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PubMed1995

Expression patterns of a glutamate-binding protein in the rat central nervous system: comparison with N-methyl-D-aspartate receptor subunit 1 in rat.

Using radioactive in situ hybridization histochemistry, we examined the topographical patterns of expression of the messenger RNA encoding a glutamate-binding protein (N-methyl-D-aspartate receptor glutamate-binding protein in rat; NMDARgbs) in the central nervous system of the rat. Expression patterns of N-methyl-D-aspartate receptor glutamate-binding protein were compared with those of N-methyl-D-aspartate receptor subunit 1 (NMDAR1) of the N-methyl-D-aspartate receptor on adjacent sections. N-methyl-D-aspartate receptor glutamate-binding protein is not expressed in glial cells. The expression of both N-methyl-D-aspartate receptor glutamate-binding protein and N-methyl-D-aspartate receptor subunit 1 was observed in virtually all neurons throughout the central nervous system. The mean level of N-methyl-D-aspartate receptor subunit 1 expression was higher than that of N-methyl-D-aspartate receptor glutamate-binding protein. Similar topographical patterns of expression of N-methyl-D-aspartate receptor glutamate-binding protein and N-methyl-D-aspartate receptor were observed in most regions, except in discrete thalamic, hypothalamic and brainstem nuclei. Concomitantly for N-methyl-D-aspartate receptor glutamate-binding protein and N-methyl-D-aspartate receptor subunit 1, the highest expression levels were distributed in the mitral layer of main and accessory olfactory bulbs, granule cell layer of the dentate gyrus, polymorphic and pyramidal layers of CA1-3 fields of Ammon's horn. A slightly less prominent expression was observed in the glomerular and granule cell layers of main and accessory olfactory bulbs, anterior olfactory nucleus, layer 2 of piriform cortex, olfactory tubercle and taenia tecta. In the cerebellum, the prominent level of N-methyl-D-aspartate receptor glutamate-binding protein expression was slightly higher in the Purkinje cell layer than in the granule cell layer, an opposite pattern being observed for N-methyl-D-aspartate receptor subunit 1. A moderately high expression level of both messenger RNAs was observed in the medial septal nucleus, nucleus of the diagonal band of Broca, dorsal part of the endopiriform nucleus, and in the anteroventral and anterolateral parts of the bed nucleus of the stria terminalis. In the neocortex, the mean expression level of N-methyl-D-aspartate receptor glutamate-binding protein is moderate, while the mean level of N-methyl-D-aspartate receptor subunit 1 expression is high. With both probes, layer IV is slightly less labeled than the other layers.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗

PubMed1996

Glutamate as a putative neurotransmitter in the mollusc, Lymnaea stagnalis.

Bath-applied glutamate (10-1000 microM) produced excitatory and inhibitory responses on numerous identified neurons of the mollusc Lymnaea stagnalis. Using both in situ and in vitro preparations, glutamate or glutamate agonists produced a depolarization in identified neurons right pedal dorsal 1 and right pedal dorsal 2 and 3. However, attempts to block glutamate-evoked responses with glutamate antagonists were unsuccessful. We examined a potential glutamatergic neuron, visceral dorsal 4. Exogenous application of the peptides (GDPFLRFamide and SDPFLRFamide) could mimic the inhibitory, but not the excitatory effects of visceral dorsal 4 on its postsynaptic cells, implying the presence of a second transmitter. We tested the possibility that glutamate is this second neurotransmitter by using excitatory synapses between visceral dorsal 4 and postsynaptic cells right pedal dorsal 2 and 3, right pedal dorsal 1, visceral F group and right parietal B group neurons. Of all the putative neurotransmitters tested, only glutamate had consistent excitatory effects on these postsynaptic cells. Also, the amplitude of the right pedal dorsal 2 and 3 excitatory postsynaptic potentials was reduced in the presence of N-methyl-D-aspartate and other glutamate agonists, suggesting desensitization of the endogenous transmitter receptor. In conclusion, some identified Lymnaea neurons respond to glutamate via a receptor with novel pharmacological properties. Furthermore, a Lymnaea interneuron may employ glutamate as a transmitter at excitatory synapses.

