Rutin improves glutamate uptake and inhibits glutamate excitotoxicity in rat brain slices

Molecular Biology Reports - Rutin is an important flavonoid consumed in the daily diet. It is also known as vitamin P and has been extensively investigated due to its pharmacological properties. On...     

Effects of a glutamate uptake inhibitor on glutamate release induced by veratridine and ischemia

It has been postulated that a reversal of glutamate reuptake (“uptake reverse”) may contribute to glutamate release during cerebral ischemia. We tested this hypothesis by studying the effect of threo-3hydroxy- -aspartic acid (THA), a glutamate uptake inhibitor, on extracellular glutamate accumulation measured by microdialysis during 4-vessel ischemia (20 min). The inhibitory effect of THA on sodium-dependent glutamate uptake was measured in vitro on rat hippocampal slices (K_i = 45 ± 11 μM). We examined in vivo the effect of THA (400 μM in the dialysis solution) on the extracellular glutamate release from the rat hippocampus, during veratridine depolarization and ischemia. THA decreased the amount of glutamate appearing in the extracellular space during veratridine depolarization (61%). In contrast, the glutamate release induced by ischemia was not affected by THA. We conclude that a reversal of the sodium-dependent uptake contributes to an increase in extracellular glutamate during veratridine depolarization. In contrast, glutamate release occurring during ischemia is not mediated by uptake reverse.     

Effects of postmortem storage by freezing on ligament tensile behavior

The purpose of this study is to examine the effect of prolonged postmortem freezing storage (between 1 1/2 and 3 months at -20 degrees C) on the structural properties of the medial collateral ligament (MCL)-bone complex as well as the mechanical properties of the MCL substance from the rabbit knee. Tensile testing of the femur-MCL-tibia specimen was performed and no statistically significant changes were noted between the fresh and stored samples in terms of the cyclic stress relaxation, the load-deformation characteristics, as well as the load, deformation and energy absorbing capability at failure. The area of hysteresis of the stored samples was significantly reduced in the first few cycles, however. The mechanical properties of the MCL substance, as represented by the stress-strain curves, tensile strength and ultimate strain also did not change following storage. We conclude, therefore, proper and careful storage by freezing would have little or no effect on the biomechanical properties of the ligaments.     

Effects of freezing on the hyperthermic survival of tissue culture cells

The survival rate of V79 Chinese hamster lung fibroblasts treated with heat and freezing was investigated. The hyperthermic behavior of these cells was similar to other reports; Thermotolerance was observed below 43 degrees C during continuous heating. There was no difference in survival rate whether the cells were heated while attached to tissue culture flasks or in suspension, although the pH of the medium during the latter situation could be around 8.2 during hyperthermic treatment. If the cells were frozen and thawed before heating, the pattern of survival did not change significantly from that of heating alone, and thermotolerance was still observed below 43 degrees C. The Arrhinius plot of heat sensitivities between 41 and 45 degrees C demonstrated a straight line, giving an activation energy of 171 kcal/mole. A break on the plot around 42.5 degrees C could not be located, probably due to the lack of experimental points. If the cells were frozen and thawed immediately after heating, the survival rate was lower than that expected from considering hyperthermia and freezing as two independent processes. Possible explanations are provided in the result and discussion section.     

Guanosine enhances glutamate uptake in brain cortical slices at normal and excitotoxic conditions

1. The effect of guanosine on L-[2,3-³H]glutamate uptake was investigated in brain cortical slices under normal or oxygen–glucose deprivation (OGD) conditions.2. In slices exposed to physiological conditions, guanosine (1–100 M) stimulated glutamate uptake (up to 100%) in a concentration-dependent manner when a high (100 M) but not a low (1 M) concentration of glutamate was used.3. In slices submitted to OGD, guanosine 1 and 100 M also increased 100 M glutamate uptake (38 and 70%, respectively).4. The increasing of glutamate and taurine released to the incubation medium in cortical slices submitted to OGD were significantly attenuated by the presence of guanosine in the incubation medium.5. Guanosine prevented the increase in propidium iodide incorporation into cortical slices induced by OGD, indicating a protective role against ischemic injury.6. These results support the hypothesis of a protective role for guanosine during brain ischemia, possibly by activating glutamate uptake into neural cells.     

