High-affinity Glutamate Uptake Research Articles

Hyperosmolar sodium chloride is toxic to cultured neurons and causes reduction of glucose metabolism and ATP levels, an increase in glutamate uptake, and a reduction in cytosolic calcium

Elevation of serum sodium, hypernatremia, which may occur during dehydration or treatment with sodium chloride, may cause brain dysfunction and damage, but toxic mechanisms are poorly understood. We found that exposure to excess NaCl, 10–100mmol/L, for 20h caused cell death in cultured cerebellar granule cells (neurons). Toxicity was due to Na+, since substituting excess Na+ with choline reduced cell death to control levels, whereas gluconate instead of excess Cl− did not. Prior to cell death from hyperosmolar NaCl, glucose consumption and lactate formation were reduced, and intracellular aspartate levels were elevated, consistent with reduced glycolysis or glucose uptake. Concomitantly, the level of ATP became reduced. Pyruvate, 10mmol/L, reduced NaCl-induced cell death. The extracellular levels of glutamate, taurine, and GABA were concentration-dependently reduced by excess NaCl; high-affinity glutamate uptake increased. High extracellular [Na+] caused reduction in intracellular free [Ca2+], but a similar effect was seen with mannitol, which was not neurotoxic. We suggest that inhibition of glucose metabolism with ensuing loss of ATP is a neurotoxic mechanism of hyperosmolar sodium, whereas increased uptake of extracellular neuroactive amino acids and reduced intracellular [Ca2+] may, if they occur in vivo, contribute to the cerebral dysfunction and delirium described in hypernatremia.

Up-regulation of P2X7 receptor-mediated inhibition of GABA uptake by nerve terminals of the human epileptic neocortex.

Thirty percent of patients with epilepsy are refractory to medication. The majority of these patients have mesial temporal lobe epilepsy (MTLE). This prompts for new pharmacologic targets, like ATP-mediated signaling pathways, since the extracellular levels of the nucleotide dramatically increase during in vitro epileptic seizures. In this study, we investigated whether sodium-dependent high-affinity γ-aminobutyric acid (GABA) and glutamate uptake by isolated nerve terminals of the human neocortex could be modulated by ATP acting via slow-desensitizing P2X7 receptor (P2X7R). Modulation of [(3) H]GABA and [(14) C]glutamate uptake by ATP, through activation of P2X7R, was investigated in isolated nerve terminals of the neocortex of cadaveric controls and patients with drug-resistant epilepsy (non-MTLE or MTLE) submitted to surgery. Tissue density and distribution of P2X7R in the human neocortex was assessed by Western blot analysis and immunofluorescence confocal microscopy. The P2X7R agonist, 2'(3')-O-(4-benzoylbenzoyl)ATP (BzATP, 3-100 μm) decreased [(3) H]GABA and [(14) C]glutamate uptake by nerve terminals of the neocortex of controls and patients with epilepsy. The inhibitory effect of BzATP (100 μm) was prevented by the selective P2X7R antagonist, A-438079 (3 μm). Down-modulation of [(14) C]glutamate uptake by BzATP (100 μm) was roughly similar in controls and patients with epilepsy, but the P2X7R agonist inhibited more effectively [(3) H]GABA uptake in the epileptic tissue. Neocortical nerve terminals of patients with epilepsy express higher amounts of the P2X7R protein than control samples. High-frequency cortical activity during epileptic seizures releases huge amounts of ATP, which by acting on low-affinity slowly desensitizing ionotropic P2X7R, leads to down-modulation of neuronal GABA and glutamate uptake. Increased P2X7R expression in neocortical nerve terminals of patients with epilepsy may, under high-frequency firing, endure GABA signaling and increase GABAergic rundown, thereby unbalancing glutamatergic neuroexcitation. This study highlights the relevance of the ATP-sensitive P2X7R as an important negative modulator of GABA and glutamate transport and prompts for novel antiepileptic therapeutic targets.

