Excitotoxic Effects Of Glutamate Research Articles

Impact of hypoxic preconditioning on the alterations in phosphate-activated glutaminase activity in the brains of offspring

The hypoxic preconditioning model is widely used in experimental studies to identify mechanisms for increasing the tolerance of organisms to subsequent hypoxic exposure. Glutaminase is the main enzyme that synthesizes glutamate, which has two important physiological roles: in the postnatal period, it acts as an excitatory neurotransmitter, and during the prenatal period, it regulates neurogenesis, synaptogenesis, and the survival of nerve cells. In our work, in 4 experimental groups, we investigated the effect of hypoxic preconditioning performed during days 16–21 of pregnancy on glutamate synthesis in the brains of 17-day-old and 6-month-old offspring. It was found that prenatal hypoxia led to a pronounced increase in the enzyme activity in various brain structures in early postnatal ontogenesis, while a decrease was observed in adult animals. In contrast, exposure to acute hypoxia resulted in a more significant increase in glutaminase activity in the brains of adult animals. Prenatal fetal hypoxic preconditioning caused a weakening effect on the increase in enzyme activity in 17-day-old rat offspring and a down-regulation in 6-month-olds, compared to the group that suffered acute hypoxia in the postnatal period. Thus, a neuroprotective adaptive-compensatory effect of prenatal preconditioning has been demonstrated, which can be associated with both the physiological and excitotoxic effects of glutamate.

Structure and Neuroprotector Properties of a Complex Compound of Lithium with Comenic Acid.

The structure, antioxidant and neuroprotective properties of lithium comenate (lithium 5-hydroxy-4-oxo-4H-pyran-2-carboxylate) were studied. Lithium comenate was obtained by reacting comenic acid (H2Com) with lithium hydroxide in an aqueous solution. The structure of lithium comenate was confirmed via thermal analysis, mass spectrometry, IR, NMR and UV spectroscopy. The crystal structure was studied in detail via X-ray diffraction. The compound crystallized in a non-centrosymmetric space group of symmetry of the orthorhombic system Pna21 in the form of a hydrate, with three water molecules entering the first coordination sphere of the cation Li+ and one molecule forming a second environment through non-valent contacts. The gross formula of the complex compound was established [Li(HCom)(H2O)3]·H2O. It has been established that lithium comenate has a pronounced neuroprotective activity under the excitotoxic effect of glutamate, increasing the survival rate of cultured rat cerebellar neurons more than two-fold. It has also been found that the pre-stress use of lithium comenate at doses of 1 and 2 mg/kg has an antioxidant effect, which is manifested in a decrease in oxidative damage to the brain tissues of mice subjected to immobilization stress. Based on the data available in the literature, we believe that the high neuroprotective and antioxidant efficacy of lithium comenate is a consequence of the mutual potentiation of the pharmacological effects of lithium and comenic acid.

Scanning Ion-Conductance Microscopy for Studying Mechanical Properties of Neuronal Cells during Local Delivery of Glutamate.

Mechanical properties of neuronal cells have a key role for growth, generation of traction forces, adhesion, migration, etc. Mechanical properties are regulated by chemical signaling, neurotransmitters, and neuronal ion exchange. Disturbance of chemical signaling is accompanied by several diseases such as ischemia, trauma, and neurodegenerative diseases. It is known that the disturbance of chemical signaling, like that caused by glutamate excitotoxicity, leads to the structural reorganization of the cytoskeleton of neuronal cells and the deviation of native mechanical properties. Thus, to investigate the mechanical properties of living neuronal cells in the presence of glutamate, it is crucial to use noncontact and low-stress methods, which are the advantages of scanning ion-conductance microscopy (SICM). Moreover, a nanopipette may be used for the local delivery of small molecules as well as for a probe. In this work, SICM was used as an advanced technique for the simultaneous local delivery of glutamate and investigation of living neuronal cell morphology and mechanical behavior caused by an excitotoxic effect of glutamate.