Methotrexate induces seizure and decreases glutamate uptake in brain slices: prevention by ionotropic glutamate receptors antagonists and adenosine

Methotrexate (MTX)-induced neurotoxicity may occur after intrathecal or systemic administration at low, intermediate and high doses for the treatment of malignant or inflammatory diseases. The mechanisms of MTX neurotoxicity are not totally understood, and appear to be multifactorial. In this study we characterized a model of MTX-induced seizures in mice to evaluate the convulsive and toxic MTX properties. Additionally, the effect of MTX-induced seizures on the activity of glutamate transporters, as well as the anticonvulsant role of MK-801, DNQX and adenosine on glutamate uptake in brain slices was investigated . MTX induced tonic-clonic seizures in approximately 95% of animals and pre-treatment with MK-801, DNQX and adenosine prevented seizure in 80%, 62% and 50% of animals, respectively. Moreover, MTX leads 59% of mice to death, which was prevented in 100% and 94% when animals received MK-801 and DNQX, respectively. Glutamate uptake decreased by 20% to 30% in cortical slices after MTX-induced seizures. Interestingly, when seizures were prevented by MK-801, DNQX or adenosine, glutamate uptake activity remained at the same level as the control group. Thus, our results demonstrate the involvement of the glutamatergic system in MTX-induced seizures.     

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 24h and 60% reduction at 5 days, P<0.001) and dose-dependent (21, 37, and 57% reduction at 1, 2.5, and 5mM 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 2.5mM NH(4)Cl, P<0.05 and 52% decrease at 5mM NH(4)Cl, P<0.001) as well as significant reductions in K(m) values (25% reduction at 2.5mM NH(4)Cl, P<0.05 and 45% reduction at 5mM NH(4)Cl, 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, 1H combined with 13C-NMR spectroscopy studies using the stable isotope [1-13C]-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.     

Effects of the putative antipsychotic alstonine on glutamate uptake in acute hippocampal slices

A dysfunctional glutamatergic system is thought to be central to the negative symptoms and cognitive deficits recognized as determinant to the poor quality of life of people with schizophrenia. Modulating glutamate uptake has, thus, been suggested as a novel target for antipsychotics. Alstonine is an indole alkaloid sharing with atypical antipsychotics the profile in animal models relevant to schizophrenia, though divergent in its mechanism of action. The aim of this study was to evaluate the effects of alstonine on glutamate uptake. Additionally, the effects on glutathione content and extracellular S100B levels were assessed. Acute hippocampal slices were incubated with haloperidol (10 μM), clozapine (10 and 100 μM) or alstonine (1–100 μM), alone or in combination with apomorphine (100 μM), and 5-HT₂ receptor antagonists (0.01 μM altanserin and 0.1 μM SB 242084). A reduction in glutamate uptake was observed with alstonine and clozapine, but not haloperidol. Apomorphine abolished the effect of clozapine, whereas 5-HT_2A and 5-HT_2C antagonists abolished the effects of alstonine. Increased levels of glutathione were observed only with alstonine, also the only compound that failed to decrease the release of S100B. This study shows that alstonine decreases glutamate uptake, which may be beneficial to the glutamatergic deficit observed in schizophrenia. Noteworthily, the decrease in glutamate uptake is compatible with the reversal of MK-801-induced social interaction and working memory deficits. An additional potential benefit of alstonine as an antipsychotic is its ability to increase glutathione, a key cellular antioxidant reported to be decreased in the brain of patients with schizophrenia. Adding to the characterization of the novel mechanism of action of alstonine, the lack of effect of apomorphine in alstonine-induced changes in glutamate uptake reinforces that D₂ receptors are not primarily implicated. Though clearly mediated by 5-HT_2A and 5-HT_2C serotonin receptors, the precise mechanisms that result in the effects of alstonine on glutamate uptake warrant elucidation.     

Monitoring single-synapse glutamate release and presynaptic calcium concentration in organised brain tissue

Brain function relies in large part on Ca²⁺-dependent release of the excitatory neurotransmitter glutamate from neuronal axons. Establishing the causal relationship between presynaptic Ca²⁺ dynamics and probabilistic glutamate release is therefore a fundamental quest across neurosciences. Its progress, however, has hitherto depended primarily on the exploration of either cultured nerve cells or giant central synapses accessible to direct experimental probing in situ. Here we show that combining patch-clamp with time-resolved imaging of Ca²⁺ −sensitive fluorescence lifetime of Oregon Green BAPTA-1 (Tornado-FLIM) enables readout of single spike-evoked presynaptic Ca²⁺ concentration dynamics, with nanomolar sensitivity, in individual neuronal axons in acute brain slices. In parallel, intensity Tornado imaging of a locally expressed extracellular optical glutamate sensor iGluSnFr provides direct monitoring of single-quantum, single-synapse glutamate releases in situ. These two methods pave the way for simultaneous registration of presynaptic Ca²⁺ dynamics and transmitter release in an intact brain at the level of individual synapses.     

