Estimating the glutamate transporter surface density in distinct sub-cellular compartments of mouse hippocampal astrocytes

Glutamate transporters preserve the spatial specificity of synaptic transmission by limiting glutamate diffusion away from the synaptic cleft, and prevent excitotoxicity by keeping the extracellular concentration of glutamate at low nanomolar levels. Glutamate transporters are abundantly expressed in astrocytes, and previous estimates have been obtained about their surface expression in astrocytes of the rat hippocampus and cerebellum. Analogous estimates for the mouse hippocampus are currently not available. In this work, we derive the surface density of astrocytic glutamate transporters in mice of different ages via quantitative dot blot. We find that the surface density of glial glutamate transporters is similar in 7-8 week old mice and rats. In mice, the levels of glutamate transporters increase until about 6 months of age and then begin to decline slowly. Our data, obtained from a combination of experimental and modeling approaches, point to the existence of stark differences in the density of expression of glutamate transporters across different sub-cellular compartments, indicating that the extent to which astrocytes limit extrasynaptic glutamate diffusion depends not only on their level of synaptic coverage, but also on the identity of the astrocyte compartment in contact with the synapse. Together, these findings provide information on how heterogeneity in the spatial distribution of glutamate transporters in the plasma membrane of hippocampal astrocytes my alter glutamate receptor activation out of the synaptic cleft.     

Cerebellar Bergmann glia: an important model to study neuron-glia interactions

The biochemical effects triggered by the action of glutamate, the main excitatory amino acid, on a specialized type of glia cells, Bergmann glial cells of the cerebellum, are a model system with which to study glia-neuronal interactions. Neuron to Bergmann glia signaling is involved in early stages of development, mainly in cell migration and synaptogenesis. Later, in adulthood, these cells have an important role in the maintenance and proper function of the synapses that they surround. Major molecular targets of this cellular interplay are glial glutamate receptors and transporters, both of which sense synaptic activity. Glutamate receptors trigger a complex network of signaling cascades that involve Ca(2+) influx and lead to a differential gene-expression pattern. In contrast, Bergmann glia glutamate transporters participate in the removal of the neurotransmitter from the synaptic cleft and act also as signal transducers that regulate, in the short term, their own activity. These exciting findings strengthen the concept of active participation of glial cells in synaptic transmission and the involvement of neuron-glia circuits in the processing of brain information.     

A trimeric quaternary structure is conserved in bacterial and human glutamate transporters

Neuronal and glial glutamate transporters play a central role in the termination of synaptic transmission and in extracellular glutamate homeostasis in the mammalian central nervous system. They are known to be multimers; however, the number of subunits forming a functional transporter is controversial. We studied the subunit stoichiometry of two distantly related glutamate transporters, the human glial glutamate transporter hEAAT2 and a bacterial glutamate transporter from Escherichia coli, ecgltP. Using blue native polyacrylamide gel electrophoresis, analysis of concatenated transporters, and chemical cross-linking, we demonstrated that human and prokaryotic glutamate transporters expressed in Xenopus laevis oocytes or in mammalian cells are assembled as trimers composed of three identical subunits. In an inducible mammalian cell line expressing hEAAT2 the glutamate uptake currents correlate to the amount of trimeric transporters. Overexpression and purification of ecgltP in E. coli resulted in a homogenous population of trimeric transporters that were functional after reconstitution in lipid vesicles. Our results indicate that an evolutionarily conserved trimeric quaternary structure represents the sole native and functional state of glutamate transporters.     

Glial glutamate transporters mediate a functional metabolic crosstalk between neurons and astrocytes in the mouse developing cortex

Neuron-glia interactions are essential for synaptic function, and glial glutamate (re)uptake plays a key role at glutamatergic synapses. In knockout mice, for either glial glutamate transporters, GLAST or GLT-1, a classical metabolic response to synaptic activation (i.e., enhancement of glucose utilization) is decreased at an early functional stage in the somatosensory barrel cortex following activation of whiskers. Investigation in vitro demonstrates that glial glutamate transport represents a critical step for triggering enhanced glucose utilization, but also lactate release from astrocytes through a mechanism involving changes in intracellular Na(+) concentration. These data suggest that a metabolic crosstalk takes place between neurons and astrocytes in the developing cortex, which would be regulated by synaptic activity and mediated by glial glutamate transporters.     