Ex vivo comparative investigation of suprachiasmatic nucleus excitotoxic resiliency

Background: Glutamate signaling in the brain is regulated by release, reuptake, and receptor responsiveness. In diseased conditions, glutamate signaling can exceed normal regulatory processes, giving rise to a condition called excitotoxicity. Although regional differences in the excitotoxic effects of glutamate in the brain have been reported, the extent and characteristics of these potential differences are not clear. Here we compared the excitotoxic resiliency of the suprachiasmatic nucleus (SCN), anterior hypothalamus (AH) and cortex. Methods: We treated acute brain slices containing either the SCN and AH or the cortex from adult male mice at different times across the diurnal cycle with varying concentrations of N-methyl-D-aspartate (NMDA), NMDA+ α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) or control medium. The extent of cell damage was assessed using propidium iodide (PI), a cell death marker. Results: The results indicate that all three brain regions exhibited increasing cell damage/death when treated with increasing concentrations of NMDA. However, higher concentrations of NMDA were needed to significantly increase cell damage in the SCN compared to the cortex and AH. All three brain regions also exhibited greater cell death/damage when treated in the nighttime compared to the daytime, although the SCN exhibited increased cell death during a more restricted time interval compared to the AH and cortex. Conclusions: Together, these data confirm previous studies showing excitotoxic resiliency in the SCN, while extending them in two ways. First, we demonstrate a dose-dependency in excitotoxic susceptibility that differentiates the SCN from the surrounding AH and the cortex using a brain slice preparation. Second, we demonstrate a diurnal rhythm in excitotoxic susceptibility with a broadly similar phase across all three brain regions. These data increase our understanding of the extent and nature of the SCN excitotoxic resiliency, which will inform future studies on the cellular mechanisms underlying this phenomenon.

Ex vivo comparative investigation of suprachiasmatic nucleus excitotoxic resiliency

Background: Glutamate signaling in the brain is regulated by release, reuptake, and receptor responsiveness. In diseased conditions, glutamate signaling can exceed normal regulatory processes, giving rise to a condition called excitotoxicity. Although regional differences in the excitotoxic effects of glutamate in the brain have been reported, the extent and characteristics of these potential differences are not clear. Here we compared the excitotoxic resiliency of the suprachiasmatic nucleus (SCN), anterior hypothalamus (AH) and cortex. Methods: We treated acute brain slices containing either the SCN and AH or the cortex from adult male mice at different times across the diurnal cycle with varying concentrations of N-methyl-D-aspartate (NMDA), NMDA+ α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) or control medium. The extent of cell damage was assessed using propidium iodide (PI), a cell death marker. Results: The results indicate that all three brain regions exhibited increasing cell damage/death when treated with increasing concentrations of NMDA. However, higher concentrations of NMDA were needed to significantly increase cell damage in the SCN compared to the cortex and AH. All three brain regions also exhibited greater cell death/damage when treated in the nighttime compared to the daytime, although the SCN exhibited increased cell death during a more restricted time interval compared to the AH and cortex. Conclusions: Together, these data confirm previous studies showing excitotoxic resiliency in the SCN, while extending them in two ways. First, we demonstrate a dose-dependency in excitotoxic susceptibility that differentiates the SCN from the surrounding AH and the cortex using a brain slice preparation. Second, we demonstrate a diurnal rhythm in excitotoxic susceptibility with a broadly similar phase across all three brain regions. These data increase our understanding of the extent and nature of the SCN excitotoxic resiliency, which will inform future studies on the cellular mechanisms underlying this phenomenon.

Ex vivo comparative investigation of suprachiasmatic nucleus excitotoxic resiliency

Background: Glutamate signaling in the brain is regulated by release, reuptake, and receptor responsiveness. In diseased conditions, glutamate signaling can exceed normal regulatory processes, giving rise to a condition called excitotoxicity. Although regional differences in the excitotoxic effects of glutamate in the brain have been reported, the extent and characteristics of these potential differences are not clear. Here we compared the excitotoxic resiliency of the suprachiasmatic nucleus (SCN), anterior hypothalamus (AH) and cortex. Methods: We treated acute brain slices containing either the SCN and AH or the cortex from adult male mice at different times across the diurnal cycle with varying concentrations of N-methyl-D-aspartate (NMDA), NMDA+ α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) or control medium. The extent of cell damage was assessed using propidium iodide (PI), a cell death marker. Results: The results indicate that all three brain regions exhibited increasing cell damage/death when treated with increasing concentrations of NMDA. However, higher concentrations of NMDA were needed to significantly increase cell damage in the SCN compared to the cortex and AH. All three brain regions also exhibited greater cell death/damage when treated in the nighttime compared to the daytime, although the SCN exhibited increased cell death during a more restricted time interval compared to the AH and cortex. Conclusions: Together, these data confirm previous studies showing excitotoxic resiliency in the SCN, while extending them in two ways. First, we demonstrate a dose-dependency in excitotoxic susceptibility that differentiates the SCN from the surrounding AH and the cortex using a brain slice preparation. Second, we demonstrate a diurnal rhythm in excitotoxic susceptibility with a broadly similar phase across all three brain regions. These data increase our understanding of the extent and nature of the SCN excitotoxic resiliency, which will inform future studies on the cellular mechanisms underlying this phenomenon.