Animals↗

PubMedDate not supplied

Glutamate and N-methyl-D-aspartate binding sites in the guinea pig hippocampus: ontogeny and effect of acute in vitro ethanol exposure.

The objectives of this study were to characterize the ontogeny of the L-glutamate (glutamate) and N-methyl-D-aspartate (NMDA) binding sites in the developing guinea pig hippocampus, and to determine the effect of acute in vitro ethanol exposure on these binding sites. Specific [3H]glutamate binding and NMDA-sensitive [3H]glutamate binding were determined using a guinea pig hippocampal synaptic membrane preparation (HSMP). To characterize the ontogeny of the density (Bmax) and affinity (Kd) of the glutamate and NMDA binding sites, saturation analysis was conducted on HSMP of guinea pigs at gestational day (GD) 50 (immature fetus; term, GD 68), GD 62 (mature, near-term fetus), postnatal day (PD) 13 (neonate), and PD > 60 (adult). To examine the effect of ethanol on the glutamate and NMDA binding sites, HSMP of guinea pigs at GD 50, GD 62, PD 13, and PD > 60 was incubated with ethanol (0-100 mM), followed by determination of specific [3H]glutamate binding and NMDA-sensitive [3H]glutamate binding. To determine the effect of 50 mM ethanol on the Bmax and Kd of the glutamate and NMDA binding sites, HSMP of guinea pigs at GD 62 and PD > 60 was incubated with 0 or 50 mM ethanol followed by saturation analysis. The Bmax values of the hippocampal glutamate and NMDA binding sites were greater at GD 62 and PD 13 compared with GD 50 and PD > 60, but there was no change in the Kd of the binding sites throughout development.(ABSTRACT TRUNCATED AT 250 WORDS)

Aging↗

PubMed2006

Dopaminergic and GABAergic modulation of glutamate release from rat subthalamic nucleus efferents to the substantia nigra.

The regulation of the glutamatergic projection from the subthalamic nucleus (STN) to the substantia nigra (SN) was investigated using dual-probe microdialysis in the awake behaving rat. Reverse dialysis of the cholinergic receptor agonist carbachol (1 mM) into the STN caused an increase in the extracellular concentrations of glutamate and dopamine in the SN. The increase in glutamate was transient and returned toward basal values despite the continued perfusions of the STN with carbachol. Carbachol-stimulated glutamate release was prolonged by perfusion of the selective D2 dopamine receptor antagonist raclopride (100 microM) into the SN and was attenuated by the perfusion of the selective D2-like receptor agonist quinpirole (10 microM). In contrast, perfusion of the D1 dopamine receptor antagonist SCH-23390 (100 microM) did not alter the carbachol-stimulated glutamate release even though it increased basal glutamate concentrations. Perfusion of the GABAA receptor antagonist bicuculline (10 microM) into the SN prolonged the carbachol-stimulated glutamate release in similar fashion as raclopride. The present findings suggest that somatodendritically released dopamine in the SN regulates glutamate release from subthalamic axon terminals by differentially activating dopamine D2 and D1 receptors. Activation of D2 heteroreceptors, located on STN axon terminals, provides a negative feedback control on stimulated subthalamic glutamate release, while D1 receptor activation preferentially regulates basal glutamate concentrations. The findings of the present study also indicate that GABA exerts an inhibitory control on glutamate release in the SN through GABAA receptors.

Analysis of Variance↗

PubMed2006

In vivo monitoring of extracellular glutamate in the brain with a microsensor.