Glial glutamate transporters: New actors in brain signaling

Glutamate, the main excitatory amino acid in the vertebrate brain, is critically involved in most of the physiological functions of the central nervous system. It has traditionally been assumed that glutamate triggers a wide array of signaling cascades through the activation of specific membrane receptors. The extracellular levels are tightly regulated to prevent neurotoxic insults. Electrogenic Na(+)-dependent glial glutamate transporters remove the bulk of the neurotransmitter from the synaptic cleft. An exquisitely ordered coupling between glutamatergic neurons and surrounding glia cells is fundamental for excitatory transmission. The glutamate/glutamine and astrocyte/neuron lactate shuttles provide the biochemical framework of this compulsory association. In this context, recent advances show that glial glutamate transporters act as signal transducers that regulate the expression of proteins involved in their compartmentalization with neurons in the so-called tripartite synapse.     

High-density presynaptic transporters are required for glutamate removal from the first visual synapse

Reliable synaptic transmission depends not only on the release machinery and the postsynaptic response mechanism but also on removal or degradation of transmitter from the synaptic cleft. Accumulating evidence indicates that postsynaptic and glial excitatory amino acid transporters (EAATs) contribute to glutamate removal. However, the role of presynaptic EAATs is unclear. Here, we show in the mouse retina that glutamate is removed from the synaptic cleft at the rod to rod bipolar cell (RBC) synapse by presynaptic EAATs rather than by postsynaptic or glial EAATs. The RBC currents evoked by electrical stimulation of rods decayed slowly after pharmacological blockade of EAATs. Recordings of the evoked RBC currents from EAAT subtype-deficient mice and the EAAT-coupled anion current reveal that functional EAATs are localized to rod terminals. Model simulations suggest that rod EAATs are densely packed near the release site and that rods are equipped with an almost self-sufficient glutamate recollecting system.     

Modulation of synaptic transmission by astrocytes in the rat supraoptic nucleus.

One of the functions of astroglial cells in the central nervous system is to clear synaptically-released glutamate from the extracellular space. This is performed thanks to specific transporters of the excitatory amino acid expressed on their surface. The way by which astrocytic glutamate uptake contributes to synaptic transmission has been investigated via numerous experimental approaches but has never been addressed under conditions where neuroglial interactions are physiologically modified. Recently, we took advantage of the neuroglial plastic properties of the hypothalamo-neurohypophysial system to examine the consequences of a physiological reduction in the astrocytic coverage of neurons on glutamatergic synaptic transmission. This experimental model has brought some insights on the physiological interactions between glial cells and neurons at the level of the synapse. In particular, it has revealed that the degree of glial coverage of neurons influences glutamate concentration at the vicinity of excitatory synapses and, as a consequence, affects the level of activation of presynaptic glutamate receptors. Astrocytes, therefore, appear to contribute to the regulation of neuronal excitability by modulating synaptic efficacy at glutamatergic nerve terminals.     

Antagonists of protein kinase C inhibit rat retinal glutamate transport activity in situ

Neuronal and glial high‐affinity transporters regulate extracellular glutamate concentration, thereby terminating synaptic transmission and preventing neuronal excitotoxicity. Glutamate transporter activity has been shown to be modulated by protein kinase C (PKC) in cell culture. This is the first study to demonstrate such modulation in situ, by following the fate of the non‐metabolisable glutamate transporter substrate, d ‐aspartate. In the rat retina, pan‐isoform PKC inhibition with chelerythrine suppressed glutamate uptake by GLAST (glutamate/aspartate transporter), the dominant excitatory amino acid transporter localized to the glial Müller cells. This effect was mimicked by rottlerin but not by Gö6976, suggesting the involvement of the PKCδ isoform, but not PKCα, β or γ. Western blotting and immunohistochemical labeling revealed that the suppression of glutamate transport was not due to a change in transporter expression. Inhibition of PKCδ selectively suppressed GLAST but not neuronal glutamate transporter activity. These data suggest that the targeting of specific glutamate transporters with isoform‐specific modulators of PKC activity may have significant implications for the understanding of neurodegenerative conditions arising from compromised glutamate homeostasis, e.g. glaucoma and amyotrophic lateral sclerosis.     