Novel Hydroxypyridine Compound Protects Brain Cells against Ischemic Damage In Vitro and In Vivo.

A non-surgical pharmacological approach to control cellular vitality and functionality during ischemic and/or reperfusion-induced phases of strokes remains extremely important. The synthesis of 2-ethyl-6-methyl-3-hydroxypyridinium gammalactone-2,3-dehydro-L-gulonate (3-EA) was performed using a topochemical reaction. The cell-protective effects of 3-EA were studied on a model of glutamate excitotoxicity (GluTox) and glucose-oxygen deprivation (OGD) in a culture of NMRI mice cortical cells. Ca2+ dynamics was studied using fluorescent bioimaging and a Fura-2 probe, cell viability was assessed using cytochemical staining with propidium iodide, and gene expression was assessed by a real-time polymerase chain reaction. The compound anti-ischemic efficacy in vivo was evaluated on a model of irreversible middle cerebral artery (MCA) occlusion in Sprague-Dawley male rats. Brain morphological changes and antioxidant capacity were assessed one week after the pathology onset. The severity of neurological disorder was evaluated dynamically. 3-EA suppressed cortical cell death in a dose-dependent manner under the excitotoxic effect of glutamate and ischemia/reoxygenation. Pre-incubation of cerebral cortex cells with 10-100 µM 3-EA led to significant stagnation in Ca2+ concentration in a cytosol ([Ca2+]i) of neurons and astrocytes suffering GluTox and OGD. Decreasing intracellular Ca2+ and establishing a lower [Ca2+]i baseline inhibited necrotic cell death in an acute experiment. The mechanism of 3-EA cytoprotective action involved changes in the baseline and ischemia/reoxygenation-induced expression of genes encoding anti-apoptotic proteins and proteins of the oxidative status; this led to inhibition of the late irreversible stages of apoptosis. Incubation of brain cortex cells with 3-EA induced an overexpression of the anti-apoptotic genes BCL-2, STAT3, and SOCS3, whereas the expression of genes regulating necrosis and inflammation (TRAIL, MLKL, Cas-1, Cas-3, IL-1β and TNFa) were suppressed. 3-EA 18.0 mg/kg intravenous daily administration for 7 days following MCA occlusion preserved rats' cortex neuron population, decreased the severity of neurological deficit, and spared antioxidant capacity of damaged tissues. 3-EA demonstrated proven short-term anti-ischemic activity in vivo and in vitro, which can be associated with antioxidant activity and the ability to target necrotic and apoptotic death. The compound may be considered a potential neuroprotective molecule for further pre-clinical investigation.

Influence of magnesium on excitability of neurons of different levels of their organization

AbstractThe article indicates that the hyper-excitability of neurons is associated with ionic imbalance of cell membranes, in which a number of trace elements are involved. The energy component of these processes is associated with the presence of magnesium (Mg) as a universal supplier of the latter. Mg supports the functioning of NMDA receptors, which does not allow the excitotoxic effects of glutamate, which triggers the mechanisms of Ca entry into the neuron and its death, to manifest itself. Mg stimulates the main excitatory neurotransmitter acetylcholine, regulates the release of serotonin, glycine, dopamine, thereby normalizing the extrapyramidal system, the spinal stem structures, and the final executive motor apparatus. Participates in the energy support of adaptation processes, the activation of central regulatory mechanisms, control the excess of catecholamines and corticosteroids, other hormones and mediators involved in stress reactions.