P2X7 receptor activation downmodulates Na+-dependent high-affinity GABA and glutamate transport into rat brain cortex synaptosomes

Sodium-dependent high-affinity amino-acid transporters play crucial roles in terminating synaptic transmission in the central nervous system (CNS). However, there is lack of information about the mechanisms underlying the regulation of amino-acid transport by fast-acting neuromodulators, like ATP. Here, we investigated whether activation of the ATP-sensitive P2X7 receptor modulates Na+-dependent high-affinity γ-aminobutyric acid (GABA) and glutamate uptake into nerve terminals (synaptosomes) of the rat cerebral cortex. Radiolabeled neurotransmitter accumulation was evaluated by liquid scintillation spectrometry. The cell-permeant sodium-selective fluorescent indicator, SBFI-AM, was used to estimate Na+ influx across plasma membrane. 2′(3′)-O-(4-benzoylbenzoyl)ATP (BzATP, 3–300μM), a prototypic P2X7 receptor agonist, concentration-dependently decreased [3H]GABA (14%) and [14C]glutamate (24%) uptake; BzATP decreased transport maximum velocity (Vmax) without affecting the Michaelis constant (Km) values. The selective P2X7 receptor antagonist, A-438079 (3μM), prevented inhibition of [3H]GABA and [14C]glutamate uptake by BzATP (100μM). The inhibitory effect of BzATP coincided with its ability to increase intracellular Na+ and was mimicked by Na+ ionophores, like gramicidin and monensin. Increases in intracellular Na+ (with veratridine or ouabain) or substitution of extracellular Na+ by N-methyl-d-glucamine (NMDG)+ all decreased [3H]GABA and [14C]glutamate uptake and attenuated BzATP effects. Uptake inhibition by BzATP (100μM) was also attenuated by calmidazolium, which selectively inhibits Na+ currents through the P2X7 receptor pore. In conclusion, disruption of the Na+ gradient by P2X7 receptor activation downmodulates high-affinity GABA and glutamate uptake into rat cortical synaptosomes. Interference with amino-acid transport efficacy may constitute a novel target for therapeutic management of cortical excitability.

Monitoring of the velocity of high-affinity glutamate uptake by isolated brain nerve terminals using amperometric glutamate biosensor

Glutamate is the major excitatory neurotransmitter in the central nervous system, which is involved in the main aspects of normal brain functioning. High-affinity Na+-dependent glutamate transporters is key proteins, which transport extracellular glutamate to the cytoplasm of nerve cells, thereby preventing continuous activation of glutamate receptors, and thus the development of neurotoxicity. Disturbance in glutamate uptake is involved in the pathogenesis of major neurological disorders. Amperometric biosensors are the most promising and successful among electrochemical biosensors. In this study, we developed (1) amperometric glutamate biosensor, (2) methodological approach for the analysis of glutamate uptake in liquid samples of isolated rat brain nerve terminals (synaptosomes). The basal level of glutamate, the initial velocity of glutamate uptake and time-dependent accumulation of glutamate by synaptosomes were determined using developed glutamate biosensor. Comparative analysis of the data with those obtained by radioactive analysis, spectrofluorimetry and ion exchange chromatography was performed. Therefore, the methodological approach for monitoring of the velocity of glutamate uptake, which takes into consideration the definite level of endogenous glutamate in nerve terminals, was developed using glutamate biosensor.

Müller glial cells induce stem cell properties in retinal progenitors in vitro and promote their further differentiation into photoreceptors

Using stem cells to replace lost neurons is a promising strategy for treating retinal neurodegenerative diseases. Among their multiple functions, Müller glial cells are retina stem cells, with a robust regenerative potential in lower vertebrates, which is much more restricted in mammals. In rodents, most retina progenitors exit the cell cycle immediately after birth, differentiate as neurons, and then cannot reenter the cell cycle. Here we demonstrate that, in mixed cultures with Müller glial cells, rat retina progenitor cells expressed stem cell properties, maintained their proliferative potential, and were able to preserve these properties and remain mitotically active after several consecutive passages. Notably, these progenitors retained the capacity to differentiate as photoreceptors, even after successive reseedings. Müller glial cells markedly stimulated differentiation of retina progenitors; these cells initially expressed Crx and then developed as mature photoreceptors that expressed characteristic markers, such as opsin and peripherin. Moreover, they were light responsive, insofar as they decreased their cGMP levels when exposed to light, and they also showed high-affinity glutamate uptake, a characteristic of mature photoreceptors. Our present findings indicate that, in addition to giving rise to new photoreceptors, Müller glial cells might instruct a pool of undifferentiated cells to develop and preserve stem cell characteristics, even after successive reseedings, and then stimulate their differentiation as functional photoreceptors. This complementary mechanism might contribute to enlarge the limited regenerative capacity of mammalian Müller cells.