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.     

GLAST Activity is Modified by Acute Manganese Exposure in Bergmann Glial Cells

Glutamate is the major excitatory amino acid neurotransmitter in the vertebrate brain. It exerts its actions through the activation of specific plasma membrane receptors expressed in neurons and glial cells. Overactivation of glutamate receptors results in neuronal death, known as excitotoxicity. A family of sodium-dependent glutamate transporters enriched in glial cells are responsible of the vast majority of the removal of this amino acid form the synaptic cleft. Therefore, a precise and exquisite regulation of these proteins is required not only for a proper glutamatergic transmission but also for the prevention of an excitotoxic insult. Manganese is a trace element essential as a cofactor for several enzymatic systems, although in high concentrations is involved in the disruption of brain glutamate homeostasis. The molecular mechanisms associated to manganese neurotoxicity have been focused on mitochondrial function, although energy depletion severely compromises the glutamate uptake process. In this context, in this contribution we analyze the effect of manganese exposure in glial glutamate transporters function. To this end, we used the well-established model of chick cerebellar Bergmann glia cultures. A time and dose dependent modulation of [³H]-d-aspartate uptake was found. An increase in the transporter catalytic efficiency, most probably linked to a discrete increase in the affinity of the transporter was detected upon manganese exposure. Interestingly, glucose uptake was reduced by this metal. These results favor the notion of a direct effect of manganese on glial cells, this in turn alters their coupling with neurons and might lead to changes in glutamatergic transmission.

     

Glial glutamate transporter and glutamine synthetase regulate GABAergic synaptic strength in the spinal dorsal horn

Decreased GABAergic synaptic strength ('disinhibition') in the spinal dorsal horn is a crucial mechanism contributing to the development and maintenance of pathological pain. However, mechanisms leading to disinhibition in the spinal dorsal horn remain elusive. We investigated the role of glial glutamate transporters (GLT-1 and GLAST) and glutamine synthetase in maintaining GABAergic synaptic activity in the spinal dorsal horn. Electrically evoked GABAergic inhibitory post-synaptic currents (eIPSCs), spontaneous IPSCs (sIPSCs) and miniature IPSCs were recorded in superficial spinal dorsal horn neurons of spinal slices from young adult rats. We used (2S,3S)-3-[3-[4-(trifluoromethyl)benzoylamino]benzyloxy]aspartate (TFB-TBOA), to block both GLT-1 and GLAST and dihydrokainic acid to block only GLT-1. We found that blockade of both GLAST and GLT-1 and blockade of only GLT-1 in the spinal dorsal horn decreased the amplitude of GABAergic eIPSCs, as well as both the amplitude and frequency of GABAergic sIPSCs or miniature IPSCs. Pharmacological inhibition of glial glutamine synthetase had similar effects on both GABAergic eIPSCs and sIPSCs. We provided evidence demonstrating that the reduction in GABAergic strength induced by the inhibition of glial glutamate transporters is due to insufficient GABA synthesis through the glutamate-glutamine cycle between astrocytes and neurons. Thus, our results indicate that deficient glial glutamate transporters and glutamine synthetase significantly attenuate GABAergic synaptic strength in the spinal dorsal horn, which may be a crucial synaptic mechanism underlying glial-neuronal interactions caused by dysfunctional astrocytes in pathological pain conditions.     

The structure of glutamate transporters shows channel-like features

Neuronal and glial glutamate transporters remove the excitatory neurotransmitter glutamate from the synaptic cleft and thus prevent neurotoxicity. The proteins belong to a large family of secondary transporters, which includes transporters from a variety of bacterial, archaeal and eukaryotic organisms. The transporters consist of eight membrane-spanning alpha-helices and two pore-loop structures, which are unique among secondary transporters but may resemble pore-loops found in ion channels. Another distinctive structural feature is the presence of a highly amphipathic membrane-spanning alpha-helix that provides a hydrophilic path through the membrane. The unusual structural features of the transporters are discussed in relation to their function.     

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.     

