptic activation, as occurs following an ischemic episode.     

Interactions between glutamate transporters and metabotropic glutamate receptors at excitatory synapses in the cerebellar cortex

Five glutamate transporter genes have been identified; two of these (EAAT3 and EAAT4) are expressed in neurons and are predominantly confined to the membranes of cell bodies and dendrites. At an ultrastructural level, glutamate transporters have been shown to surround excitatory synapses in hippocampus and cerebellum [J. Neurosci. 18 (1998) 3606; J. Comp. Neurol. 418 (2000) 255]. This pattern of localization overlaps the well-described perisynaptic distribution of Group I metabotropic glutamate receptors or mGluRs [Neuron 11 (1993) 771; J. Chem. Neuroanat. 13 (1997) 77]. Both of the principal excitatory synaptic inputs to cerebellar Purkinje neurons, the parallel fiber (PF) and climbing fiber (CF) synapses, express mGluR-dependent forms of synaptic plasticity [Nat. Neurosci. 4 (2001) 467]. Prompted by the colocalization of postsynaptic glutamate transporters and mGluRs, we have examined whether glutamate uptake limits mGluR-mediated signals and mGluR-dependent forms of plasticity at PF and CF synapses in cerebellar slices. We find that, at PF and, surprisingly also at CF synapses, mGluR activation generates a slow synaptic current and triggers intracellular calcium release. At both PF and CF synapses, mGluR responses are strongly limited by glutamate transporters under resting conditions and are facilitated by short trains of stimuli. Nearly every Purkinje neuron expresses an mGluR-mediated synaptic current upon inhibition of glutamate transport. Global applications of glutamate achieved by photolysis of chemically caged glutamate yield similar results and argue that the colocalized transporters can effectively limit glutamate access to the mGluRs even in the face of such a large amount of transmitter. We hypothesize that neuronal glutamate transporters and Group I mGluRs located in the perisynaptic space interact to sense and then regulate the amount of glutamate escaping excitatory synapses. This hypothesis is currently being tested using electrophysiological methods and the introduction of optically tagged glutamate transporter proteins. In the brain, synaptic signals are terminated mainly by neurotransmitter transporters. Families of genes encoding transporters for the major neurotransmitters (dopamine, GABA, glutamate, glycine, norepinephrine and 5-HT) have been identified. Although transporters serve as targets for important classes of therapeutic drugs (e.g. selective serotonin reuptake inhibitors) and drugs of abuse (amphetamine, cocaine), little is known about how they operate at a molecular level or contribute to synaptic transmission.     

Presynaptic kainate receptors in the hippocampus: slowly emerging from obscurity.

Kainate receptor agonists depress transmitter release at several synapses in the hippocampus. Distinct mechanisms appear to underlie this phenomenon at different synapses. Recently, it has emerged that presynaptic kainate receptors can also potentiate the release of both GABA and glutamate and that axonal kainate receptors can trigger ectopic action potentials in interneurons. Because synaptically released glutamate mimics many of the actions of exogenous agonists, presynaptic kainate receptors potentially play an extensive role in hippocampal signaling.     

Extracellular glutamate diffusion determines the occupancy of glutamate receptors at CA1 synapses in the hippocampus

Following exocytosis at excitatory synapses in the brain, glutamate binds to several subtypes of postsynaptic receptors. The degree of occupancy of AMPA and NMDA receptors at hippocampal synapses is, however, not known. One approach to estimate receptor occupancy is to examine quantal amplitude fluctuations of postsynaptic signals in hippocampal neurons studied in vitro. The results of such experiments suggest that NMDA receptors at CA1 synapses are activated not only by glutamate released from the immediately apposed presynaptic terminals, but also by glutamate spillover from neighbouring terminals. Numerical simulations point to the extracellular diffusion coefficient as a critical parameter that determines the extent of activation of receptors positioned at different distances from the release site. We have shown that raising the viscosity of the extracellular medium can modulate the diffusion coefficient, providing an experimental tool to investigate the role of diffusion in activation of synaptic and extrasynaptic receptors. Whether intersynaptic cross-talk mediated by NMDA receptors occurs in vivo remains to be determined. The theoretical and experimental approaches described here also promise to shed light on the roles of metabotropic and kainate receptors, which often occur in an extrasynaptic distribution, and are therefore positioned to sense glutamate escaping from the synaptic cleft.     