Energy substrates to support glutamatergic and GABAergic synaptic function: Role of glycogen, glucose and lactate

Maintenance of glutamatergic and GABAergic activity requires a continuous supply of energy since the exocytotic processes as well as high affinity glutamate and GABA uptake and subsequent metabolism of glutamate to glutamine are energy demanding processes. The main energy substrate for the brain under normal conditions is glucose but at the cellular level, i.e., neurons and astrocytes, lactate may play an important role as well. In addition to this the possibility exists that glycogen, which functions as a glucose storage molecule and which is only present in astrocytes, could play a role not only during aglycemia but also during normoglycemia. These issues are discussed and it is concluded that both glucose and lactate are of importance for the maintenance of normal glutamatergic and GABAergic activity. However, with regard to maintenance of an adequate capacity for glutamate transport, it appears that glucose metabolism via the glycolytic pathway plays a fundamental role. Additionally, evidence is presented to support the notion that glycogen turnover may play an important role in this context. Moreover, it should be noted that the amino acid neurotransmitters can be used as metabolic substrates. This requires pyruvate recycling, a process that is discussed as well.

High-affinity glycine and glutamate transport in pig forebrain white and gray matter: A quantitative study

High-affinity uptake of glycine and glutamate modulates glutamatergic neurotransmission in gray matter. N-Methyl- d-aspartate (NMDA) receptors were recently described on white matter oligodendrocytes, therefore uptake of glutamate and glycine in white matter may also modulate NMDA receptor function. We found that glycine uptake in white structures of pig forebrain (corpus callosum, fimbria, subcortical pyramidal tracts, and occipital subcortical white matter) was similar to that in gray structures (frontal and temporal cortices and hippocampus), and that it was sensitive to sarcosine, a GLYT1 inhibitor (IC 50 15 μM). Glutamate uptake in white matter was ∼10% of that in gray; it was sensitive to dihydrokainate, an EAAT2 inhibitor. The levels of glycine and its precursor serine were similar in white and gray matter: approximately 2 and 1 nmol/mg tissue, respectively. The white matter level of glutamate was ∼7.6 nmol/mg tissue, or ∼74% of gray matter levels. The activity of serine hydroxymethyl transferase, which converts serine into glycine, was similar in white and gray matter (11–18 pmol/(mg tissue) min), whereas the white matter activity of phosphate-activated glutaminase, which converts glutamine into glutamate, was ∼100 pmol/(mg tissue) min, or ∼34% of gray matter activity. The white matter activity of glutamine synthetase, the glial enzyme that converts glutamate into glutamine, was 20–40 nmol/(mg tissue) min in neocortex and 5–6 nmol/(mg tissue) min in white matter. The data show that forebrain white matter is equipped to regulate extracellular levels of glycine and glutamate, functions that may modulate white matter NMDA receptor function.

Inhibitors of glutamate transport modulate distinct patterns in brain metabolism

High affinity uptake of glutamate plays a major role in the termination of excitatory neurotransmission. Identification of the ramifications of transporter function is essential to understand the diseases in which defective excitatory amino acid transporters (EAAT) have been implicated. In this work we incubated Guinea pig cortical tissue slices with [3-(13)C]pyruvate and major currently available glutamate uptake inhibitors and studied the resultant metabolic sequelae by (13)C and (1)H NMR spectroscopy using a multivariate statistical approach. Perturbation of glutamate uptake produced significant effects on metabolic flux through the Krebs cycle, and on glutamate/glutamine cycling rates, with this effect accounting for 76% of the variation in the total data set. The effects of all inhibitors were separable from each other along three major principal components. The competitive inhibitor L-CCG III ((2S,1'S,2'R)-2-carboxycyclopropyl)glycine) differed most from the other inhibitors, showing negative weightings on both the first and second principal components, whereas the EAAT2-specific inhibitor dihydrokainate (DHK) showed metabolic patterns similar to that of anti-endo-3,4-methanopyrolidine dicarboxylate but separate from those of DL-threo-beta-benzyloxyaspartate (TBOA) and L-trans-pyrrolidine-2,4-dicarboxylate (L-tPDC). This indicates that different inhibition mechanisms or different colocalisation of the separate transporter subtypes with glutamate receptors can produce significantly different metabolic and functional outcomes for the brain.

