Model Of Glutamate Excitotoxicity Research Articles

Bioenergetics of mitochondria in cultured neurons and their role in glutamate excitotoxicity

The pathologic activation of NMDA receptors by glutamate is a major contributor to neuronal cell death after stroke. Receptor activation causes a massive influx of calcium into the neuron that is accumulated by the mitochondria. The favored hypothesis is that the calcium loaded mitochondria generate reactive oxygen species that damage and ultimately killed the neuron. In this review this hypothesis is critically re-examined with an emphasis on the role played by deficits in ATP generation. Novel techniques are developed to monitor the bioenergetic status of in situ mitochondria in cultured neurons. Applying these techniques to a model of glutamate excitotoxicity suggests that enhanced reactive oxygen species are a consequence rather than a cause of failed cytoplasmic calcium homeostasis (delayed calcium deregulation, [DCD]), but that prior oxidative damage facilitates DCD by damaging mitochondrial ATP generation. This impacts on current hypotheses relating to the neuroprotective effects of mild mitochondrial uncoupling.

17beta-estradiol effect on the extracellular concentration of amino acids in the glutamate excitotoxicity model in the rat.

Estrogen has demonstrated a neuroprotective role in a rat model of glutamate excitotoxicity and other neurodegenerative disorders. We studied the effect of 17beta-estradiol on glutamate-induced increases in amino acids levels (aspartate, histidine, taurine and GABA) in the rat cortex. Local perfusion of glutamate produced a transient increase of aspartate, histidine, taurine and GABA in the extracellular fluid. Pretreatment with 17beta-estradiol significantly reduced the increases of taurine and moderately attenuated that of histidine, whereas aspartate and GABA releases were not modified. The effect of 17beta-estradiol on histidine release was reversed by the antiestrogen tamoxifen, suggesting a receptor-dependent mechanism. Good correlations between the volumes of the glutamate-induced lesions and the extracellular concentrations of taurine and aspartate were observed. These findings suggest that the attenuation of the glutamate-induced release of taurine by 17beta-estradiol may participate in the neuroprotective effects of 17beta-estradiol and that increased levels of aspartate and taurine are markers for the severity of the glutamate-induced cortical lesions.

17β-Estradiol reduces cortical lesion size in the glutamate excitotoxicity model by enhancing extracellular lactate: a new neuroprotective pathway

Estrogens play an important role in neuronal function and in protecting neurones in the cerebral cortex against pathological conditions. An in vivo model of glutamate excitotoxicity in which glutamate is applied to the cortex of rats through a microdialysis probe has been used to investigate the neuroprotective processes initiated by 17β-estradiol. Rats were pre-treated with 17β-estradiol (i.v.) before local application of 100 mM glutamate into the cortex through a microdialysis probe. Pre-treatment with 17β-estradiol significantly reduced the size of the glutamate-induced cortical lesion. In the cortical microdialysates collected from the probe, a peak of lactate was observed immediately after glutamate application. After 17β-estradiol pre-treatment this peak of lactate was significantly higher with estradiol than without 120 min after glutamate application, reaching 700% basal level at the end of measurement. The level of extracellular glucose was markedly decreased with and without 17β-estradiol pre-treatment. Local blockage of neuronal lactate transporters with α-cyano-4-hydroxycinnamate (4-CIN) completely abolished the neuroprotective effect of 17β-estradiol and induced a larger cortical lesion. An accumulation of extracellular lactate was observed after inhibition of the lactate transporters suggesting that transport of lactate into neurones is necessary for the neuroprotective effect of 17β-estradiol. The anti-estrogen tamoxifen also abolished the neuroprotective effect of 17β-estradiol on the lesion size and inhibited the production of lactate. These results suggest a new neuroprotective mechanism of 17β-estradiol by activating glutamate-stimulated lactate production, which is estrogen receptor-dependent.

Effect of 17 beta-estradiol on the brain damage and metabolic changes in rats.

An in vivo model of glutamate excitotoxicity in which glutamate is applied to the cortex of rats through a microdialysis probe has been used to investigate the neuroprotective processes initiated by 17 beta-estradiol. Rats were pre-treated with 17 beta-estradiol i.v. before local application of glutamate. The experimental results showed that pre-treatment with 17 beta-estradiol significantly reduced the size of the glutamate-induced lesion. In the microdialysates, the peak of lactate observed immediately after glutamate application was significantly higher and longer lasting after 17 beta-estradiol pre-treatment. The level of extracellular glucose was markedly decreased concomitantly to the increase in lactate, but no difference could be observed with and without 17 beta-estradiol pre-treatment. These suggest a new neuroprotective mechanism of 17 beta-estradiol by activating glutamate-induced lactate production. This effect on lactate production and lesion reduction is estrogen receptor dependent and is abolished totally by estrogen antagonist tamoxifen. It was also demonstrated here that high lactate subserves estrogen neuroprotection during glutamate toxicity.

Mitochondria and calcium: from cell signalling to cell death.