Recent discoveries have revealed that glutamatergic neurotransmission in the central nervous system is mediated by a dynamic interplay between neurons and astrocytes. To enhance our understanding of this process, the study of extracellular glutamate is crucial. At present, microdialysis is the most frequently used analytical technique to monitor extracellular glutamate levels directly in the brain. However, the neuronal and physiological origin of the detected glutamate levels is questioned as they do not fulfil the classical release criteria for exocytotic release, such as calcium dependency or response to the sodium channel blocker tetrodotoxine (TTX). It is hypothesized that an analytical technique with a higher spatial and temporal resolution is required. Glutamate microsensors provide a promising analytical solution to meet this requirement. In the present study, we applied a 10 micro m diameter hydrogel-coated glutamate microsensor to monitor extracellular glutamate levels in the striatum of anesthetized rats. To explore the potential of the microsensor, different pharmacological agents were injected in the vicinity of the sensor at an approximate distance of 100 micro m. It was observed that KCl, exogenous glutamate, kainate and the reuptake inhibitor DL-threo-beta-benzyloxyaspartate (DL-TBOA) increased the extracellular glutamate levels significantly. TTX decreased the basal extracellular glutamate levels approximately 90%, which indicates that the microsensor is capable of detecting neuronally derived glutamate. This is one of the first studies in which a microsensor is applied in vivo on a routine base, and it is concluded that microsensor research can contribute significantly to improve our understanding of the physiology of glutamatergic neurotransmission in the brain.

Animals↗

PubMed2006

Involvement of TNF-alpha in glutamate-induced apoptosis in a differentiated neuronal cell line.

We examined the involvement of tumor necrosis factor (TNF)-alpha on glutamate-induced cytotoxicity in a differentiated neuronal cell line. In this study, we used nerve growth factor (NGF)-differentiated PC12h cells. Glutamate cytotoxicity was assessed using the MTS and TUNEL assays. To detect TNF-alpha levels in culture supernatants after glutamate exposure, we used ELISA methods. The involvement of caspase-8, which is downstream from TNF receptor 1 (TNF-R1) in glutamate-induced cytotoxicity, was determined by Western blot analysis. The MTS assay showed that the addition of glutamate resulted in dose-dependent cell death, while the TUNEL assay showed that glutamate induced apoptosis in differentiated PC12h cells in a dose-dependent manner. TNF-alpha levels in the supernatant of glutamate-exposed cells were significantly increased compared with those in unexposed cells. In addition, glutamate caused increases in the levels of caspase-8 protein. The increases in caspase-8 levels were ameliorated by pretreatment with soluble TNF-R1. Moreover, soluble TNF-R1 significantly ameliorated the cell death induced by glutamate. These results suggest that TNF-alpha released from neuronal cells may be associated with glutamate-induced neuronal cell death.

Analysis of Variance↗

PubMed2006

Glutamate-related gene expression changes with age in the mouse auditory midbrain.

Glutamate is the main excitatory neurotransmitter in both the peripheral and central auditory systems. Changes of glutamate and glutamate-related genes with age may be an important factor in the pathogenesis of age-related hearing loss-presbycusis. In this study, changes in glutamate-related mRNA gene expression in the CBA mouse inferior colliculus with age and hearing loss were examined and correlations were sought between these changes and functional hearing measures, such as the auditory brainstem response (ABR) and distortion product otoacoustic emissions (DPOAEs). Gene expression of 68 glutamate-related genes was investigated using both genechip microarray and real-time PCR (qPCR) molecular techniques for four different age/hearing loss CBA mouse subject groups. Two genes showed consistent differences between groups for both the genechip and qPCR. Pyrroline-5-carboxylate synthetase enzyme (Pycs) showed down-regulation with age and a high-affinity glutamate transporter (Slc1a3) showed up-regulation with age and hearing loss. Since Pycs plays a role in converting glutamate to proline, its deficiency in old age may lead to both glutamate increases and proline deficiencies in the auditory midbrain, playing a role in the subsequent inducement of glutamate toxicity and loss of proline neuroprotective effects. The up-regulation of Slc1a3 gene expression may reflect a cellular compensatory mechanism to protect against age-related glutamate or calcium excitoxicity.