Neurotoxicity of reactive aldehydes: The concept of “aldehyde load” as demonstrated by neuroprotection with hydroxylamines

The concept of “oxidative stress” has become a mainstay in the field of neurodegeneration but has failed to differentiate critical events from epiphenomena and sequalae. Furthermore, the translation of current concepts of neurodegenerative mechanisms into effective therapeutics for neurodegenerative diseases has been meager and disappointing. A corollary of current concepts of “oxidative stress” is that of “aldehyde load”. This relates to the production of reactive aldehydes that covalently modify proteins, nucleic acids, lipids and carbohydrates and activate apoptotic pathways. However, reactive aldehydes can also be generated by mechanisms other than “oxidative stress”. We therefore hypothesized that agents that can chemically neutralize reactive aldehydes should demonstrate superior neuroprotective actions to those of free radical scavengers. To this end, we evaluated hydroxylamines as aldehyde-trapping agents in an in vitro model of neurodegeneration induced by the reactive aldehyde, 3-aminopropanal (3-AP), a product of polyamine oxidase metabolism of spermine and spermidine. In this model, the hydroxylamines N-benzylhydroxylamine, cyclohexylhydroxylamine and t-butylhydroxylamine were shown to protect, in a concentration-dependent manner, against 3-AP neurotoxicity. Additionally, a therapeutic window of 3 h was demonstrated for delayed administration of the hydroxylamines. In contrast, the free radical scavengers TEMPO and TEMPONE and the anti-oxidant ascorbic acid were ineffective in this model. Extending these tissue culture findings in vivo, we examined the actions of N-benzylhydroxylamine in the trimethyltin (TMT) rat model of hippocampal CA3 neurodegeneration. This model involves augmented polyamine metabolism resulting in the generation of reactive aldehydes that compromise mitochondrial integrity. In the rat TMT model, NBHA (50 mg/kg, sc, daily) provided 100% protection against neurodegeneration, as reflected by measurements of KCl-evoked glutamate release from hippocampal brain slices and septal high affinity glutamate uptake. In contrast, ascorbic acid (100 mg/kg, sc, daily) failed to protect CA3 neurons from TMT toxicity. In summary, our data support further evaluation of the concept of “aldehyde load” in neurodegeneration and the potential clinical investigation of agents that are effective traps for reactive aldehydes.

Cortical Glutamatergic Markers in Schizophrenia

Post-mortem studies have yet to produce consistent findings on cortical glutamatergic markers in schizophrenia; therefore, it is not possible to fully understand the role of abnormal glutamatergic function in the pathology of the disorder. To better understand the changes in cortical glutamatergic markers in schizophrenia, we measured the binding of radioligands to the ionotropic glutamate receptors (N-methyl D-aspartate, [3H]CGP39653, [3H]MK-801), amino-3-hydroxy-5-methyl-4-isoxazole ([3H]AMPA), kainate ([3H]kainate), and the high-affinity glutamate uptake site ([3H]aspartate) using in situ radioligand binding with autoradiography and levels of mRNA for kainate receptors using in situ hybridization in the dorsolateral prefrontal cortex from 20 subjects with schizophrenia and 20 controls matched for age and sex. Levels of [3H]kainate binding were significantly decreased in cortical laminae I-II (p = 0.01), III-IV (p < 0.05), and V-VI (p < 0.01) from subjects with schizophrenia. By contrast, levels of [3H]MK-801, [3H]AMPA, [3H]aspartate, or [3H]CGP39653 binding did not differ between the diagnostic cohorts. Levels of mRNA for the GluR5 subunit were decreased overall (p < 0.05), with no changes in levels of mRNA for GluR6, GluR7, KA1, or KA2 in tissue from subjects with schizophrenia. These data indicate that the decreased number of kainate receptors in the dorsolateral prefrontal cortex in schizophrenia may result, in part, from reduced expression of the GluR5 receptor subunits.

Valproate is Neuroprotective against Malonate Toxicity in Rat Striatum: An Association with Augmentation of High-Affinity Glutamate Uptake