While a pathway for Ca2+ accumulation into mitochondria has long been established, its functional significance is only now becoming clear in relation to cell physiology and pathophysiology. The observation that mitochondria take up Ca2+ during physiological Ca2+ signalling in a variety of cell types leads to four questions: (i) 'What is the impact of mitochondrial Ca2+ uptake on mitochondrial function?' (ii) 'What is the impact of mitochondrial Ca2+ uptake on Ca2+ signalling?' (iii) 'What are the consequences of impaired mitochondrial Ca2+ uptake for cell function?' and finally (iv) 'What are the consequences of pathological [Ca2+]c signalling for mitochondrial function?' These will be addressed in turn. Thus: (i) accumulation of Ca2+ into mitochondria regulates mitochondrial metabolism and causes a transient depolarisation of mitochondrial membrane potential. (ii) Mitochondria may act as a spatial Ca2+ buffer in many cells, regulating the local Ca2+ concentration in cellular microdomains. This process regulates processes dependent on local cytoplasmic Ca2+ concentration ([Ca2+]c), particularly the flux of Ca2+ through IP3-gated channels of the endoplasmic reticulum (ER) and the channels mediating capacitative Ca2+ influx through the plasma membrane. Consequently, mitochondrial Ca2+ uptake plays a substantial role in shaping [Ca2+]c signals in many cell types. (iii) Impaired mitochondrial Ca2+ uptake alters the spatiotemporal characteristics of cellular [Ca2+]c signalling and downregulates mitochondrial metabolism. (iv) Under pathological conditions of cellular [Ca2+]c overload, particularly in association with oxidative stress, mitochondrial Ca2+ uptake may trigger pathological states that lead to cell death. In the model of glutamate excitotoxicity, microdomains of [Ca2+]c are apparently central, as the pathway to cell death seems to require the local activation of neuronal nitric oxide synthase (nNOS), itself held by scaffolding proteins in close association with the NMDA receptor. Mitochondrial Ca2+ uptake in combination with NO production triggers the collapse of mitochondrial membrane potential, culminating in delayed cell death.

Lysosphingomyelin prevents behavioral aberrations and hippocampal neuron loss induced by the metabotropic glutamate receptor agonist quisqualate

1. 1. Excessive excitation of brain neurons by the excitatory neurotransmitter, glutamate, induces a cascade of events leading to increased intracellular Ca ++, neuronal degeneration and death. 2. 2. Recent in vitro research has demonstrated that a natural cationic amphiphile in the brain, lysosphingomyelin, may be able to prevent neuronal degeneration by repressing phosphosinositidase-C overactivation induced by excessive excitation of the metabotropic glutamate receptor. 3. 3. This research tested the latter finding in vivo in a rat model of glutamate excitotoxicity. Intracerebroventricular (i.c.v) administration of the Group 1 metabotropic glutamate receptor (mGluR) agonist, quisqualate, produced seizures, akinesia, destruction of hippocampal pyramidal cell dendritic microtubule-associated protein-2, and major loss of hippocampal CA sector neurons. 4. 4. Prophylactic i.c.v. infusion of lysosphingomyelin powerfully attenuates these quisqualate-induced behaviors and prevents neuronal degeneration. 5. 5. Lysosphingomyelin may be of clinical use in allaying progressive Group 1 mGluR-induced hippocampal cognitive and motor disorders including Alzheimer's disease, brain seizure, and stroke.

Psychosine blocks quisqualate-induced glutamate excitotoxicity in hippocampal CA sector neurons

Quisqualate is a potent specific agonist for Group 1 metabotropic glutamate receptors (mGluR's), that activate G protein-coupled phospholipase C (PLC) in a molecular signal-transduction mechanism that raises cytoplasmic Ca 2+ and, when excessive, damages hippocampal neurons. Psychosine ( β-galactosylsphingosine), a cationic lysosphingolipid occurring naturally in nervous tissues, dose-dependently inhibited PLC activation induced by metabotropic α 1-adrenergic receptor signaling in cultured rat brain astrocytes in vitro. In the present study, we have tested neuroprotective efficacy of psychosine in vivo, in a rat model of glutamate excitotoxicity induced by intracerebroventricular (i.c.v.) administration of quisqualate. A sublethal i.c.v. dose of quisqualate caused episodes of prolonged akinesia and convulsions, and major damage to pyramidal neurons of the hippocampal CA1 and CA3 sector, but not to granule cell neurons of the dentate gyrus. Prior infusion of psychosine greatly attenuated quisqualate-induced behaviors, and fully prevented destruction by quisqualate of vulnerable hippocampal neurons. Psychosine may prove useful in prophylaxis of neurodegenerative disorders that arise from intensive hippocampal Group 1 mGluR stimulation.

Pharmacological modification of glutamate neurotoxicity in vivo

The ability of five agents (dizocilpine [MK-801], 2,3-dihydroxy-6-nitro-7-sulfamoyl-benzo(F)-quinoxaline [NBQX], enadoline [CI-977], l-nitroarginine methyl ester [ l-NAME] and BW 1003c87) with well defined, distinct pharmacological profiles and with established anti-ischemic efficacy, to modify neuronal damage has been examined in a simple in vivo model of glutamate excitotoxicity. Cortical lesions were produced in physiologically-monitored, halothane-anesthestised rats by reverse dialysis of glutamate. The volume of the lesion was quantified histologically by image analysis of approximately 20 sections taken at 200 μm intervals throughout the lesion. The AMPA and NMDA receptor antagonists (NBQX and MK-801) and the inhibitor of nitric oxide synthase ( l-NAME) significantly reduced the lesion volume by a similar extent (by approximately 30% from vehicle). Two agents (the kappa opioid agonist, CI-977 and the sodium channel blocker, BW 1003c87) which putatively inhibit the release of endogenous glutamate presynaptically, had dissimilar effects on lesion size. CI-977 failed to alter the amount of damage produced by exogenous glutamate, whereas BW 1003c87 reduced the lesion size by approximately 50%. Using this model, the neuroprotective effects of anti-ischemic drugs can be explored in vivo, uncomplicated in contrast to experimental ischemia by reduced oxygen delivery, drug effects on tissue blood flow and compromised energy generation. In consequence, additional mechanistic insight into anti-ischemic drug action in vivo can be obtained.