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PubMed1993

Opioid-glutamate interactions in rat locus coeruleus neurons.

1. The effect of mu-opioids on the glutamate response was investigated in rat locus coeruleus (LC) neurons by intracellular recording in the brain slice preparation. Glutamate responses were evoked by bath application of selective glutamate agonists, glutamate iontophoresis, and stimulation of excitatory afferents. 2. The mu-opioid agonist D-Ala2-MePhe4-Gly-ol5-enkephalin (DAMGO; 1 microM) potentiated the response to bath application of N-methyl-D-aspartate (NMDA) and alpha-amino-3-hydroxy-5-methyl-4-isoxazole proprionic acid by 91 and 142%, respectively, in slices cut in the horizontal plane. The mechanism of action of this effect was investigated under conditions that limited the DAMGO-induced hyperpolarization and improved the space clamp of the neuron through 1) addition of barium, 2) increase in extracellular potassium concentration, 3) sectioning of the LC in the coronal plane, and 4) addition of carbenoxolone. Each experimental manipulation decreased the DAMGO outward current and reduced the mu-opioid potentiation of the glutamate response. The results suggest that the mu-opioid-mediated potentiation of the glutamate response is dependent on membrane hyperpolarization. 3. Neither forskolin nor the phorbol ester 4b-phorbol 12,13-dibutyrate (PDBu) altered the glutamate-mediated inward currents. The potentiation of the glutamate response by DAMGO was not affected by PDBu. 4. The mu-opioids DAMGO and [met]5enkephalin (10 microM) did not significantly affect the NMDA receptor-mediated depolarization (mean 14%) evoked by local application of glutamate but inhibited the NMDA receptor-mediated synaptic potential (mean 25%).(ABSTRACT TRUNCATED AT 400 WORDS)

Afferent Pathways↗
PubMed2000

Immunocytochemical localization of ionotropic glutamate receptors subunits in the adult quail forebrain.

The excitatory amino acid glutamate is implicated in the central control of many neuroendocrine and behavioral processes. The ionotropic glutamate receptors are usually divided into the N-methyl-D-aspartate (NMDA) and non-NMDA (kainate and AMPA) subtypes. Subunits of these receptors have been cloned in a few mammalian species. Information available in birds is more limited. In quail, we recently demonstrated that glutamate agonists (kainate, AMPA, and NMDA) rapidly (within minutes) and reversibly decrease in vitro aromatase activity like several other manipulations affecting intracellular HCa(2+) pools. Aromatase catalyzes the conversion of androgens into estrogens which is a limiting step in the control by testosterone of many behavioral and physiologic processes. Therefore, glutamate could control estrogen production in the brain, but the anatomic substrate supporting this effect is poorly understood. In quail, aromatase is mainly localized in the preoptic-hypothalamic-limbic system. We visualized here the distribution of the major ionotropic glutamate receptors in quail by immunocytochemical methods by using commercial primary antibodies raised against rat glutamate receptor 1 and receptors 2-3 (GluR1, GluR2/3: AMPA subtype, Chemicon, CA), rat glutamate receptors 5-7 (GluR5-7: kainate subtype, Pharmingen, CA), and rat NMDA receptors (NMDAR1, Pharmingen, CA). Dense and specific signals were obtained with all antibodies. The four types of receptors are broadly distributed in the brain, and, in particular, immunoreactive cells are identified within the major aromatase cell groups located in the medial preoptic nucleus, ventromedial hypothalamus, nucleus striae terminalis, and nucleus taeniae. Dense specific populations of glutamate receptor-immunoreactive cells are also present with a receptor subtype-specific distribution in broad areas of the telencephalon. The distribution of glutamate receptors, therefore, is consistent with the idea that these receptors could be located at the surface of aromatase-containing cells and mediate the rapid regulation of aromatase activity in a direct manner.

Animals↗
PubMed1992

A further evaluation of the effects of K+ depolarization on glutamate-evoked [3H]dopamine release from striatal slices.