Aging↗

PubMed2005

Modeling of slow glutamate diffusion and AMPA receptor activation in the cerebellar glomerulus.

Synaptic conductances are influenced markedly by the geometry of the space surrounding the synapse since the transient glutamate concentration in the synaptic cleft is determined by this geometry. Our paper is an attempt to understand the reasons for slow glutamate diffusion in the cerebellar glomerulus, a structure situated around the enlarged mossy fiber terminal in the cerebellum and surrounded by a glial sheath. For this purpose, analytical expressions for glutamate diffusion in the glomerulus were considered in models with two-, three-, and fractional two-three-dimensional (2D-3D) geometry with an absorbing boundary. The time course of average glutamate concentration in the synaptic cleft of the mossy fiber-granule cell connection was calculated for both direct release of glutamate from the same synaptic unit, and for cumulative spillover of glutamate from neighboring release sites. Several kinetic schemes were examined, and the parameters of the diffusion models were estimated by identifying theoretical activation of AMPA receptors with direct release and spillover components of published experimental AMPA receptor-mediated EPSCs. For model selection, the correspondence of simulated paired-pulse ratio and EPSC increase after prevention of desensitization to experimental values were also taken into consideration. Our results suggest at least a 7- to 10-fold lower apparent diffusion coefficient of glutamate in the porous medium of the glomerulus than in water. The modeling of glutamate diffusion in the 2D-3D geometry gives the best fit of experimental EPSCs. We show that it could be only partly explained by normal diffusion of glutamate in the complex geometry of the glomerulus. We assume that anomalous diffusion of glutamate occurs in the glomerulus. A good match of experimental estimations and theoretical parameters, obtained in the simulations that use an approximation of anomalous diffusion by a solution for fractional Brownian motion, confirms our assumption.

Cerebellar Cortex↗

PubMed2006

N-acetylaspartate as a reservoir for glutamate.

N-acetylaspartate (NAA) is an intermediary metabolite that is found in relatively high concentrations in the human brain. More specifically, NAA is so concentrated in the neurons that it generates one of the most visible peaks in nuclear magnetic resonance (NMR) spectra, thus allowing NAA to serve as "a neuronal marker". However, to date there is no generally accepted physiological (primary) role for NAA. Another molecule that is found at similar concentrations in the brain is glutamate. Glutamate is an amino acid and neurotransmitter with numerous functions in the brain. We propose that NAA, a six-carbon amino acid derivative, is converted to glutamate (five carbons) in an energetically favorable set of reactions. This set of reactions starts when aspartoacylase converts the six carbons of NAA to aspartate and acetate, which are subsequently converted to oxaloacetate and acetyl CoA, respectively. Aspartylacylase is found in astrocytes and oligodendrocytes. In the mitochondria, oxaloacetate and acetyl CoA are combined to form citrate. Requiring two steps, the citrate is oxidized in the Kreb's cycle to alpha-ketoglutarate, producing NADH. Finally, alpha-ketoglutarate is readily converted to glutamate by transaminating the alpha-keto to an amine. The resulting glutamate can be used by multiple cells types to provide optimal brain functional and structural needs. Thus, the abundant NAA in neuronal tissue can serve as a large reservoir for replenishing glutamate in times of rapid or dynamic signaling demands and stress. This is beneficial in that proper levels of glutamate serve critical functions for neurons, astrocytes, and oligodendrocytes including their survival. In conclusion, we hypothesize that NAA conversion to glutamate is a logical and favorable use of this highly concentrated metabolite. It is important for normal brain function because of the brain's relatively unique metabolic demands and metabolite fluxes. Knowing that NAA is converted to glutamate will be important for better understanding myriad neurodegenerative diseases such as Canavan's Disease and Multiple Sclerosis, to name a few. Future studies to demonstrate the chemical, metabolic and pathological links between NAA and glutamate will support this hypothesis.