The antiepileptic drug valproate (VPA) may be neuroprotective. We treated rats with VPA for 14 days (300 mg/kg twice daily) before intrastriatal injection of 1.5 micromol (1 M) of the succinate dehydrogenase inhibitor malonate. VPA-treated animals developed smaller lesions than control animals: 10 +/- 2 mm(3) versus 26 +/- 8 mm(3) (means +/- SD; P = 10(-4). Injection of NaCl that was equiosmolar with 1 M malonate caused lesions of only 1.2 +/- 0.4 mm(3) in control animals, whereas physiologic saline produced no lesion. VPA pretreatment reduced the malonate-induced extracellular accumulation of glutamate. This effect paralleled an increase in the striatal level of the glutamate transporter GLT, which augmented high-affinity glutamate uptake by 25%, as determined from the uptake of [(3)H] glutamate into striatal proteoliposomes. Malonate caused a 76% reduction in striatal adenosine triphosphate (ATP) content, but the glial, ATP-dependent formation of glutamine from radiolabeled glucose or glutamate was intact, indicating that glial ATP production supported uptake of glutamate. Striatal levels of HSP-70 and fos were reduced, and the levels of bcl-2 and phosphorylated extracellular signal-regulated kinase remained unaffected, but histone acetylation was increased by VPA treatment. The results suggest that augmentation of glutamate uptake may contribute importantly to VPA-mediated neuroprotection in striatum.

Glutamate transporters in platelets: EAAT1 decrease in aging and in Alzheimer’s disease

Platelets release glutamate upon activation and are an important clearance system of the amino acid from blood, through high-affinity glutamate uptake, similar to that described in brain synaptosomes. Since platelet glutamate uptake is decreased in neurodegenerative disorders, we performed a morphological and molecular characterization of platelet glutamate transporters. The three major brain glutamate transporters EAAT1, EAAT2 and EAAT3 are expressed in platelets, with similar molecular weight, although at lower density than brain. A Na +-dependent-high-affinity glutamate uptake was competitively inhibited by known inhibitors but not by dihydrokainic acid, suggesting platelet EAAT2 does not play a major role in glutamate uptake at physiological conditions. We observed decreased glutamate uptake V max, without modification of transporter affinity, in aging, which could be linked to the selective decrease of EAAT1 expression and mRNA. Moreover, in AD patients we found a further EAAT1 reduction compared to age-matched controls, which could explain the decrease of platelet uptake previously described. Platelet glutamate transporters may be used as peripheral markers to investigate the role of glutamate in patients with neuropsychiatric disorders.

Content of ileal EAAC1 and hepatic GLT-1 high-affinity glutamate transporters is increased in growing vs. nongrowing lambs, paralleling increased tissue D- and L-glutamate, plasma glutamine, and alanine concentrations.

Glutamate is a central metabolite for whole-animal energy and N metabolism. This study tested the hypothesis that ileal epithelium, liver, and kidney content of system X-(AG) glutamate transporters EAAC1 and GLT-1 would be up-regulated to support growth of wethers (30 +/- 1.2 kg) fed a forage-based diet for at least 14 d to gain (2.0 x NEm; n = 9) vs. maintain (1.2 x NEm; n = 9) BW. We have previously demonstrated that two high-affinity glutamate transporters (EAAC1, GLT-1) are expressed by these extensive glutamate metabolizing epithelial tissues. Wethers fed at 2.0 x NEm gained (P < 0.001; 0.26 kg/d) BW, whereas those fed 1.2 x NEm did not. Although plasma concentrations (microM) of glucose and L- or D-glutamate did not differ, plasma glutamine (precursor of glutamate) and alanine concentrations (transamination product of glutamate) were 28% (P < 0.007) and 22% (P < 0.072) greater for growing lambs than nongrowing lambs. In tissues, the concentration of L-glutamate in ileum epithelia and D-glutamate of liver was 49% (P < 0.015) and 181% (P < 0.042) greater, respectively, in growing vs. nongrowing animals, whereas concentrations of glutamate isoforms did not differ in kidney. Paralleling these increased amino acid concentrations, ileal epithelium contained 313% more (P < 0.038) EAAC1 protein and liver contained 240% more (P < 0.001) GLT-1 protein, whereas kidney transporter content did not differ between growing and nongrowing wethers. In contrast to increased EAAC1 and GLT-1 protein content in ileal and liver tissue of growing lambs, messenger RNA levels did not differ. These results indicate that the increased capacity for high-affinity glutamate uptake in growing vs. nongrowing lambs is achieved through increased expression of EAAC1 by ileal epithelium and GLT1 by liver, which parallel increased tissue concentrations of glutamate and plasma concentrations of two major interorgan N carriers, glutamine and alanine.