Exogenous glutamate will evoke dopamine (DA) release from striatal slices in vitro. To further characterize glutamate-evoked DA release from striatal slices, experiments were designed to: 1) determine if sufficient endogenous glutamate can be released in vitro to presynaptically mediate [3H]DA release in the absence of Mg++ and 2) reevaluate how K+ depolarization affects glutamate-evoked [3H]DA release. Removal of Mg++ to potentiate N-methyl-D-aspartate (NMDA) receptor-mediated DA release increased 15 mM K(+)-evoked [3H]DA release to about 200% of control. The potentiation of this release was probably not mediated by NMDA receptors because it was not blocked by the glutamate receptor antagonists MK-801, 6,7-dinitroquinoxalinedione (DNQX) or kynurenate. Furthermore, the removal of Mg++ increased DA release substantially (200%) in the presence of 5 microM sulpiride and 10 microM nomifensine, indicating that DA reuptake and DA D2 autoreceptors are not primarily responsible for increased DA release. In the absence of Mg++, depolarization produced by 20 mM or greater [K+] inhibited DA released by exogenous glutamate, whereas a much higher [K+] was necessary to evoke endogenous glutamate release. In the presence of 1.5 mM Mg++, a reduction of the "Mg++ blockade" of NMDA receptors by 15 mM K+ depolarization during glutamate-evoked DA release was evaluated with and without the DA reuptake inhibitor nomifensine and the DA D2 antagonist sulpiride. DA released by K+ depolarization (Mg++ present) was markedly increased by 1 mM glutamate, but this effect was only partially reversed by kynurenate or high concentrations of either MK-801 (25 microM) or DNQX (100 microM).(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1988

Glutamate biosynthesis in Bacillus azotofixans. 15N NMR and enzymatic studies.

Pathways of ammonia assimilation into glutamic acid in Bacillus azotofixans, a recently characterized nitrogen-fixing species of Bacillus, were investigated through observation by NMR spectroscopy of in vivo incorporation of 15N into glutamine and glutamic acid in the absence and presence of inhibitors of ammonia-assimilating enzymes, in combination with measurements of the specific activities of glutamate dehydrogenase, glutamine synthetase, glutamate synthase, and alanine dehydrogenase. In ammonia-grown cells, both the glutamine synthetase/glutamate synthase and the glutamate dehydrogenase pathways contribute to the assimilation of ammonia into glutamic acid. In nitrate-grown and nitrogen-fixing cells, the glutamine synthetase/glutamate synthase pathway was found to be predominant. NADPH-dependent glutamate dehydrogenase activity was detectable at low levels only in ammonia-grown and glutamate-grown cells. Thus, B. azotofixans differs from Bacillus polymyxa and Bacillus macerans, but resembles other N2-fixing prokaryotes studied previously, as to the pathway of ammonia assimilation during ammonia limitation. Implications of the results for an emerging pattern of ammonia assimilation by alternative pathways among nitrogen-fixing prokaryotes are discussed, as well as the utility of 15N NMR for measuring in vivo glutamate synthase activity in the cell.

Ammonia↗
PubMed1995

Glutamate receptor (gamma-amino-3-hydroxy-5-methyl-4-isoxazole propionate and kainate subtype) activity enhanced by dizocilpine (MK801) in rat hippocampus. Rapid chemical kinetic measurements of sodium flux and receptor desensitization with native membranes.