The regulation of glutamate receptor trafficking and function by TARPs and other transmembrane auxiliary subunits

At excitatory synapses in the brain, glutamate released from nerve terminals binds to glutamate receptors to mediate signaling between neurons. Glutamate receptors expressed in heterologous cells show ion channel activity. Recently, native glutamate receptors were shown to contain auxiliary subunits that modulate the trafficking and/or channel properties. The AMPA receptor (AMPAR) can contain TARP and CNIHs as the auxiliary subunits, whereas kainate receptor (KAR) can contain the Neto auxiliary subunit. Each of these auxiliary subunits uniquely modulates the glutamate receptors, and determines properties of native glutamate receptors. A thorough elucidation of the properties of native glutamate receptor complexes is indispensable for the understanding of the molecular machinery that regulates glutamate receptors and excitatory synaptic transmission in the brain.     

Synthesis and characterization of 4-methoxy-7-nitroindolinyl-D-aspartate, a caged compound for selective activation of glutamate transporters and N-methyl-D-aspartate receptors in brain tissue

The D-isomer of aspartate is efficiently transported by high-affinity Na(+)/K(+)-dependent glutamate transporters and is an effective ligand of N-methyl-d-aspartate (NMDA) receptors. To facilitate analysis of the regulation of these proteins in their native membranes, we synthesized a photolabile analogue of D-aspartate, 4-methoxy-7-nitroindolinyl-D-aspartate (MNI-D-aspartate). This compound was photolyzed with a quantum efficiency of 0.09 at pH 7.4. Photorelease of d-aspartate in acute hippocampal slices through brief (1 ms) UV laser illumination of MNI-d-aspartate triggered rapidly activating currents in astrocytes that were inhibited by the glutamate transporter antagonist DL-threo-beta-benzyloxyaspartic acid (TBOA), indicating that they resulted from electrogenic uptake of D-aspartate. These transporter currents exhibited a distinct tail component that was approximately 2% of the peak current, which may result from the release of K(+) into the extracellular space during counter transport. MNI-D-aspartate was neither an agonist nor an antagonist of glutamate transporters at concentrations up to 500 muM and was stable in aqueous solution for several days. Glutamate transporter currents were also elicited in Bergmann glial cells and Purkinje neurons of the cerebellum in response to photolysis of MNI-D-aspartate, indicating that this compound can be used for monitoring the occupancy and regulation of glutamate transporters in different brain regions. Photorelease of D-aspartate did not activate alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)/kainate receptors or metabotropic glutamate receptors (mGluRs) in neurons, but resulted in the selective, but transient, activation of NMDA receptors in hippocampal pyramidal neurons; MNI-D-aspartate was not an antagonist of NMDA receptors. These results indicate that MNI-D-aspartate also may be useful for studying the regulation of NMDA receptors at excitatory synapses.     

Glutamate escape from a tortuous synaptic cleft of the hippocampal mossy fibre synapse

The time course of neurotransmitter in the synaptic cleft contributes substantially to the fast kinetics of synaptic signalling. Hippocampal mossy fibres (MFs), a well-characterised excitatory pathway from dentate granule cells to the hippocampus proper, form large glutamatergic synapses at branched spiny structures in CA3 pyramidal cell dendrites. To what extent transmission at these synapses is affected by retarded glutamate clearance from the large tortuous synaptic cleft is not known. Here, we propose a simple geometrical approximation representing the 'typical' geometry of thorny excrescences that form the tortuous cleft interface at a MF synapse. We then employ Monte Carlo simulations to monitor movements of 3000 individual glutamate molecules released within the cleft. The results predict that, in the absence of neuronal glutamate transporters, it should take approximately 10 ms for 50% and 60-70 ms for 90% of glutamate molecules to escape the MF synapse.     

Modulation of sensory input to the spinal cord by presynaptic ionotropic glutamate receptors

Sensory input from peripheral nerves to the dorsal horn of the spinal cord is mediated by a variety of agents released by the central terminals of dorsal root ganglion (DRG) neurons. These include, but are not limited to, amino acids, especially glutamate, peptides and purines. The unraveling of the mechanisms of synaptic transmission by central terminals of DRG neurons has to take into account various ways in which the message from the periphery can be modulated at the level of the first central synapse. These include postsynaptic and presynaptic mechanisms. Homomeric and heteromeric complexes of receptor subunits for the different transmitters released by DRG neurons and interneurons, clustered at the postsynaptic site of central synapses, can be expressed in different combinations and their rate of insertion into the postsynaptic membrane is activity-regulated. Inhibitory mechanisms are an important part of central modulation, especially via presynaptic inhibition, currently believed to involve GABA released by inhibitory intrinsic neurons. Recent work has established the occurrence of another way by which sensory input can be modulated, i.e. the expression of presynaptic ionotropic and metabotropic receptors in central terminals of DRG neurons. Microscopic evidence for the expression, in these terminals, of various subunits of ionotropic glutamate receptors documents the selective expression of glutamate receptors in functionally different DRG afferents. Electrophysiological and pharmacological data suggest that activation of presynaptic ionotropic glutamate receptors in central terminals of DRG neurons may result in inhibition of release of glutamate by the same terminals. Glutamate activating presynaptic receptors may spill over from the same or adjacent synapses, or may be released by processes of astroglial cells surrounding synaptic terminals. The wide expression of presynaptic ionotropic glutamate receptors, especially in superficial laminae of the dorsal horn, where Adelta- and C fibers terminate, provides an additional or alternative mechanism, besides GABA-mediated presynaptic inhibition, for the modulation of glutamate release by these fibers. Since, however, presynaptic ionotropic glutamate receptors are also expressed in terminals of GABAergic intrinsic interneurons, a decrease of GABA release resulting from activation of these receptors in the same laminae, may also play a role in central sensitization and hyperalgesia.     