Influence of the age of rats treated with the sodium salt of glutamate acid on the reactivity of astroglia of the infundibular nucleus

The aim of the study was the immunohistochemical evaluation of the impact of the age of animals treated with the sodium salt of glutamic acid on the behaviour of astrocytes of the infundibular nucleus (IN). Immunohistochemical peroxidase-antiperoxidase reactions were conducted on brain sections of 10-day-old (I) and 63-day-old (II) rats treated s.c with monosodium glutamate (MSG) in a dose of 4g/kg b.w. for three consecutive days. The staining was performed using specific antibodies against glial fibrillary acidic protein (GFAP), S-100β protein and Ki-67 antigen. Cells immunopositive for the proteins under investigation were assessed morphologically and morphometrically in an Olympus BX51 light microscope with the Cell ^ D program. Statistically significant differences were tested using ANOVA and the non-parametric Kruskal-Wallis test. In the infundibular nucleus of 10-day-old rats treated with MSG, there was an increase in the number of GFAP, S-100β and Ki-67 immunopositive astrocytes without any changes in their morphology, which was typical of immature glia. In adult rats treated with MSG, a decrease in the number of cells expressing GFAP and S-100β was found. Most astrocytes had thick and weakly branched processes, in contrast to those observed in control animals. The results of our study showed a diverse behaviour of astroglia of IN in young and adult rats treated with MSG. In 10-day-old rats, hyperplasia of glia occurred, whereas in 63-day-old individuals there was a loss and hypertrophy of astrocytes, which may indicate a late stage of their reactivity. This information may contribute to targeting the therapy of diseases of the nervous system induced by the excitotoxic effects of glutamate.

Excitotoxic effects of glutamate on cochlear organotypic cultures.

Glutamate (Glu) is the major afferent excitatory neurotransmitter in the auditory system, and excessive Glu may play an important role in cochlear dysfunction. It is unclear how excessive Glu plays roles in cochlear dysfunction in cochlear organotypic cultures. In this study neonatal rat cochlear organotypic cultures were prepared, and then the cochlear tissues were incubated with a new medium containing specific concentrations of Glu (0.1, 0.5, 1, 10 or 20 mmol/L) for 24 h, or incubated with the medium containing a concentration of 20 mmol/L Glu for 6, 12, 24 or 72 h, respectively. It was found that when the cochlear tissues were cultured for 24 h, the inner hair cells (IHCs) were damaged at the concentration of 0.5 mmol/L Glu, and with the increases of the concentrations, the injury was gradually aggravated, and 20 mmol/L Glu resulted in the significant loss of IHCs. In the 20 mmol/L Glu groups, the stereocilia bundles were missing or disarrayed on a few IHCs after culture for 6 h and the damage effect was time-dependent. The missing of IHCs was more significant in the basal turn of the cochlea than in the middle turn of the cochlea under the same concentration of Glu exposure. These results suggest that excessive exogenous Glu affects the morphology of IHCs, but not affects the outer hair cells (OHCs) in cochlear organotypic cultures, and the excitotoxic effects are different on IHCs of different parts of the cochlea under the same concentration of Glu exposure.

Astrocytic Dynamin-Like Protein 1 Regulates Neuronal Protection against Excitotoxicity in Parkinson Disease

Mitochondrial dynamics has recently become an area of piqued interest in neurodegenerative disorders, including Parkinson disease (PD); however, the contribution of astrocytes to these disorders remains unclear. Here, we show that the level of dynamin-like protein 1 (Dlp1; official name DNM1L), which promotes mitochondrial fission, is lower in astrocytes from the brains of PD patients, and that decreased astrocytic Dlp1 likely represents a relatively early event in PD pathogenesis. In support of this conclusion, we show that Dlp1 knockdown dramatically affects mitochondrial morphological characteristics and localization in astrocytes, impairs the ability of astrocytes to adequately protect neurons from the excitotoxic effects of glutamate, and increases intracellular Ca(2+) in response to extracellular glutamate, resulting from compromised intracellular Ca(2+) buffering. Taken together, our results suggest that astrocytic mitochondrial Dlp1 is a key protein in mitochondrial dynamics and decreased Dlp1 may interfere with neuron survival in PD by disrupting Ca(2+)-coupled glutamate uptake.