高亲和力谷氨酸转运体

高亲和力谷氨酸转运体主要位于神经元和胶质细胞的细胞膜上,能逆浓度梯度从胞外向胞内摄取谷氨酸,中止谷氨酸能传递,使胞外谷氨酸浓度保持在较低水平,以保护神经元不受谷氨酸的毒性影响。近年来,随着高亲和力谷氨酸转运体的克隆,有关研究迅速发展。本文从高亲和力谷氨酸转运体的克隆、分子结构特征、表达分布、生理功能、结构－功能关系等方面对近年的进展加以综述。     

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.     

Electrogenic Glutamate Transporters in the CNS: Molecular Mechanism,Pre-steady-state Kinetics,and their Impact on Synaptic Signaling

Glutamate is the major excitatory neurotransmitter in the mammalian CNS. The spatiotemporal profile of the glutamate concentration in the synapse is critical for excitatory synaptic signalling. The control of this spatiotemporal concentration profile requires the presence of large numbers of synaptically localized glutamate transporters that remove pre-synaptically released glutamate by uptake into neurons and adjacent glia cells. These glutamate transporters are electrogenic and utilize energy stored in the transmembrane potential and the Na⁺/K⁺-ion concentration gradients to accumulate glutamate in the cell. This review focuses on the kinetic and electrogenic properties of glutamate transporters, as well as on the molecular mechanism of transport. Recent results are discussed that demonstrate the multistep nature of the transporter reaction cycle. Results from pre-steady-state kinetic experiments suggest that at least four of the individual transporter reaction steps are electrogenic, including reactions associated with the glutamate-dependent transporter halfcycle. Furthermore, the kinetic similarities and differences between some of the glutamate transporter subtypes and splice variants are discussed. A molecular mechanism of glutamate transport is presented that accounts for most of the available kinetic data. Finally, we discuss how synaptic glutamate transporters impact on glutamate receptor activity and how transporters may shape excitatory synaptic transmission.     

Gliosis alters expression and uptake of spinal glial amino acid transporters in a mouse neuropathic pain model

Gliosis is strongly implicated in the development and maintenance of persistent pain states following chronic constriction injury of the sciatic nerve. Here we demonstrate that in the dorsal horn of the spinal cord, gliosis is accompanied by changes in glial amino acid transporters examined by immunoblot, immunohistochemistry and RT-PCR. Cytokines, proinflammatory mediators and microglia increase up to postoperative day (pd) 3 before decreasing on pd 7. Then, spinal glial fibrillary acidic protein increases on pd 7, lasting until pd 14 and later. Simultaneously, the expression of glial amino acid transporters for glycine and glutamate (GlyT1 and GLT1) is reduced on pd 7 and pd 14. Consistent with a reduced expression of GlyT1 and GLT1, high performance liquid chromatography reveals a net increase in the concentration of glutamate and glycine on pd 7 and pd 14 in tissue from the lumbar spinal cord of neuropathic mice. In this study we have confirmed that microglial activation precedes astrogliosis. Such a glial cytoskeletal rearrangement correlates with a marked decrease in glycine and glutamate transporters, which might, in turn, be responsible for the increased concentration of these neurotransmitters in the spinal cord. We speculate that these phenomena might contribute, via over-stimulation of NMDA receptors, to the changes in synaptic functioning that are responsible for the maintenance of persistent pain.     

Glutamate uptake in synaptic plasticity: from mollusc to mammal

A great deal of research has been directed toward understanding the cellular mechanisms underlying synaptic plasticity and memory formation. To this point, most research has focused on the more "active" components of synaptic transmission: presynaptic transmitter release and postsynaptic transmitter receptors. Little work has been done characterizing the role neurotransmitter transporters might play during changes in synaptic efficacy. We review several new experiments that demonstrate glutamate transporters are regulated during changes in the efficacy of glutamatergic synapses. This regulation occurred during long-term facilitation of the sensorimotor synapse of Aplysia and long-term potentiation of the Schaffer-collateral synapse of the rat. We propose that glutamate transporters are "co-regulated" with other molecules/processes involved in synaptic plasticity, and that this process is phylogenetically conserved. These new findings indicate that glutamate transporters most likely play a more active role in neurotransmission than previously believed.