Blockade Of Glutamate Uptake Research Articles

Hydrogen sulfide protects astrocytes against H 2O 2-induced neural injury via enhancing glutamate uptake

Excess extracellular glutamate, the main excitatory neurotransmitter, may result in excitotoxicity and neural injury. The present study was designed to study the effect of hydrogen sulfide (H 2S), a novel neuromodulator, on hydrogen peroxide (H 2O 2) -induced glutamate uptake impairment and cellular injuries in primary cultured rat cortical astrocytes. We found that NaHS (an H 2S donor, 0.1-1000 μM) reversed H 2O 2-induced cellular injury in a concentration-dependent manner. This effect was attenuated by L-trans-pyrrolidine-2,4-dicarboxylic (PDC), a specific glutamate uptake inhibitor. Moreover, NaHS significantly increased [ 3H]glutamate transport in astrocytes treated with H 2O 2, suggesting that H 2S may protect astrocytes via enhancing glutamate uptake function. NaHS also reversed H 2O 2-impaired glutathione (GSH) production. Blockade of glutamate uptake with PDC attenuated this effect, indicating that the effect of H 2S on GSH production is secondary to the stimulation of glutamate uptake. In addition, it was also found that H 2S may promote glutamate uptake activity via decreasing ROS generation, enhancing ATP production and suppressing ERK1/2 activation. In conclusion, our findings provide direct evidence that H 2S has potential therapeutic value for oxidative stress-induced brain damage via a mechanism involving enhancing glutamate uptake function.

Generation of Slow Network Oscillations in the Developing Rat Hippocampus After Blockade of Glutamate Uptake

Cell-surface glutamate transporters are essential for the proper function of early cortical networks because their dysfunction induces seizures in the newborn rat in vivo. We have now analyzed the consequences of their inhibition by DL-TBOA on the activity of the developing CA1 rat hippocampal network in vitro. DL-TBOA generated a pattern of recurrent depolarization with an onset and decay of several seconds' duration in interneurons and pyramidal cells. These slow network oscillations (SNOs) were mostly mediated by gamma-aminobutyric acid (GABA) in pyramidal cells and by GABA and N-methyl-D-aspartate (NMDA) receptors in interneurons. However, in both cell types SNOs were blocked by NMDA receptor antagonists, suggesting that their generation requires a glutamatergic drive. Moreover, in interneurons, SNOs were still generated after the blockade of NMDA-mediated synaptic currents with MK-801, suggesting that SNOs are expressed by the activation of extrasynaptic NMDA receptors. Long-lasting bath application of glutamate or NMDA failed to induce SNOs, indicating that they are generated by periodic but not sustained activation of NMDA receptors. In addition, SNOs were observed in interneurons recorded in slices with or without the strata pyramidale and oriens, suggesting that the glutamatergic drive may originate from the radiatum and pyramidale strata. We propose that in the absence of an efficient transport of glutamate, the transmitter diffuses in the extracellular space to activate extrasynaptic NMDA receptors preferentially present on interneurons that in turn activate other interneurons and pyramidal cells. This periodic neuronal coactivation may contribute to the generation of seizures when glutamate transport dysfunction is present.

Behavioral Stress Enhances Hippocampal CA1 Long-Term Depression through the Blockade of the Glutamate Uptake

Behavioral stress has been shown to enhance long-term depression (LTD) in the CA1 region of the hippocampus, but the underlying mechanisms remain unclear. In the present study, we found that selectively blocking NR2B-containing NMDA receptors (NMDARs) abolishes the induction of LTD by prolonged low-frequency stimulation (LFS) in slices from stressed animals. Additionally, there is no need to activate NR2A-containing or synaptic NMDARs to induce this LTD, suggesting that LTD observed in slices from stressed animals is triggered primarily by extrasynaptic NMDAR activation. In contrast, stress has no effect on LTD induced by either a brief bath application of NMDA or a combination of LFS with the glutamate-uptake inhibitor DL-threo-beta-benzyloxyaspartate (DL-TBOA). Furthermore, saturation of LFS-induced LTD in slices from stressed animals occludes the subsequent induction of LTD by LFS in the presence of dl-TBOA. We also found that stress induces a profound decrease in the glutamate uptake in the synaptosomal fraction of the hippocampal CA1 region. These effects were prevented when the animals were given a glucocorticoid receptor antagonist, 11beta,17beta-11[4-(dimethylamino)phenyl]-17-hydroxy-17-(1-(propynyl)-estra-4,9-dien-3-one, before experiencing stress. These results suggest that the blockade of glutamate uptake is a potential mechanism underlying the stress-induced enhancement of LTD and point to a novel role for glutamate-uptake machinery in the regulation of synaptic plasticity induction.