L-Glutamate-mediated transmembrane influx of 22Na+ into a native membrane vesicle preparation from rat hippocampus was resolved into components due to glutamate receptor and Na(+)-glutamate symport, by kinetic analysis and pharmacological specificity. Measurements made with a quench-flow technique showed that receptor-mediated cation exchange proceeded in two phases, the faster phase progressively attenuated by a desensitization process with a half time of approximately 90 ms. The influx of 22Na+ continued in the second phase with a half time of 6 s. Receptor-mediated 22Na+ influx was replicated with the glutamate mimetics, alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate and kainate but not with N-methyl-D-aspartate and was inhibited by 6-cyano-7-nitroquinoxaline-2,3-dione but not by 2-amino-5-phosphonovaleric acid. Receptor-mediated 22Na+ influx did not require glycine and was pH dependent; influx was inhibited at pH values less than pH 5.0. Thus, the receptor-mediated activity was of the gamma-amino-3-hydroxy-5-methyl-4-isoxazole propionate-kainate subtype. The N-methyl-D-aspartate receptor inhibitor 5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine (dizocilpine or MK801), increased receptor-mediated transmembrane 22Na+ flux in both phases, but did not accelerate receptor desensitization. Receptor-mediated radiotracer exchange was the major contribution to 22Na+ influx in times less than 5 s and the receptor-mediated responses could be measured with a small correction for sodium-glutamate symport or in the presence of a glutamate-uptake inhibitor. A third phase of 22Na+ influx accessed a different internal volume and proceeded with the same first-order rate constant as [3H]glutamate uptake when measured simultaneously. This was shown to be mediated by the glutamate uptake mechanism, with a half time of 20-40 s varying with the preparation. The Na(+)-glutamate symport became the largest cause of 22Na+ influx after at least 5 s, the final magnitude being, on average, twofold larger than the receptor-mediated 22Na+ influx.

Amino Acid Transport System X-AG↗
PubMed2005

Glutamate transporter function of rat hippocampal astrocytes is impaired following the global ischemia.

Astroglial glutamate transporters, GLT-1 and GLAST, play an essential role in removing released glutamate from the extracellular space and are essential for maintaining a low concentration of extracellular glutamate in the brain. It was hypothesized that impaired function of glial glutamate transporters induced by transient global ischemia may lead to an elevated level of extracellular glutamate and subsequent excitotoxic neuronal death. To test this hypothesis, in the present study, we performed whole-cell patch-clamp recording of hippocampal CA1 astrocytes in control or postischemic slices, and measured glutamate transporter activity by recording glutamate-evoked transporter currents. Six to 24 h after global ischemia, maximal amplitude of glutamate transporter currents recorded from postischemic CA1 astrocytes was significantly reduced. Western blotting analysis indicated that transient global ischemia decreased the protein level of GLT-1 in the hippocampal CA1 area without affecting GLAST protein level. Further real-time quantitative RT-PCR assays showed that global ischemia resulted in a decrease in GLT-1 mRNA level of hippocampal CA1 region. Global ischemia-induced reduction in GLT-1 expression and glutamate transporter function of CA1 astrocytes precedes the initiation of delayed neuronal death in CA1 pyramidal layer. The present study provides the evidence that transient global ischemia downregulates glutamate transporter function of hippocampal CA1 astrocytes by decreasing mRNA and protein levels of GLT-1.

Amino Acid Transport System X-AG↗
PubMedDate not supplied

Beta-amyloid and brain-derived neurotrophic factor, BDNF, up-regulate the expression of glutamate transporter GLT-1/EAAT2 via different signaling pathways utilizing transcription factor NF-kappaB.