Cell-selective effects of ammonia on glutamate transporter and receptor function in the mammalian brain

Increased brain ammonia concentrations are a hallmark feature of several neurological disorders including congenital urea cycle disorders, Reye’s syndrome and hepatic encephalopathy (HE) associated with liver failure. Over the last decade, increasing evidence suggests that hyperammonemia leads to alterations in the glutamatergic neurotransmitter system. Studies utilizing in vivo and in vitro models of hyperammonemia reveal significant changes in brain glutamate levels, glutamate uptake and glutamate receptor function. Extracellular brain glutamate levels are consistently increased in rat models of acute liver failure. Furthermore, glutamate transport studies in both cultured neurons and astrocytes demonstrate a significant suppression in the high affinity uptake of glutamate following exposure to ammonia. Reductions in NMDA and non-NMDA glutamate receptor sites in animal models of acute liver failure suggest a compensatory decrease in receptor levels in the wake of rising extracellular levels of glutamate. Ammonia exposure also has significant effects on metabotropic glutamate receptor activation with implications, although less clear, that may relate to the brain edema and seizures associated with clinical hyperammonemic pathologies. Therapeutic measures aimed at these targets could result in effective measures for the prevention of CNS consequences in hyperammonemic syndromes.

Effects of ammonia on high affinity glutamate uptake and glutamate transporter EAAT3 expression in cultured rat cerebellar granule cells

Increased levels of extracellular glutamate are a consistent feature of hepatic encephalopathy (HE) associated with liver failure and other hyperammonemic pathologies. Reduction of glutamate uptake has been described in ammonia-exposed cultured astrocytes, synaptosomes, and in animal models of hyperammonemia. In the present study, we examine the effects of pathophysiological concentrations of ammonia on d-aspartate (a non-metabolizable analog of glutamate) uptake by cultured rat cerebellar granule neurons. Exposure of these cells to ammonia resulted in time-dependent (24% reduction at 24 h and 60% reduction at 5 days, P<0.001) and dose-dependent (21, 37, and 57% reduction at 1, 2.5, and 5 mM for 5 days, P<0.01) suppression of d-aspartate uptake. Kinetic analyses revealed significant decreases in the velocity of uptake ( V max) (37% decrease at 2.5 mM NH 4Cl, P<0.05 and 52% decrease at 5 mM NH 4Cl, P<0.001) as well as significant reductions in K m values (25% reduction at 2.5 mM NH 4Cl, P<0.05 and 45% reduction at 5 mM NH 4Cl, P<0.001). Western blotting, on the other hand, showed no significant changes in the neuronal glutamate transporter EAAC1/EAAT3 protein, the only glutamate transporter currently known to be expressed by these cells. In addition, 1 H combined with 13 C -NMR spectroscopy studies using the stable isotope [1- 13 C ]-glucose demonstrated a significant increase in intracellular glutamate levels derived from the oxidative metabolism of glucose, rather than from the deamidation of exogenous glutamine in cultured granule neurons exposed to ammonia. The present study provides evidence that the effects of ammonia on glutamate uptake are not solely an astrocytic phenomenon and that unlike the astrocytic glutamate transporter counterpart, EAAT3 protein expression in cultured cerebellar granule cells is not down-regulated when exposed to ammonia. Decrease of glutamate uptake in these cellular preparations may afford an additional regulatory mechanism aimed at controlling intracellular levels of glutamate and ultimately the releasable pool of glutamate in neurons.

The 'glial' glutamate transporter, EAAT2 (Glt-1) accounts for high affinity glutamate uptake into adult rodent nerve endings.

The excitatory amino acid transporters (EAAT) removes neurotransmitters glutamate and aspartate from the synaptic cleft. Most CNS glutamate uptake is mediated by EAAT2 into glia, though nerve terminals show evidence for uptake, through an unknown transporter. Reverse-transcriptase PCR identified the expression of EAAT1, EAAT2, EAAT3 and EAAT4 mRNAs in primary cultures of mouse cortical or striatal neurones. We have used synaptosomes and glial plasmalemmal vesicles (GPV) from adult mouse and rat CNS to identify the nerve terminal transporter. Western blotting showed detectable levels of the transporters EAAT1 (GLAST) and EAAT2 (Glt-1) in both synaptosomes and GPVs. Uptake of [3H]D-aspartate or [3H]L-glutamate into these preparations revealed sodium-dependent uptake in GPV and synaptosomes which was inhibited by a range of EAAT blockers: dihydrokainate, serine-o-sulfate, l-trans-2,4-pyrrolidine dicarboxylate (PDC) (+/-)-threo-3-methylglutamate and (2S,4R )-4-methylglutamate. The IC50 values found for these compounds suggested functional expression of the 'glial, transporter, EAAT2 in nerve terminals. Additionally blockade of the majority EAAT2 uptake sites with 100 micro m dihydrokainate, failed to unmask any functional non-EAAT2 uptake sites. The data presented in this study indicate that EAAT2 is the predominant nerve terminal glutamate transporter in the adult rodent CNS.