Aspartic Acid↗

PubMed2004

Expression of the ionotropic glutamate receptor subunits and NMDA receptor-associated intracellular proteins in the substantia nigra in schizophrenia.

Multiple neurotransmitter systems have been implicated in the pathophysiology of schizophrenia. Dopamine hyperactivity has often been implicated in this illness. More recently, the glutamate hypothesis of schizophrenia suggests that NMDA receptor (NMDAR) hypofunction may also play a role in this illness. This is based primarily on studies showing that phencyclidine, an NMDAR antagonist, can induce a schizophreniform psychosis. While NMDAR dysfunction is most often implicated in schizophrenia, other components of the glutamate system, such as the AMPA and kainate receptors, as well as NMDAR-associated intracellular proteins, may also play a role in regulating NMDA receptor activity and glutamate neurotransmission. There is growing interest in the hypothesis that the pathophysiology of schizophrenia involves alterations in dopamine-glutamate interactions. The glutamate system is anatomically and functionally linked to the dopamine system, and glutamate can modulate dopaminergic activity and release by stimulating various glutamate receptor subtypes expressed by dopaminergic neurons in the substantia nigra/ventral tegmental area. In this study, we investigated dopamine-glutamate interactions by measuring the expression of transcripts encoding the subunits for the ionotropic glutamate receptors (NMDA, AMPA and kainate) and five NMDAR-associated intracellular proteins, PSD-93, PSD-95, SAP102, NF-L and yotiao in the dopaminergic neurons in the substantia nigra pars compacta (SNc) of subjects with schizophrenia and a comparison group. Tyrosine hydroxylase (TH, a marker of dopamine-synthesizing cells), NR1 (an NMDA receptor subunit) and GluR5 (a kainate subunit) transcript levels were significantly increased in the SNc in schizophrenia. These data support the hypothesis that schizophrenia may involve alterations in dopamine-glutamate interactions.

A Kinase Anchor Proteins↗

PubMed2004

Interleukin-6 protects cultured cerebellar granule neurons against glutamate-induced neurotoxicity.

Cytokine interleukin-6 (IL-6) has been showed to be an important mediator of neuroimmune responses. However, effects of IL-6 in the central nervous system (CNS) are quite complex and diverse, and mechanisms through which IL-6 influences neuronal functions are primarily unknown. In the present study, we explored protective effect of IL-6 that was chronically applied to cerebellar granule neurons (CGNs) in culture against neurodamage induced by glutamate and mechanisms involved in the neuroprotective effect of IL-6. The chronic IL-6 exposure significantly prevented the CGNs from the glutamate-induced attenuation of neuronal vitality. This neuroprotective effect of IL-6 depended on its concentrations. IL-6 at 2.5 ng/ml did not markedly improve the neuronal vitality, but IL-6 at 5 and 10 ng/ml notably improved the neuronal vitality. The glutamate-evoked neuronal apoptosis also was strikingly inhibited by the chronic IL-6 pretreatment. Intracellular Ca2+ in the CGNs lacking IL-6 pretreatment acutely rose as soon as these neurons were stimulated by glutamate and were maintained at higher levels during the whole 18-min period of glutamate attack. Although intracellular Ca2+ in the IL-6-pretreated CGNs also produced an acute and transient elevation in response to the glutamate insult, they quickly dropped and recovered to basal levels before the glutamate application. Anti-gp130 monoclonal antibody (mAb) blocked the suppressive effect of IL-6 on the glutamate-induced intracellular Ca2+ overload. These results reveal that IL-6 can protect neurons against glutamate-induced neurotoxicity, and suggest that the neuroprotective effect of IL-6 may be via gp130 signal transducing pathway to suppress the glutamate-evoked intracellular Ca2+ overload.

Animals↗

PubMed2005

l-dopa-induced reversal in striatal glutamate following partial depletion of nigrostriatal dopamine with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine.