Malfunctioning of high-affinity glutamate transporters is believed to contribute to the accumulation of toxic concentrations of glutamate and, thus, trigger the cellular mechanisms of neurodegeneration. Emerging data point to the presence of excitotoxic component in Alzheimer's disease (AD) and aberrant expression of glutamate transporters in this neurodegenerative malady. Neuronal soluble factors are essential for differential expression and fine tuning of the astroglial glutamate transporters, GLT-1/EAAT2 and GLAST/EAAT1. However, the nature of factors specifically affecting glutamate uptake in AD is largely unknown. The overproduction of neurotoxic beta-amyloid peptide (Abeta), a major constituent of amyloid plaques, and marked down-regulation of BDNF, a neuroprotective factor, are hallmarks of AD pathophysiology. None of these typically neuronal factors was capable of changing the pattern of glutamate transporter expression in undifferentiated rat astrocytes that predominantly expressed GLAST. In differentiated astrocytes, BDNF and, to a lesser extent, subtoxic concentrations of Abeta 1-42 (1-5 microM) induced the expression of GLT-1 and increased glutamate uptake, whereas the GLAST levels were unaltered by these factors. The BDNF-dependent up-regulation of GLT-1 in differentiated astrocytes was partially antagonized by the activation of metabotropic glutamate receptor 4 (mGluR4), but not by group I or II mGluRs. Activation of transcription factor NF-kappaB appeared to be a shared essential, but not a sufficient molecular event in the BDNF- or Abeta-dependent induction of GLT-1. The BDNF-dependent activation of NF-kappaB and up-regulation of GLT-1 was critically dependent on the upstream activation of p42/p44 MAP kinase signaling, whereas the inhibition of these MAP kinases dramatically increased the Abeta-dependent activation of NF-kappaB and production of GLT-1. The capacity to up-regulate astroglial glutamate uptake system, that apparently represents a novel element in the neuroprotective repertoire of BDNF, can, however, provide adverse effect under certain insults when glutamate transporters start operating in reverse direction. The Abeta-dependent up-regulation of GLT-1/EAAT2, more pronounced under the deficit of MAP kinase signaling, may attenuate synaptic efficacy and, thus contribute to the impairment of neuroplasticity in AD.

Amyloid beta-Peptides↗
PubMedDate not supplied

Neurotoxic and neuroprotective effects of the glutamate transporter inhibitor DL-threo-beta-benzyloxyaspartate (DL-TBOA) during physiological and ischemia-like conditions.

Maintenance of low extracellular glutamate ([Glu](O)) preventing excitotoxic cell death requires fast removal of glutamate from the synaptic cleft. This clearance is mainly provided by high affinity sodium-dependent glutamate transporters. These transporters can, however, also be reversed and release glutamate to the extracellular space in situations with energy failure. In this study the cellular localisation of the glutamate transporters GLAST and GLT-1 in organotypic hippocampal slice cultures was studied by immunofluorescence confocal microscopy, under normal culture conditions, and after a simulated ischemic insult, achieved by oxygen and glucose deprivation (OGD). In accordance with in vivo findings, GLAST and GLT-1 were primarily expressed by astrocytes under normal culture conditions, but after OGD some damaged neurons also expressed GLAST and GLT-1. The potential damaging effect of inhibition of the glutamate transporters by DL-threo-beta-benzyloxyaspartate (DL-TBOA) was studied using cellular uptake of propidium iodide (PI) as a quantitative marker for the cell death. Addition of DL-TBOA for 48 h was found to induce significant cell death in all hippocampal regions, with EC(50) values ranging from 38 to 48 microM for the different hippocampal subregions. The cell death was prevented by addition of the glutamate receptor antagonists NBQX and MK-801, together with an otherwise saturating concentration of DL-TBOA (100 microM). Finally, the effect of inhibition of glutamate release, via reverse operating transporters during OGD, was investigated. Addition of a sub-toxic (10 microM) dose of DL-TBOA during OGD, but not during the subsequent 48 h recovery period, significantly reduced the OGD-induced PI uptake. It is concluded: (1) that the cellular expression of the glutamate transporters GLAST and GLT-1 in hippocampal slice cultures in general corresponds to the expression in vivo, (2) that inhibition of the glutamate transporters induces cell death in the slice cultures, and (3) that partial inhibition during simulation of ischemia by OGD protects against the induced PI uptake, most likely by blocking the reverse operating transporters otherwise triggered by the energy failure.

Amino Acid Transport System X-AG↗
PubMed2004

Noncompetitive inhibition by L-cysteine and activation by L-glutamate of the iron-oxidizing activity of a mixotrophic iron-oxidizing bacterium strain OKM-9.