The NMDA receptor antagonist MK-801 abolishes the increase in both p53 and Bax/Bcl2 index induced by adult-onset hypothyroidism in rat.

Hypothyroidism affects neuron population dynamics in the hippocampus of the adult rat, with neuronal damage as the main feature of its effect. This effect is prevented by the blockade of NMDA receptors, which suggests that glutamatergic activity mediates cell death in this condition. Glutamate can also stimulate cell proliferation and survival of newborn neurons, indicating that it can affect different stages of the cell cycle. In this work we measured the expression of specific proteins that control cell proliferation (cycline-D1), cell arrest (p21), damage (p53) or apoptosis (Bax and Bcl2) in the hippocampus of hypothyroid rats treated with the NMDA receptor (NMDAR) blocker MK-801 during the induction of hypothyroidism. The results show that hypothyroidism increases the expression of markers of DNA damage, cell arrest, and apoptosis, but does not affect the marker of cell proliferation. NMDAR blockade prevents the increase on markers of DNA damage and apoptosis, but does not influence cell arrest or cell proliferation. This suggests that hypothyroidism promotes cell death mainly by an excitotoxic effect of glutamate.

Neuroprotection of rat retinal ganglion cells mediated through alpha7 nicotinic acetylcholine receptors

Glutamate-induced excitotoxicity is thought to play an important role in several neurodegenerative diseases in the central nervous system (CNS). In this study, neuroprotection against glutamate-induced excitotoxicity was analyzed using acetylcholine (ACh), nicotine and the α7 specific nicotinic acetylcholine receptor (α7 nAChR) agonist, N-[(3R)-1-azabicyclo[2.2.2]oct-3-yl]-4-chlorobenzamide hydrochloride (PNU-282987), in cultured adult rat retinal neurons. Adult Long Evans rat retinas were dissociated and retinal ganglion cells (RGCs) were isolated from all other retinal tissue using a two-step panning technique. Once isolated, RGCs were cultured under various pharmacological conditions to demonstrate excitotoxicity and neuroprotection against excitotoxicity. After 3days, RGCs were immunostained with antibodies against the glycoprotein, Thy 1.1, counted and cell survival was assessed relative to control untreated conditions. 500μM glutamate induced excitotoxicity in large and small RGCs in an adult rat dissociated culture. After 3days in culture with glutamate, the cell survival of large RGCs decreased by an average of 48.16% while the cell survival of small RGCs decreased by an average of 42.03%. Using specific glutamate receptor agonists and antagonists, we provide evidence that the excitotoxic response was mediated through α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)/kainic acid (KA) and N-methyl-d-aspartate (NMDA) glutamate receptors through an apoptotic mechanism. However, the excitotoxic effect of glutamate on all RGCs was eliminated if cells were cultured for an hour with 10μM ACh, 100μM nicotine or 100nM of the α7 nAChR agonist, PNU-282987, before the glutamate insult. Inhibition studies using 10nM methyllycaconitine (MLA) or α-bungarotoxin (α-Bgt) supported the hypothesis that neuroprotection against glutamate-induced excitotoxicity on rat RGCs was mediated through α7 nAChRs. In immunocytochemical studies, double-labeled experiments using antibodies against Thy 1.1 and α7 nAChR subunits demonstrated that both large and small RGCs contained α7 nAChR subunits. The data presented in this study support the hypothesis that ACh and nicotinic acetylcholine receptor (nAChR) agonists provide neuroprotection against glutamate-induced excitotoxicity in adult rat RGCs through activation of α7 nAChR subunits. These studies lay the groundwork required for analyzing the effect of specific α7 nAChR agonists using in vivo models of excitotoxicity. Understanding the type of ACh receptors involved in neuroprotection in the rat retina could ultimately lead to therapeutic treatment for any CNS disease that involves excitotoxicity.