We have reported that 1 month following acute (20mg/kg x 4) or subchronic (30 mg/kg/day x 7d) administration of the neurotoxin, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine, there is an increase or decrease, respectively, in the extracellular level of striatal glutamate as determined by in vivo microdialysis [Robinson S, Freeman P, Moore C, Touchon JC, Krentz L, Meshul CK (2003) Acute and subchronic MPTP administration differentially affects striatal glutamate synaptic function. Exp Neurol 180:73-86]. The goal of this study was to determine the effects of treatment with l-dopa (15 mg/kg) for 21 days on striatal glutamate starting on day 8 after the first dose of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine was administered to mice. Following acute administration of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine, the increase in extracellular striatal glutamate due to lesion of the nigrostriatal pathway was completely reversed to a level below that found in the vehicle-treated group after l-dopa treatment. Subchronic 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine treatment resulted in a decrease in striatal extracellular glutamate that was reversed to the level close to that observed in the vehicle-treated group. There was no change in the density of nerve terminal glutamate immunolabeling associated with the synaptic vesicle pool, suggesting that the alterations in extracellular glutamate most likely originated from the calcium-independent pool. There was a similar decrease in the relative density of tyrosine hydroxylase immunolabeling, a marker for dopamine terminals, within the dorsolateral striatum in both the acute and subchronic 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-treated groups that had been administered l-dopa. There was a decrease in the relative density of immunolabeling within the dorsolateral striatum for the glutamate transporter, GLT-1, following acute 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine treatment in the groups administered either vehicle or l-dopa. There was no change in GLT-1 immunolabeling following subchronic 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine. The results demonstrate that the reversal in the extracellular level of striatal glutamate following l-dopa treatment in both the acute and subchronic 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-treated groups is not due to changes in either striatal dopamine nerve terminals or in the density of the glutamate transporter, GLT-1.

1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine↗

PubMed2004

Inhibition of short-term memory formation in the chick by blockade of extracellular glutamate uptake.

Injection of monosodium glutamate (40nmol/hemisphere) into the intermediate hyperstriatum ventrale of the day-old chick inhibits the formation of short-term memory for a single trial learning that discriminates between colours of beads. These experiments showed that an excess of glutamate close to learning could be damaging to memory. In the present experiments we have blocked the normal reuptake of glutamate and suggest that glutamate release plays a role in normal learning. Removal of glutamate, released from presynaptic neurones during learning, is achieved by various neuronal and astrocytic glutamate transporters. By blocking the primarily astrocytic removal of glutamate by the injection of L-aspartic acid beta-hydroxamate, we effectively increased extrasynaptic levels of glutamate and inhibited short-term memory in a similar manner to central injection of 40nmol glutamate per hemisphere. These experiments suggest that glutamate release within 2.5min of the learning experience is an important feature of short-term memory formation.

Animals↗

PubMed1999

Decreased glutamate metabolism in cultured astrocytes in the presence of thiopental.

The effect of thiopental on glutamate metabolism was studied by 13C magnetic resonance spectroscopy. Cerebral cortical astrocytes were incubated with 0.5 mM [U-13C]glutamate for 2 hr in the presence of 0.5 or 1 mM thiopental. Labeled glutamate, glutamine, aspartate, and glutathione were observed in cell extracts, and glutamine, aspartate, and lactate in the medium. Not only present in the medium was uniformly labeled glutamate, but also glutamate derived from the tricarboxylic acid (TCA) cycle, and thus glutamate release could be detected. The amounts of [U-13C]glutamate and unlabeled glucose taken up by astrocytes were unchanged in the presence of 0.5 mM thiopental and decreased to about 50% and 80%, respectively when the concentration was increased to 1 mM. The amounts of most metabolites synthesized from [U-13C]glutamate were unchanged in the presence of 0.5 mM thiopental, but decreased [U-13C]glutamine, [U-13C]aspartate, and [U-13C]lactate were observed in the 1 mM group. Surprisingly, the amounts of [1,2,3-13C]glutamate, [2,3-13C]aspartate, and [3,4-13C]aspartate (2nd turn via the TCA cycle) were unchanged. However, this was not the case for [1,2-13C]lactate and [2,3-13C]lactate. Such variations indicate cellular compartmentation, possibly caused by a heterogeneous glutamate concentration within the cells affecting TCA cycle turnover rates differently.