A mesophilic, mixotrophic iron-oxidizing bacterium strain OKM-9 uses ferrous iron as a sole source of energy and L-glutamate as a sole source of cellular carbon. Uptake of L-glutamate into OKM-9 cells is absolutely dependent on ferrous iron oxidation. Thus, the Fe(2+)-dependent L-glutamate uptake system of strain OKM-9 is crucial for the bacterium to grow mixotrophically in iron medium with L-glutamate. The relationship between iron oxidation and L-glutamate transport activities was studied. Iron oxidase containing cytochrome a was purified 9-fold from the plasma membrane of OKM-9. A purified iron oxidase showed one rust-colored band following disc gel electrophoresis after incubation with Fe(2+). The Fe(2+)-dependent L-glutamate transport system was also purified 14.5-fold from the plasma membrane using the same purification steps as for iron oxidase. Fe(2+)-dependent L-glutamate and L-cysteine uptake activities of OKM-9 were 0.36 and 0.24 nmol/mg/min, respectively, when a concentration of 18 mM of these amino acids was used as a substrate. Both uptake activities were completely inhibited by potassium cyanide (KCN), suggesting that cytochrome a in the iron oxidase is involved in the transport process. The iron-oxidizing activity of strain OKM-9 was activated 1.7-fold by 80 mM L-glutamate. In contrast, the activity was noncompetitively inhibited by L-cysteine. The Michaelis constant of iron oxidase for Fe(2+) was 12.6 mM and the inhibition constant for L-cysteine was 41.6 mM. A marked inhibition of iron oxidase by 50 mM L-cysteine was completely reversed by the addition of 60 mM L-glutamate. The results suggest the possibility that iron oxidase has a binding site for L-cysteine and the cysteine first bound to the iron oxidase was replaced by the added L-glutamate.

Journal Article↗
PubMed1998

Molecular pharmacology and physiology of glutamate transporters in the central nervous system.

1. Glutamate is the predominant excitatory neurotransmitter in the brain, but it is also a potent neurotoxin. Following release of glutamate from presynaptic vesicles into the synapse and activation of a variety of ionotropic and metabotropic glutamate receptors, glutamate is removed from the synapse. This is achieved through active uptake of glutamate by transporters located pre- and also post-synaptically or, alternatively, glutamate can diffuse out of the synapse and be taken up by transporters located on the cell surface of glial cells. 2. Complementary DNA encoding a number of glutamate transporters have recently been cloned and form a family of structurally related membrane proteins with a high degree of amino acid sequence conservation. Expression of the cloned glutamate transporters in various cell types has aided in the characterization of the functional properties of the different transporter subtypes. 3. Glutamate transport is coupled to sodium, potassium and pH gradients across the cell membrane creating an electrogenic process. This allows transport to be measured using electrophysiological techniques, which has greatly aided in understanding some of the basic mechanisms of the transport process and has also allowed a detailed understanding of the molecular pharmacology of the different transporter subtypes. 4. In the present review I shall discuss some of the recent advances in understanding the molecular basis for glutamate transporter function and then highlight some of the unanswered questions concerning the physiological roles of these proteins and suggest possible strategies for pharmacological manipulation of transporters for the treatment of neurological disorders.

ATP-Binding Cassette Transporters↗
PubMed2004

Roles of glutamate transporters in shaping excitatory synaptic currents in cerebellar Purkinje cells.

Several subtypes of glutamate transporters are abundantly expressed near the excitatory synapses on cerebellar Purkinje cells. We investigated the roles of the glutamate transporters in shaping the excitatory postsynaptic currents (EPSCs) and regulating the levels of extracellular glutamate in the mouse cerebellum using a potent blocker of glutamate transporters, dl-threo-beta-benzyloxyaspartate (dl-TBOA). This drug markedly prolonged AMPA receptor-mediated EPSCs in Purkinje cells evoked by stimulating both parallel fibres and climbing fibres. The decay phase of the prolonged EPSCs was fitted by double exponentials, of which the slower component was preferentially inhibited by a low-affinity competitive antagonist of AMPA receptors, gamma-d-glutamyl-glycine, indicating that the slow component induced by dl-TBOA was the AMPA receptor-mediated current activated by lower concentrations of glutamate than those contributing to the peak of the EPSC. This result suggests that dl-TBOA prolongs the stay of synaptically released glutamate in the synaptic cleft and also induces glutamate spillover to extrasynaptic targets as well as neighbouring synapses. Furthermore, high concentrations of dl-TBOA in the presence of cyclothiazide generated a continuous inward current in Purkinje cells, of which the amplitude reached the peak level of the climbing-fibre EPSC. This continuous inward current was abolished by the blocker of AMPA receptors, indicating that the strong inhibition of glutamate uptake causes the rapid accumulation of glutamate in the extracellular space. These results highlight the importance of glutamate transporters in maintaining the proper glutamatergic transmission in Purkinje cell synapses.