Targeted Over-Expression of Glutamate Transporter 1 (GLT-1) Reduces Ischemic Brain Injury in a Rat Model of Stroke

Following the onset of an ischemic brain injury, the excitatory neurotransmitter glutamate is released. The excitotoxic effects of glutamate are a major contributor to the pathogenesis of a stroke. The aim of this study was to examine if overexpression of a glutamate transporter (GLT-1) reduces ischemic brain injury in a rat model of stroke. We generated an adeno-associated viral (AAV) vector expressing the rat GLT-1 cDNA (AAV-GLT1). Functional expression of AAV-GLT1 was confirmed by increased glutamate clearance rate in non-stroke rat brain as measured by in vivo amperometry. AAV-GLT1 was injected into future cortical region of infarction 3 weeks prior to 60 min middle cerebral artery occlusion (MCAo). Tissue damage was assessed at one and two days after MCAo using TUNEL and TTC staining, respectively. Behavioral testing was performed at 2, 8 and 14 days post-stroke. Animals receiving AAV-GLT1, compared to AAV-GFP, showed significant decreases in the duration and magnitude of extracellular glutamate, measured by microdialysis, during the 60 minute MCAo. A significant reduction in brain infarction and DNA fragmentation was observed in the region of AAV-GLT1 injection. Animals that received AAV-GLT1 showed significant improvement in behavioral recovery following stroke compared to the AAV-GFP group. We demonstrate that focal overexpression of the glutamate transporter, GLT-1, significantly reduces ischemia-induced glutamate overflow, decreases cell death and improves behavioral recovery. These data further support the role of glutamate in the pathogenesis of ischemic damage in brain and demonstrate that targeted gene delivery to decrease the ischemia-induced glutamate overflow reduces the cellular and behavioral deficits caused by stroke.

Amyloid-β and aluminum ions enhance neuronal damage mediated by NMDA-activated glutamate receptors

Effects of N-methyl-D-aspartate (NMDA), aluminum and amyloid-β (Aβ) on mouse cerebellar granule cells were studied. In the presence of all three compounds, reactive oxygen species levels and cell necrosis were increased dramatically. Mg2+ ions and D-AP5, which are known to prevent ligand binding to NMDA-activated glutamate receptors, were effective in attenuating the neurotoxic effect induced by the presence of all three compounds. All substances tested induced activation of p42/44 MAPK (mitogen activated protein kinase) with no cumulative effects between them. We conclude that neurotoxicity induced by aluminum and Aβ appears at outer cell membranes and NMDA receptors take part in this process. Increase in excitotoxic effect of glutamate in the presence of aluminum and Aβ is suggested to be a factor which provokes Alzheimer’s disease in brain neurons.

Effects of pituitary adenylate cyclase activating polypeptide in retinal degeneration induced by monosodium-glutamate

Pituitary adenylate cyclase activating polypeptide (PACAP) is a pleiotropic neuropeptide with a wide range of effects in the central and peripheral nervous systems. PACAP has well-documented neurotrophic and neuroprotective actions in both in vitro and in vivo models of different neuronal injuries. The aim of the present study was to investigate the possible neuroprotective effect of PACAP in retinal degeneration induced by monosodium-glutamate (MSG) in neonatal rats. Preceding the MSG treatment, PACAP (1 or 100 pmol/5 μl) was injected unilaterally into the vitreous body on postnatal days 1, 5 and 9. Immediately after the PACAP treatment, pups were treated with 2 mg/g body weight MSG subcutaneously. At 3 weeks of age, rats were sacrificed and retinas were removed and processed for histological examination. Our results show that MSG treatment caused severe degeneration, primarily of the inner retinal layers. The thickness of the entire retina was only approximately half of that of the normal retinas, and the inner nuclear layer seemed to be fused with the ganglionic cell layer, with no discernible inner plexiform layer. Retinas of animals treated with 1 pmol PACAP showed a similar degree of degeneration. However, retinas of rats treated with 100 pmol PACAP showed significantly less damage, with clearly distinguishable inner retinal layers. In summary, our present study shows that local PACAP treatment could attenuate the retinal degeneration induced by the excitotoxic effects of glutamate.