Anesthetics, Intravenous↗

PubMed2000

Transporter reversal as a mechanism of glutamate release from the ischemic rat cerebral cortex: studies with DL-threo-beta-benzyloxyaspartate.

Elevated levels of the excitotoxic amino acids, glutamate and aspartate, have been implicated in the pathogenesis of neuronal injury and death induced by cerebral ischemia. This study evaluated the contribution of reversed high-affinity, Na(+)-dependent, glutamate transport to the ischemia-evoked release of glutamate and aspartate using DL-threo-beta-benzyloxyaspartate (DL-TBOA), a newly developed competitive, non-transported blocker of the EAAT 1-3 transporters. Changes in the extracellular levels of these and other amino acids, and of glucose and lactate in cerebral cortical superfusates during four-vessel occlusion-elicited global cerebral ischemia were examined using a cortical window technique. Basal and ischemia-evoked amino acid, glucose and lactate efflux were compared in control versus DL-TBOA (100 microM; applied topically for 35 min prior to ischemia) animals. Twenty minutes of ischemia caused large increases in aspartate, glutamate, GABA and taurine effluxes into cortical superfusates, with non-significant effects on the efflux of glycine, glutamine, alanine and serine. Application of DL-TBOA caused a 2-fold increase in basal, preischemic, extracellular glutamate levels, but did not affect those of the other compounds. In the presence of DL-TBOA, ischemia-evoked release of aspartate, glutamate, taurine and glutamine was significantly reduced; that of the other amino acids was not affected. The ischemia-evoked declines in glucose were significantly attenuated, and lactate release was enhanced above that in control animals. The amino acid data are interpreted as indicating that aspartate and glutamate releases were reduced as a consequence of DL-TBOA inhibition of reversed transport by high-affinity, Na-dependent carriers, predominantly involving the glial EAAT 2 transporter. The reduction in ischemia-evoked taurine release is interpreted as being due to a decrease in cell swelling prior to and during the initial phase of ischemia due to reduced entry of the Na(+), and other ions, associated with a decreased glutamate uptake. Glucose-sparing and availability for lactate formation would also result from a reduced glutamate/Na(+) uptake. These results indicate that reversed transport, primarily from glial cells by the EAAT 2 carrier, is responsible for a substantial (42 and 56%) portion of the ischemia-evoked increase in extracellular glutamate and aspartate levels, respectively. As a potent, competitive, non-transported blocker of high-affinity, Na(+)-dependent, glutamate transporters, DL-TBOA promises to be a valuable new compound for the study of glutamatergic mechanisms.

ATP-Binding Cassette Transporters↗

PubMed2000

delta-, but not mu- and kappa-, opioid receptor activation protects neocortical neurons from glutamate-induced excitotoxic injury.

Recent observations from our laboratory have led us to hypothesize that delta-opioid receptors may play a role in neuronal protection against hypoxic/ischemic or glutamate excitotocity. To test our hypothesis in this work, we used two independent methods, i.e., "same field quantification" of morphologic criteria and a biochemical assay of lactate dehydrogenase (LDH) release (an index of cellular injury). We used neuronal cultures from rat neocortex and studied whether (1) glutamate induces neuronal injury as a function of age and (2) activation of opioid receptors (delta, mu and kappa subtypes) protects neurons from glutamate-induced injury. Our results show that glutamate induced neuronal injury and cell death and this was dependent on glutamate concentration, exposure period and days in culture. At 4 days, glutamate (up to 10 mM, 4 h-exposure) did not cause apparent injury. After 8-10 days in culture, neurons exposed to a much lower dose of glutamate (100 microM, 4 h) showed substantial neuronal injury as assessed by morphologic criteria (>65%, n=23, P<0.01) and LDH release (n=16, P<0. 001). Activation of delta-opioid receptors with 10 microM DADLE reduced glutamate-induced injury by almost half as assessed by the same criteria (morphologic criteria, n=21, P<0.01; LDH release, n=16, P<0.01). Naltrindole (10 microM), a delta-opioid receptor antagonist, completely blocked the DADLE protective effect. Administration of mu- and kappa-opioid receptor agonists (DAMGO and U50488H respectively, 5-10 microM) did not induce appreciable neuroprotection. Also, mu- or kappa-opioid receptor antagonists had no appreciable effect on the glutamate-induced injury. This study demonstrates that activation of neuronal delta-opioid receptors, but not mu- and kappa-opioid receptors, protect neocortical neurons from glutamate excitotoxicity.