Amino Acid Transport System X-AG↗
PubMed2002

Glutamate transporters in bone.

In the central nervous system Na(+)-dependent glutamate transporters bind extracellular glutamate and transport it into cells surrounding the synapse, terminating excitatory signals. These glutamate transporters also function as ion channels. The glutamate transporter, GLAST-1, is expressed in the plasma membrane of osteoblasts and osteocytes and is the same molecular weight as in brain. Thus in bone cells GLAST-1 may transport glutamate or operate as a glutamate gated ion channel. A splice variant, GLAST-1a, is also expressed in bone. Hydropathy and Western blot analysis suggest GLAST-1a adopts a reversed orientation within the cell membrane. Sodium and potassium ion gradients drive glutamate transport but glycosylation, oxidation and phosphorylation modulate transporter activity. Reversal of GLAST-1a would alter these modifications varying its transport activity under the same ionic gradients. The significance of GLAST-1/1a in bone in vivo is unknown. GLAST-1 knockout mice show no major disruption of skeletal development but have not been investigated in detail. Glutamate affects both osteoclast and osteoblast biology and the regulation of GLAST-1 by mechanical loading in bone suggests a role for glutamate transporters in osteogenesis. Differential regulation and modification of GLAST variants may provide an intricate mechanism controlling extracellular glutamate levels and thus its downstream signalling effects in bone.

Journal Article↗
PubMed2002

Osteoblastic glutamate receptor function regulates bone formation and resorption.

Previous studies showed that a variety of bone cells express protein components necessary for neuronal-like glutamatergic signaling and implicated glutamate as having a role in mechanically induced bone remodeling. Initial functional studies concentrated on the role of glutamate signaling in bone resorption and provided compelling evidence to suggest that glutamate signaling through functional NMDA type ionotropic glutamate receptors (iGluRs) is a prerequisite for in vitro osteoclastogenesis. Originally, effects of iGluR antagonists seen in co-cultures were attributed to antagonists acting directly on osteoclast precursors. However, in the light of recent osteoblast studies it now seems likely that the observed effects on osteoclastogenesis are an indirect effect of modulating the function of pre-osteoblast present within these cultures. The presence of iGluRs in osteoblasts suggests a role for them in bone formation and this paper reviews and discusses the emerging data relating to the role of glutamate signaling in osteoblasts. A number of recently published studies have shown that osteoblasts not only express a wide number of 'pre-synaptic' glutamatergic proteins but also possess the ability to both regulate glutamate release and actively recycle extracellular glutamate. The functionality of osteoblastic 'post-synaptic' glutamatergic components has also been shown as both primary and clonal osteoblasts express electrophysiologically active iGluRs, metabotropic type glutamate receptors (mGluRs) along with a variety of glutamate receptor associated signaling proteins. There is, however, little published data regarding the actual role of glutamatergic signaling in osteoblastic bone formation. In vivo and in vitro studies performed provide evidence that glutamatergic signaling is a necessity for normal osteoblast function. In a number of different models of in vitro bone formation, the addition of non-competitive antagonists of iGluRs prevents the formation of mineralized bone, moreover antagonizing some sub-types of iGluR mediates the differentiation of pre-osteoblasts. iGluR antagonists modulate osteoblast function in a manner that correlates with the previously reported data regarding in vitro osteoclastogenesis. Interestingly iGluR mediated glutamate signaling appears to function differently in osteoblasts derived from flat and long bones. This implies the components of osteoblastic glutamatergic signaling may be adapted in vivo possibly to reflect the differential function of osteoblasts in those regions of the skeleton.