Neuroprotective effects of the antifungal drug clotrimazole

Pretreatment with 10 μM of the antifungal drug clotrimazole potently reduced the death of cultured rat cerebellar granule cells induced by oxygen/glucose deprivation, and the excitotoxic effect of glutamate on cultured hippocampal neurons and cerebellar granule cells. In patch-clamped hippocampal pyramidal neurons, 10–50 μM clotrimazole caused a decrease in the amplitude of N-methyl- D-aspartate (NMDA) receptor-mediated currents. Glutamate induced intracellular Ca 2+ overload, as measured by Fluo-3 confocal fluorescence imaging, while clotrimazole reduced Ca 2+ overload and promoted the recovery of intracellular calcium homeostasis after glutamate treatment. Using tetramethylrhodamine ethyl ester fluorescence as a marker of mitochondrial membrane potential we found that clotrimazole prevented the glutamate-induced loss of mitochondrial membrane potential. Our data provide evidence that the protective effect of clotrimazole against oxygen/glucose deprivation and excitotoxicity is due to the ability of this drug to partially block NMDA receptor-gated channel, thus causing both reduced calcium overload and lower probability of the mitochondrial potential collapse.

AMPA prevents glutamate-induced neurotoxicity and apoptosis in cultured cerebellar granule cell neurons.

Exposure of cultured cerebellar neurons to alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) in the presence of aniracetam protects all of the vulnerable neurons against the excitotoxic actions of glutamate acting on N-methyl-D-aspartate receptors. The protective effect of AMPA was both time- and concentration-dependent. Aniracetam alone did not protect the neurons against the excitotoxic effects of glutamate. Pretreatment of cerebellar neurons with the AMPA antagonist, 6-cyano-7-nitroquinoxaline-2,3-dione blocked the neuroprotective effect mediated by AMPA indicating that the neuroprotective effect is mediated specifically by AMPA receptors. An excitotoxic concentration of glutamate, which killed between 60-80% of granule cell neurons on day 8 in vitro, mediated its toxic effect via a time-dependent apoptotic pathway. Pretreatment of cerebellar granule cell neurons with AMPA (500 microM) completely blocked glutamate-mediated apoptosis. Our results suggest that AMPA receptors may play an important role in neuronal survival.

The potassium channel opener (-)-cromakalim prevents glutamate-induced cell death in hippocampal neurons.

(-)-Cromakalim, a typical K+-channel opener, prevents neuronal death induced by either glucose and oxygen privation or by high (100 microM) extracellular glutamate in primary cultures of hippocampus. (-)-Cromakalim has no effect on the earliest events associated with exposure to glutamate. It does not prevent the rapid rise of intracellular Ca2+, the initial swelling of neurons, or the induction of c-fos mRNA transcription. (-)-Cromakalim inhibits all delayed effects associated with the excitotoxic effect of glutamate: (a) (-)-cromakalim inhibits the late and major phase of intracellular Ca2+ increase occurring up to hours after glutamate application; and (b) although (-)-cromakalim cannot prevent the initial cellular swelling induced by glutamate, cells that have been pretreated with (-)-cromakalim return to their original size in a few hours, whereas non-(-)-cromakalim-treated cells remain swollen for more prolonged periods. Many neurons surviving the initial necrotic phase of glutamate-induced cell death undergo progressive DNA cleavage leading to apoptosis. This apoptotic process is prevented completely by (-)-cromakalim. Glibenclamide, a potent blocker of the ATP-sensitive K+ channel, abolishes all the beneficial effects of (-)-cromakalim. These findings strongly suggest that (-)-cromakalim has postsynaptic effects that are closely related to the regulation of Ca2+ homeostasis and cell volume.

Contributing mechanisms for cysteine excitotoxicity in cultured cerebellar granule cells.

Several possible mechanisms for cysteine toxicity on rat cerebellar granule cells were studied and compared with the excitotoxic effect of glutamate. It was shown that the excitotoxic potency of both cysteine and glutamate increased in the presence of elevated concentrations of bicarbonate or increased pH. Pharmacological studies showed that the cysteine toxicity was specifically coupled to the NMDA receptor, whereas the glutamate toxicity was mediated to a smaller extent also by non-NMDA receptors. Treatment of cerebellar granule cells with cysteine led to an increased extracellular level of glutamate. In addition, cysteine sensitized NMDA receptors by reducing disulfide bonds in the receptor to sulfhydryl groups. A mechanism for cysteine excitotoxicity may therefore be formation of cysteine-sensitized NMDA receptors that are stimulated either by cysteine and/or by endogenous glutamate. This mechanism may also be important for the effects observed during regulated physiological release of cysteine.