Inhibition of non-vesicular glutamate release by group III metabotropic glutamate receptors in the nucleus accumbens.

Previous in vitro studies have shown that group III metabotropic glutamate receptors (mGluRs) regulate synaptic glutamate release. The present study used microdialysis to characterize this regulation in vivo in rat nucleus accumbens. Reverse dialysis of the group III mGluR agonist l-(+)-2-amino-4-phosphonobutyric acid (L-AP4) decreased, whereas the antagonist (R,S)-alpha-methylserine-O-phosphate (MSOP) increased the extracellular level of glutamate. The decrease by L-AP4 or the increase by MSOP was antagonized by co-administration of MSOP or L-AP4, respectively. Activation of mGluR4a by (1S,3R,4S)-1-aminocyclopentane-1,2,4-tricarboxylic acid or mGluR6 by 2-amino-4-(3-hydroxy-5-methylisoxazol-4-yl)butyric acid had no effect on extracellular glutamate. (R,S)-4-Phosphonophenylglycine (PPG), another group III agonist with high affinity for mGluR4/6/8, reduced extracellular glutamate only at high concentrations capable of binding to mGluR7. The increase in extracellular glutamate by MSOP was tetrodotoxin-independent, and resistant to both the L-type and N-type Ca2+ channel blockers. L-AP4 failed to block 30 mm K+-induced vesicular glutamate release. Blockade of glutamate uptake by d,l-threo-beta-benzyloxyaspartate caused a Ca2+-independent elevation in extracellular glutamate that was reversed by L-AP4. Finally, (S)-4-carboxyphenylglycine, an inhibitor of cystine-glutamate antiporters, attenuated the L-AP4-induced reduction in extracellular glutamate. Together, these data indicate that group III mGluRs regulate in vivo extracellular glutamate in the nucleus accumbens by inhibiting non-vesicular glutamate release.

Beta-Amyloid-induced cytotoxicity, peroxide generation and blockade of glutamate uptake in cultured astrocytes.

beta-Amyloid (betaA) is cytotoxic to neurons in culture by increasing hydrogen peroxide and altering calcium homeostasis. We have evaluated betaA-induced cytotoxicity, peroxide generation and glutamate (Glu) uptake in cultured astrocytes. Twenty-four hours after a single addition of either betaA25-35 or betaA1-40there was a concentration-dependent decrease in viability. Catalase or vitamin E showed no protective effect against betaA25-35 Dithiothreitol (DTT), N-acetylcysteine (NAC) and cyclosporine A significantly prevented the toxic effects of both betaA25-35 and peroxide, while inhibition of peroxide detoxifying enzymes enhanced toxicity. Exposure to betaA25-35 or betaA1-40 increased peroxides at 2 h and 24 h, which was prevented by DTT and NAC, but not vitamin E. betaA25-35 inhibited Glu uptake in astrocytes and neurons in culture. Following exposure of neurons to betaA for 24 h there was decreased uptake and increased Glu levels in the culture medium, that resulted in gradual excitotoxicity.

Glutamate Spillover Mediates Excitatory Transmission in the Rat Olfactory Bulb

In the CNS, glutamate typically mediates excitatory transmission via local actions at synaptic contacts. In the olfactory bulb, mitral cell dendrites release glutamate at synapses formed only onto the dendrites of inhibitory granule cells. Here, I show excitatory transmission mediated solely by transmitter spillover between mitral cells in olfactory bulb slices. Dendritic glutamate release from individual mitral cells causes self-excitation via local activation of N-methyl-D-aspartate (NMDA) receptors. Paired recordings reveal that glutamate release from one cell generates NMDA receptor-mediated responses in neighboring mitral cells that are enhanced by blockade of glutamate uptake. Furthermore, spillover generates spontaneous NMDA receptor-mediated population responses. This simultaneous activation of neighboring mitral cells by a diffuse action of glutamate provides a mechanism for synchronizing olfactory principal cells.

Prolonged enhancement of AP-1 DNA binding by blockade of glutamate uptake in cultured neurons.

Prolonged blockade of glutamate reuptake by the specific inhibitor of glutamate transporters, L-transpyrrolidine-2,4-dicarboxylate (PDC), produces a dramatic decrease in NMDA-induced neurotoxicity in cerebellar granule cell cultures, and is accompanied by a down-regulation of NMDA receptors. We now report that cultured cerebellar granule cells treated with 100 microM PDC for 1, 2, 4, 8, 16 and 24h, respectively, show increased AP-1 DNA-binding activity as measured by electrophoretic mobility shift assay. This effect was blocked by the NMDA receptor antagonist, CGP 37849, indicative of a pivotal role of NMDA receptors in the PDC-evoked enhancement of AP-1 DNA-binding. Our results suggest that AP-1 may be involved in the transcriptional regulation of neuronal adaptation initiated by prolonged inhibition of glutamate reuptake.