The role of glutamate transporters in bone cell signalling

The amino acid L-glutamate mediates signals at excitatory synapses in the CNS where its effects are controlled by co-ordinated activities of various types of glutamate receptor and transporter. This signalling mechanism has proved to be far more ubiquitous with many different cell types responding to glutamate. The glutamate transporter GLAST-1 was the first component of this pathway identified in bone where its expression was found to be mechanoresponsive in osteocytes. There is now a wealth of evidence supporting a role for this signalling mechanism in bone. Osteoblasts can release glutamate in a regulated manner and express functional glutamate receptors that influence their differentiation and osteogenic activity. Likewise, osteoclasts express functional glutamate receptors that influence their bone resorbing capacity. This article considers the various functions of glutamate transporters in this signalling pathway, and the evidence supporting an important role of glutamate signalling in regulating bone cell activities.     

Importance of astrocytic inactivation of synaptically released glutamate for cell survival in the central nervous system--are astrocytes vulnerable to low intracellular glutamate concentrations?

Multiple levels of neuron-astrocyte interactions do exist at glutamatergic synapses, glial glutamate transporters being involved in most of them. Inactivation of synaptically released glutamate is not only important for the phasic aspect of glutamatergic transmission but also for astrocyte metabolism, which supply neurons with different metabolic precursors, and for cell survival in the central nervous system. Alteration of glutamate transport, which leads to abnormally high extracellular glutamate levels, has been involved in numerous neurodegenerative diseases. There are different ways by which elevated extracellular levels of glutamate can be toxic. Excitotoxic mechanisms, involving overstimulation of glutamate receptors, have been shown to induce the death of neurons and oligodendrocytes, but not of astrocytes. Oxidative glutamate toxicity, which can affect every cell type of the central nervous system, is currently viewed as the consequence of altered cystine transport, leading in turn to reduced glutathione synthesis and oxidative stress. This review summarizes the functional implications of astroglial glutamate transport and the consequences of its alteration. Emphasis is laid on our recent finding that alteration of glutamate transport, by depleting intracellular stores of glutamate, can induce oxidative toxicity in astrocytes. The consequences for the other cell types of the central nervous system are discussed in terms of neuron dependency on astrocytes for glutathione synthesis and therefore oxidative stress protection.     

Glutamate transporters: confining runaway excitation by shaping synaptic transmission

Traditionally, glutamate transporters have been viewed as membrane proteins that harness the electrochemical gradient to slowly transport glutamate from the extracellular space into glial cells. However, recent studies have shown that glutamate transporters on glial and neuronal membranes also rapidly bind released glutamate to shape synaptic transmission. In this Review, we summarize the properties of glutamate transporters that influence synaptic transmission and are subject to regulation and plasticity. We highlight how the diversity of glutamate-transporter function relates to transporter location, density and affinity.     

Motor Axon Synapses on Renshaw Cells Contain Higher Levels of Aspartate than Glutamate

Motoneuron synapses on spinal cord interneurons known as Renshaw cells activate nicotinic, AMPA and NMDA receptors consistent with co-release of acetylcholine and excitatory amino acids (EAA). However, whether these synapses express vesicular glutamate transporters (VGLUTs) capable of accumulating glutamate into synaptic vesicles is controversial. An alternative possibility is that these synapses release other EAAs, like aspartate, not dependent on VGLUTs. To clarify the exact EAA concentrated at motor axon synapses we performed a quantitative postembedding colloidal gold immunoelectron analysis for aspartate and glutamate on motor axon synapses (identified by immunoreactivity to the vesicular acetylcholine transporter; VAChT) contacting calbindin-immunoreactive (-IR) Renshaw cell dendrites. The results show that 71% to 80% of motor axon synaptic boutons on Renshaw cells contained aspartate immunolabeling two standard deviations above average neuropil labeling. Moreover, VAChT-IR synapses on Renshaw cells contained, on average, aspartate immunolabeling at 2.5 to 2.8 times above the average neuropil level. In contrast, glutamate enrichment was lower; 21% to 44% of VAChT-IR synapses showed glutamate-IR two standard deviations above average neuropil labeling and average glutamate immunogold density was 1.7 to 2.0 times the neuropil level. The results were not influenced by antibody affinities because glutamate antibodies detected glutamate-enriched brain homogenates more efficiently than aspartate antibodies detecting aspartate-enriched brain homogenates. Furthermore, synaptic boutons with ultrastructural features of Type I excitatory synapses were always labeled by glutamate antibodies at higher density than motor axon synapses. We conclude that motor axon synapses co-express aspartate and glutamate, but aspartate is concentrated at higher levels than glutamate.     