A microdialysis study in rat brain of dihydrokainate,a glutamate uptake inhibitor

Microdialysis in neostriatum of anaesthetized rats was performed to study effects on amino acid efflux of the glutamate uptake-inhibitor dihydrokainate (DHK). Both basal and K⁺-evoked (100 mM) efflux of glutamate increased in the presence of DHK. The increase in the basal glutamate efflux occurred at lower DHK concentrations than during K⁺-depolarization (when the extracellular glutamate concentration was several-fold higher), confirming that DHK is a competitive inhibitor. The increase in basal efflux caused by DHK did not exhibit Ca²⁺-dependency, whereas ∼50% of the increase in glutamate efflux during K⁺-depolarization was Ca²⁺-dependent. The Ca²⁺-dependent efflux is related to transmitter release, whereas the Ca²⁺-independent efflux is probably due to metabolic events and/or transport of DHK into cells in exchange for glutamate. Taurine efflux in response to DHK increased both during basal conditions and K⁺-depolarization, probably secondary to the increase in glutamate concentration, whereas aspartate, GABA, glutamine and alanine effluxes did not change.     

Effects of pressure on uptake and release of calcium by brain synaptosomes

Uptake of radioactive calcium from guinea pig brain fractions enriched in synaptosomes could be significantly and reproducibly decreased by exposure to high pressure. Calcium efflux from preloaded synaptosomes was unaffected by pressure exposure. It was hypothesized that the development of pressure-induced encephalopathy may be related to an effect of pressure on the central nervous system calcium transport system.     

3H-dopamine uptake by synaptic storage vesicles of rat whole brain and brain regions

A crude preparation of neurotransmitter storage vesicles was obtained by differential centrifugation and the ability to take up ³H-dopamine was evaluated $\text{in}$$\text{vitro}$. The uptake was highly dependent on temperature, had an absolute requirement for ATP and Mg²⁺ and was inhibited totally by reserpine. The uptake displayed saturation kinetics, with a Km of 0.26 μM at 20°. ³H-dopamine uptake was inhibited competitively by norepinephrine, with a Ki of 0.69 μM. Vesicles derived from a primarily dopaminergic region (corpus striatum) exhibited the same ratio of uptakes of ³H-dopamine/³H-norepinephrine as did those from a primarily noradrenergic region (cerebral cortex). These results indicate that viable rat brain storage vesicles can be readily prepared and used for evaluation of pharmacologic effects on ³H-dopamine uptake, and that dopaminergic and noradrenergic storage vesicles exhibit identical uptake properties.     

Properties of the uptake and release of neurotransmitter glutamate in cerebral cortical tissue of guinea pigs

In order to investigate the dynamics of glutamate as a neurotransmitter and to avoid a complication by its metabolism, we studied the uptake and release of labeled non-metabolizabled-isomers of aspartate and glutamate in cerebral cortical slices and synaptosome preparation from guinea-pigs. The rate of uptake ofd-aspartate and glutamate was mutually inhibited in a non-competitive fashion, indicating that their uptake mechanisms are not exactly the same. By ouabain (0.05 mM), the uptake ofd-aspartate and glutamate into synaptosome preparation was less inhibited than that into cerebral slices. In synaptosome preparation most of the preloadedd-aspartate and glutamate was released by high-potassium (50 mM) stimulation, whereas in cerebral slices only a slight release was observed. However, when the slices were superfused with a medium free of sodium ions, which are absolutely necessary for the uptake, after preloaded with the labeled amino acids in the standard medium, a distinct release of radioactivity was induced by high-potassium stimulation. This potassium-induced release corresponded to only about 20% of the radioactivity accumulated in the slices. The accumulation ofd-aspartate and glutamate into cerebral slices was much larger on the basis of their protein content than that into synaptosome preparation, when a high concentration (1 mM) of the amino acids was added to the medium. These observations suggest that the uptake system ofd-aspartate and glutamate in cerebral slices is quite different from that in synaptosome preparation, and that the accumulation into cerebral slices is mainly localized in glial cells. In vivo the glial cell uptake is probably more important in removing the released neurotransmitter glutamate.Dedicated to Professor Yasuzo Tsukada.