Selective suppression of rod signal transmission by cobalt ions of low levels in carp retina

Selective suppression of rod signal transmission by cobalt ions was reported in carp retina. Using 10 munol/L Co(2+), rod-driven horizontal cells were hyperpolarized and light responses were completely suppressed in superfused, isolated retina, while cone-driven horizontal cells were almost unaffected. Similarly, scotopic electroretinographic bwave was suppressed by 10 munol/L Co(2+), while the photopic b-wave remained unaffected. Furthermore, the glutamate-isolated receptor potential (PIII) was not altered by low Co(2+) under dark-adapted conditions. Other divalent ions with high affinity to calcium channels, such as cadmium and manganese ions, did not show similar suppressive effect on the rod horizontal cells. When rod horizontal cells were hyperpolarized by 10 munol/L Co(2+), the use of 3 mmol/L glutamate caused a significant depolarization of the cells, indicating that Co(2+) application did not impair the ability of these cells to respond to glutamate. On the other hand, application of 200 munol/L beta-hydroxyaspartate, a glutamate transport blocker, mimicked the effect of low Co(2+), suggesting a possibility that the low Co(2+) effect might be related to a blockade of glutamate uptake by rods.

Complex Roles of Glutamate in the Gibbs—Ng Model of One-trial Aversive Learning in the New-born Chick

NG, K. T., B. S. O'DOWD, N. S. RICKARD, S. R. ROBINSON, M. E. GIBBS, C. RAINEY, W.-Q. ZHAO, G. L. SEDMAN AND L. HERTZ. Complex roles of glutamate in the Gibbs-Ng Model of one-trial aversive learning in the newborn chick. NEUROSCI BIOBEHAV REV 21 [1]45–54, 1997.—Glutamate is the most widespread excitatory transmitter in the CNS and is probably involved in LTP, a neural phenomenon which may be associated with learning and memory formation. Intracerebral injection of large amounts of glutamate between 5 min before and 2.5 min after passive avoidance learning in young chicks inhibits short-term memory, which occurs between 0 and 10 min post-learning in a three-stage model of memory formation first established by Gibbs and Ng [25][Physiol. Behav. 23:369–375; 1979]. This effect may be attributed to non-specific excitation. Blockade of glutamate uptake by l-aspartic acid β-hydroxamate also abolishes this stage of memory, provided the drug is administered within 2.5 min of learning. Interference with either the production of precursors for transmitter glutamate in astrocytes or with glutamate receptors is also detrimental to memory formation, but the effects appear much later. After its release from glutamatergic neurons, glutamate is, to a large extent, accumulated into astrocytes where it is converted to glutamine, which can be returned to glutamatergic neurons and reutilized for synthesis of transmitter glutamate, and partly oxidized as a metabolic substrate. The latter process leads to a net loss of transmitter glutamate which can be compensated for by de novo synthesis of a glutamate precursor α-ketoglutarate (αKG) in astrocytes, a process which is inhibited by the astrocyte-specific toxin fluoroacetate (R. A. Swanson, personal communication). Intracerebral injection of this toxin abolishes memory during an intermediate stage of memory processing occurring between 20 and 30 min post-training [50][Cog. Brain Res. 2:93–102; 1994]. Injection of methionine sulfoximine (MSO), a specific inhibitor of glutamine synthetase, which interferes with the re-supply of transmitter glutamate to neurons by inhibition of glutamine synthesis in astrocytes, has a similar effect. This effect of MSO is prevented by intracerebral injection of glutamate, glutamine, or a combination and αKG and alanine. MSO must be administered before learning, but does not interfere with acquisition since short-term memory remains intact. Administration of either the NMDA antagonist AP5, the AMPA antagonist DNQX, or the metabotropic receptor antagonist MCPF, also induces amnesia. Memory loss in each case does not occur until after 70 min post-training, during a protein synthesis-dependent long-term memory stage which begins at 60 min following learning. However, to be effective, AP5 must be administered within 60 s following learning, MCPG before 15 min post-learning, and DNQX between 15 and 25 min after learning. Together, these findings suggest that learning results in an immediate release of glutamate, followed by a secondary release of this transmitter at later stages of processing of the memory trace, and that one or both of these increases in extracellular glutamate concentration are essential for the consolidation of long-term memory. Since both fluoroacetate and MSO act exclusively on glial cells, the findings also show that neuronal-glial interactions are necessary during the establishment of memory. Copyright © 1996 Elsevier Science Ltd.