Long-term plasticity mediated by mGluR1 at a retinal reciprocal synapse

The flow of information across the retina is controlled by reciprocal synapses between bipolar cell terminals and amacrine cells. However, the synaptic delays and properties of plasticity at these synapses are not known. Here we report that glutamate release from goldfish Mb-type bipolar cell terminals can trigger fast (delay of 2-3 ms) and transient GABA(A) IPSCs and a much slower and more sustained GABA(C) feedback. Synaptically released glutamate activated mGluR1 receptors on amacrine cells and, depending on the strength of presynaptic activity, potentiated subsequent feedback. This poststimulus enhancement of GABAergic feedback lasted for up to 10 min. This form of mGluR1-mediated long-term synaptic plasticity may provide retinal reciprocal synapses with adaptive capabilities.     

Control of Cleft Glutamate Concentration and Glutamate Spill-Out by Perisynaptic Glia: Uptake and Diffusion Barriers

Most glutamatergic synapses in the mammalian central nervous system are covered by thin astroglial processes that exert a dual action on synaptically released glutamate: they form physical barriers that oppose diffusion and they carry specific transporters that remove glutamate from the extracellular space. The present study was undertaken to investigate the dual action of glia by means of computer simulation. A realistic synapse model based on electron microscope data and Monte Carlo algorithms were used for this purpose. Results show (1) that physical obstacles formed by glial processes delay glutamate exit from the cleft and (2) that this effect is efficiently counteracted by glutamate uptake. Thus, depending on transporter densities, the presence of perisynaptic glia may result in increased or decreased glutamate transient in the synaptic cleft. Changes in temporal profiles of cleft glutamate concentration induced by glia differentially impact the response of the various synaptic and perisynaptic receptor subtypes. In particular, GluN2B- and GluN2C-NMDA receptor responses are strongly modified while GluN2A-NMDA receptor responses are almost unaffected. Thus, variations in glial transporter expression may allow differential tuning of NMDA receptors according to their subunit composition. In addition, simulation data suggest that the sink effect generated by transporters accumulation in the vicinity of the release site is the main mechanism limiting glutamate spill-out. Physical obstacles formed by glial processes play a comparatively minor role.     

The Protective Effects of Riluzole on Manganese-Induced Disruption of Glutamate Transporters and Glutamine Synthetase in the Cultured Astrocytes

Chronic exposure to excessive manganese (Mn) can lead to manganism, a type of neurotoxicity accomplished with extracellular glutamate (Glu) accumulation. To investigate this accumulation, this study focused on the role of astrocyte glutamate transporters (GluTs) and glutamine synthetase (GS), which have roles in Glu transport and metabolism, respectively. And the possible protective effects of riluzole (a glutamatergic modulator) were studied in relation to Mn exposure. At first, the astrocytes were exposed to 0, 125, 250, and 500 μM MnCl(2) for 24 h, and 100 μM riluzole was pretreated to astrocytes for 6 h before 500 μM MnCl(2) exposure. Then, [(3)H]-glutamate uptake was measured by liquid scintillation counting; Na(+)-K(+) ATPase and GS activities were determined by a colorimetric method; glutamate/aspartate transporter (GLAST), glutamate transporter-1 (GLT-1), and GS mRNA expression were determined by RT-PCR and protein levels were measured by western blotting. The results showed that Mn inhibited Glu uptake, Na(+)-K(+) ATPase and GS activities, GLAST, GLT-1, and GS mRNA, and protein in a concentration-dependent manner. And they were significantly higher for astrocytes pretreated with 100 μM riluzole than the group exposed to 500 μM MnCl(2). The results suggested that Mn disrupted Glu transport and metabolism by inhibiting GluTs and GS. Riluzole activated protective effects on enhancing GluTs and GS to reverse Glu accumulation. In conclusion, Mn exposure results in the disruption of GLAST, GLT-1, and GS expression and function. Furthermore, riluzole attenuates this Mn toxicity.