Journal Article↗
PubMed1981

Glutamate metabolism in malnutrition and sepsis in man.

The metabolism of intravenous glutamate has been studied for its usefulness as a nutrient in intravenous feeding regimens. Intravenously infused monosodium L-glutamate was rapidly removed from the blood with a half-life of 10.7 and 9.1 min in healthy control and malnourished septic patients, respectively. Under the same conditions, alanine was metabolized much more slowly, with a half-life of 35 min (1). After the infusion of glutamate, blood lactate fell and glucose was increased in the control patients but not in the sick patients. Blood pyruvate, ketone bodies, glutamine, aspartate and alanine levels did not change after the infusion of glutamate in either group of patients. Increased urinary excretion of aspartate was observed in the control patients following the infusion of glutamate, but in the sick patients there was an increased excretion of glutamine. Insulin concentration in the blood rose after the infusion of glutamate in the control patients but not in the sick patients. Although the glutamate half-life life was similar in the two groups of patients, glutamate was metabolized differently in the sick. Deamination predominates over transamination. We conclude that glutamate, in the amounts provided in intravenous feeding, is safe, although its effectiveness may be less than previously thought.

Adult↗
PubMed1992

Direct observations of synapses between L-glutamate-immunoreactive boutons and identified spinocervical tract neurones in the spinal cord of the cat.

Four spinocervical tract cells in lumbosacral spinal cords of adult cats were physiologically characterized and intracellularly labelled with horseradish peroxidase. The neurones were examined with a light microscope and reconstructed. Selected regions were chosen for ultrastructural analysis. Thin sections were treated to reveal the presence of L-glutamate by using the postembedding immunogold method. Two antisera, which specifically recognise the presence of fixed glutamate in tissue, were used in the study. Somata, proximal, and distal dendrites of all four neurones received synaptic contacts from boutons which displayed an obvious immunogold reaction. These boutons formed between 35% and 48% of all synaptic contacts onto spinocervical tract cells. Glutamate-enriched boutons were associated with gold particle densities which were 2-3 times greater than the average densities associated with the surrounding neuropil. Their profiles had a mean diameter of 1.68 microns, contained round agranular synaptic vesicles, and formed asymmetrical synaptic junctions. However, not all boutons displaying these characteristics were enriched with glutamate. Immunogold studies of alternate thin sections, which were incubated with glutamate or GABA antiserum, demonstrated that synaptic boutons on spinocervical tract cells were either enriched with GABA or with glutamate and formed two separate populations which had distinct morphological characteristics. GABA-containing boutons contained irregularly shaped agranular vesicles and formed symmetrical synaptic junctions, whereas glutamate-enriched boutons corresponded to those described above. A further population of boutons, containing highly flattened vesicles, was not immunoreactive for GABA or glutamate. The evidence supports the idea that much of the excitatory transmission into the SCT is mediated by L-glutamate.

Animals↗
PubMed2006

Glutamate-mediated glial injury: mechanisms and clinical importance.

Primary and/or secondary glial cell death can cause and/or aggravate human diseases of the central nervous system (CNS). Like neurons, glial cells are vulnerable to glutamate insults. Astrocytes, microglia, and oligodendrocytes express a wide variety of glutamate receptors and transporters that mediate many of the deleterious effects of glutamate. Astrocytes are responsible for most glutamate uptake in synaptic and nonsynaptic areas and consequently, are the major regulators of glutamate homeostasis. Microglia in turn may secrete cytokines, which can impair glutamate uptake and reduce the expression of glutamate transporters. Finally, oligodendrocytes, the myelinating cells of the CNS, are very sensitive to excessive glutamate signaling, which can lead to the apoptosis or necrosis of these cells. This review aims at summarizing the mechanisms leading to glial cell death as a consequence of alterations in glutamate signaling, and their clinical relevance. A thorough understanding of these events will undoubtedly lead to better therapeutic strategies to treat CNS diseases affecting glia and in particular, those that involve damage to white matter tracts.