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.

Inhibitor of glutamate transport alters synaptic transmission at sensorimotor synapses in Aplysia.

Aplysia sensory neurons possess high-affinity glutamate uptake activity that is regulated by serotonin. To gain insight into the physiological role of glutamate uptake in sensory neurons, we examined whether blockade of glutamate transport altered synaptic transmission. We also examined whether glutamate transport affected homosynaptic depression and posttetanic potentiation (PTP). In the presence of DL-threo-beta-hydroxyaspartic acid (THA), previously shown to block glutamate uptake in Aplysia, the duration of unitary excitatory postsynaptic potentials (EPSPs) was significantly increased and their amplitude was significantly reduced. Similar effects were observed in the properties of summated EPSPs. However, no effect on the induction of homosynaptic depression or PTP was observed. Although it is unclear whether THA exerted its effect by modulating neuronal and/or glial mechanisms, at least one target of THA was neuronal, as the duration of unitary EPSPs measured in cultured sensorimotor synapses was also increased in the presence of THA. These results support the hypotheses that glutamate is the transmitter released by the sensory neurons and that glutamate transport plays an important role in regulating features of synaptic transmission in Aplysia.

Strategies for metabolic exchange between glial cells and neurons

The brain is a major energy consumer and dependent on carbohydrate and oxygen supply. Electrical and synaptic activity of neurons can only be sustained given sufficient availability of ATP. Glial cells, which have long been assigned trophic functions, seem to play a pivotal role in meeting the energy requirements of active neurons. Under conditions of high neuronal activity, a number of glial functions, such as the maintenance of ion homeostasis, neurotransmitter clearance from synaptic domains, the supply of energetic compounds and calcium signalling, are challenged. In the vertebrate brain, astrocytes may increase glucose utilization and release lactate, which is taken up and consumed by neurons to generate ATP by oxidative metabolism. The CO 2 produced is processed primarily in astrocytes, which display the major activity of carboanhydrase in the brain. Protons and bicarbonate in turn may contribute to drive acid/base-coupled transporters. In the present article a scenario is discussed which couples the transfer of energy and the conversion of CO 2 with the high-affinity glutamate uptake and other transport processes at glial and neuronal cell membranes. The transporters can be linked to glial signalling and may cooperate with each other at the cellular level. This could save energy, and would render energy exchange processes between glial cells and neurons more effective. Functions implications and physiological responses, in particular in chemosensitive brain areas, are discussed.

Endothelin-1 decreases glutamate uptake in primary cultured rat astrocytes.

Endothelin-1 (ET-1) is a potent vasoconstrictor peptide that is also known to induce a wide spectrum of biological responses in nonvascular tissue. In this study, we found that ET-1 (100 nM) inhibited the glutamate uptake in cultured astrocytes expressing the glutamate/aspartate transporter (GLAST); astrocytes did not express the glutamate transporter-1 (GLT-1). The V(max) and the K(m) of the glutamate uptake were reduced by 57% and 47%, respectively. Application of the ET(A) and ET(B) receptor antagonists BQ-123 and BQ-788 partly inhibited the ET-1-evoked decrease in the glutamate uptake, whereas the nonspecific ET receptor antagonist bosentan completely inhibited this decrease. Incubation of the cultures with pertussis toxin abolished the effect of ET-1 on the uptake. The ET-1-induced decrease in the glutamate uptake was independent of extracellular free Ca(2+) concentration, whereas the intracellular Ca(2+) antagonists thapsigargin and 3,4,5-trimethoxybenzoic acid 8-(diethylamino)octyl ester abolished the effect of ET-1 on the glutamate uptake. Incubation with the protein kinase C (PKC) antagonist staurosporine, but not with the fatty acid-binding protein bovine serum albumin, prevented the ET-1-induced decrease in the glutamate uptake. These results suggest that ET-1 impairs the high-affinity glutamate uptake in cultured astrocytes through a G protein-coupled mechanism, involving PKC and changes in intracellular Ca(2+).