3,4-Dichloro-N-methyl-N-(2-(1-pyrrolidinyl)-cycloh↗

PubMed1997

Immediate and delayed effects of in vitro ischemia on glutamate efflux from guinea-pig cerebral cortex slices.

Immediate and delayed effects of glucose deprivation, oxygen deprivation (hypoxia) and both oxygen and glucose deprivation (in vitro ischemia) on glutamate efflux from guinea pig cerebral cortex slices were studied. Immediate effects were evaluated by measuring changes of glutamate efflux during the metabolic insults. Delayed effects were evaluated by measuring the response of the tissue to a 50 mM KCI pulse applied 60 min after the metabolic insults. Deprivation of glucose in the medium did not induce either immediate or delayed effects, while hypoxic condition produced an immediate slight stimulation of glutamate efflux without any delayed effect. Conversely, in vitro ischemia produced both immediate and delayed effects on glutamate efflux. During in vitro ischemia glutamate efflux dramatically increased in a calcium-independent and tetrodotoxin-sensitive manner; this effect was potentiated by a low sodium containing medium. The blockade of the sodium/potassium ATPase exchanger by ouabain caused a glutamate outflow similar to that induced by in vitro ischemia. On the whole, these data demonstrate the central role played by the sodium electrochemical gradient and by the membrane glutamate uptake system in the glutamate overflow induced by in vitro ischemia. Moreover, in slices previously exposed to both oxygen and glucose deprivation the effect of KCI on glutamate efflux was potentiated. This in vitro ischemia-induced delayed potentiation of neurotransmitter efflux, until now unreported in the literature, was found to be selectively restricted to glutamatergic structures and to be mainly due to an enhancement of the exocytotic component of glutamate release.

Animals↗

PubMed1997

Extracellular glutamate and dopamine measured by microdialysis in the rat striatum during blockade of synaptic transmission in anesthetized and awake rats.

We investigated the effect of high dose tetrodotoxin (TTX) on microdialysis measurements of extracellular striatal glutamate and dopamine in normal female rats. Both halothane-anesthetized rats with acutely implanted microdialysis probes and awake rats with microdialysis probes implanted for 24 h were tested. Glutamate levels in awake rats were 45% higher than those of anesthetized rats. Extracellular glutamate remained TTX-insensitive regardless of TTX concentration, anesthesia, or time lapsed after probe implantation. In contrast, TTX reduced dialysate dopamine in all TTX concentrations tested. We speculate that the lower glutamate levels in anesthetized rats reflect the effect of anesthesia. Because glutamate is involved, either as a reactant or a product in a variety of reactions critical to intermediary metabolism in the brain, basal dialysate glutamate levels might indirectly reflect brain metabolism. Further, we conclude that extracellular glutamate collected during non-stimulated conditions is TTX-insensitive. The fact that glutamate levels are TTX-independent does not rule out that glutamate is synaptic in origin but rather demonstrates that it is not nerve impulse-dependent. However, the brain interstitial glutamate pool accessible to the microdialysis probe during control conditions is most likely isolated from the synapse, and therefore does not impose a neurotoxic potential.

Anesthesia↗