Amino Acid Transport System X-AG↗
PubMed1991

Characterization of glutamate uptake systems in astrocyte primary cultures from rat brain.

The dependence of 3[H]-L-glutamate uptake on the presence of sodium, chloride, or calcium ions or on a combination of the three was investigated in astrocyte primary cultures. A stimulating effect on glutamate uptake by each of the ions tested was found. In addition to the comparably small effect by calcium alone, calcium exhibits a synergistic effect on the sodium- and chloride-dependent uptake. The sodium-dependent transport accumulates the glutamate analogue D-aspartate as well as L-glutamate. L-aspartate is taken up by about 50% of the values observed for L-glutamate transport. Sodium-dependent glutamate uptake is strongly inhibited by aspartate-beta-hydroxamate (A beta H) and threo-beta-hydroxyaspartate (T beta H). Quisqualate is less potent in inhibiting this uptake. In contrast, the chloride- and the calcium-dependent uptake systems do not handle D- and L-aspartate as substrates. A beta H and T beta H are only poor inhibitors of these transporters while quisqualate reduces glutamate uptake almost completely. Kinetic data of all uptake systems were estimated. High and low affinity components of each individual system are demonstrated by Eadie-Hofstee analysis. Hill plots indicate that high and low affinity uptake may be due either to two respective uptake sites for Na(+)-, Cl(-)-, and Ca(++)-dependent glutamate transport, or to two glutamate binding sites for each single transport system with negative cooperativity.

Animals↗
PubMed1995

Intracellular acidification and Ca2+ transients in cultured rat cerebellar astrocytes evoked by glutamate agonists and noradrenaline.

The effect of different neurotransmitters on the intracellular pH (pHi) and intracellular calcium (Ca2+i) was studied in cultured astrocytes from neonatal rat cerebellum, using the fluorescent dyes 2,7'-bis(carboxyethyl)-5,6-carboxy-fluorescein (BCECF) and Fura-2. Application of glutamate or kainate (100 microM) in a HEPES-buffered, CO2/HCO3(-) -free saline induced a decrease in pHi and an increase in Ca2+i. Amplitude and time course of the pHi and Ca2+i transients were different. Glutamate and kainate evoked a mean acidification of 0.22 +/- 0.05 (n = 29) and 0.20 +/- 0.09 (n = 12) pH units, respectively. The changes in pHi and Ca2+i induced by kainate, but not by glutamate, were inhibited by 6-cyano-7-dinitroquinozalin-2,3-dion (CNQX; 50 microM). In order to elucidate the mechanism of the agonist-induced acidification, whether the pHi changes were secondary to the Ca2+ rises was tested. In the absence of extracellular Ca2+, the kainate-induced Ca2+i transient was suppressed, while the intracellular acidification was only reduced by 13%. Removal of extracellular Ca2+ reduced the glutamate-induced pHi change by 8%, while the second component of the Ca2+i transient was abolished. Application of trans-( +/- )-1-amino-(1S,3R)-cyclopentadicarboxylic acid (t-ACPD, 100 microM), a metabotropic glutamate receptor agonist, and of noradrenaline (20 microM) evoked a Ca2+i increase, but no change of pHi. D-aspartate, which has a low affinity to glutamate receptors, but is known to be transported by the glutamate uptake system in some astrocytes, evoked an intracellular acidification, similar to that induced by glutamate, but no Ca2+i transient. The results suggest that the kainate-induced acidification is only partly due to the concomitant Ca2+i rise, while the glutamate/aspartate-induced acidification is mainly due to the activation of the glutamate uptake system.

Animals↗