Delayed clearance of transmitter and the role of glutamate transporters at synapses with multiple release sites

The roles of glutamate diffusion, uptake, and channel kinetics in shaping the AMPA receptor EPSC were examined at a calyceal synapse. The EPSC decay was described by three exponential components: two matching desensitizing channel kinetics, and a third component at least 10 times slower. The slowest component had identical voltage dependence to the steady-state AMPA current and was selectively increased and prolonged by blockade of glutamate uptake, indicating that the slow EPSC represented rebinding of glutamate at partially desensitized AMPA receptors. The data were in strong agreement with the predictions of a model of transmitter diffusion from multiple release sites into a large synaptic cleft. Within the first millisecond after release, transmitter concentrations in the cleft fell below millimolar levels, causing the fastest parts of the EPSC to be shaped by channel kinetics. The slowest component was determined by the removal over tens of milliseconds of the final 10-100 microM glutamate by diffusion and uptake. The data and modeling indicate that transmitter uptake and cooperation between release sites are significant determinants of a slow "tail" of glutamate in the synaptic cleft. This slow clearance of glutamate is likely to limit postsynaptic receptor availability through desensitization.

Amino acid neurotransmitters in nucleus tractus solitarius: an in vivo microdialysis study.

Amino acid neurotransmitters in the nucleus tractus solitarius (NTS) are thought to play a key role in the mediation of visceral reflexes and glutamate has been proposed as the neurotransmitter of visceral afferent nerves projecting to this region. The present studies sought to characterize the use of in vivo microdialysis to examine extracellular fluid levels of amino acids in the NTS of anesthetized rats. Using a microdialysis probe that was 450 microns in length and a sensitive HPLC assay for amino acids, amino acids could be measured in dialysate samples collected from the NTS. Perfusion of the microdialysis probe with 60 mM K+, to elicit depolarization of nerve terminals in the vicinity of the probe, resulted in increased dialysate fluid levels of aspartate, glutamate, glycine, taurine, and GABA. In contrast, glutamine and tyrosine were decreased and other amino acids were not significantly affected. Prior removal of the ipsilateral nodose ganglion did not alter the K(+)-evoked changes in dialysate levels of any of these amino acids. Electrical stimulation of the vagus nerves, using a variety of stimulus parameters, did not significantly alter dialysate levels of glutamate or any of the other amino acids that were measured. Blockade of glutamate uptake with dihydrokainate increased dialysate levels of glutamate, aspartate, and GABA, but in the presence of dihydrokainate vagal stimulation did not alter dialysate levels of these amino acids. The results show that in vivo microdialysis can be used to examine amino acid efflux in the rat NTS and provide further evidence for amino acidergic neural transmission in the NTS. However, these studies fail to support the hypothesis that vagal afferents release glutamate or aspartate.

Electrophysiological properties of neurones following mild and acute retinal ischaemia

Early electrophysiological changes following acute retinal ischaemia were studied by recording single or multiunit retinal ganglion cells and the electroretinogram (ERG) in barbiturate anaesthetized cats. Retinal ischaemia was initiated photochemically by platelet aggregation in retinal vessels which had been irradiated with monochromatic green light following an intravenous injection of Rose Bengal dye. No physiologically active ganglion cells were found within, or close to, the irradiated sites with chorioretinal oedema. On the other hand, in the areas 5–20° away from the irradiation sites, ganglion cells had abnormally raised spontaneous (background) firing which obscured visually driven firing. The retinal areas where no physiologically active ganglion cells were found showed histopathological changes which are similar to those described for glutamate-induced retinal damage. Retinal areas where depolarized retinal ganglion cells were located, however, showed only minor vacuolation of the ganglion cell fibre layer. Early global electrophysiological changes following photochemically induced retinal vascular lesion were consistent with those predicted from the findings in the single cell study. Vascular lesions produced with high irradiation energy (10–30 J), which promote extensive chorioretinal oedema, resulted in gradual loss of visually responsive ganglion cells. Lesions produced by low-energy irradiation (2 J), causing slight narrowing of the blood columns in the vessels, on the other hand, resulted in significant increases in the amplitude and the implicit time of the ERG b-wave and the background firing of multiunit retinal ganglion cells. Electrophysiological changes associated with mild retinal ischaemia are analogous to physiological effects associated with exogenous glutamate or blockade of glutamate uptake. It is suggested that early ischaemic challenge causes extracellular glutamate accumulation in the retina.