Neurotoxic Effects Of Glutamate Research Articles

The relationship between cannabis use and taurine: A MRS and metabolomics study.

Taurine is an essential amino acid. It has been shown to be neuroprotective including protecting against the neurotoxic effects of glutamate. The goal of the current study was to examine the relationship between CB use and taurine measured in brain using magnetic resonance spectroscopy (MRS), and peripherally from a urine sample. Two experiments are presented. The first is a reanalysis of published data that examined taurine and glutamate in the dorsal anterior cingulate of a CB user group and non-user group using MRS. The second experiment, in a separate CB user group, used metabolomics analysis to measure taurine levels in urine. Because body composition has been associated with the pharmacokinetics of cannabis and taurine levels, a moderation model was examined with body composition included as the covariate. The MRS study found taurine levels were correlated with glutamate in both groups and taurine was correlated with frequency of CB use in the CB user group. The moderation model demonstrated significant effects of CB use and BMI; the interaction was marginally significant with lower BMI individuals showing a positive relationship between CB use and taurine. A similar finding was observed for the urine analysis. Both CB use and weight, as well as the interaction were significant. In this case, individuals with higher weight showed an association between CB use and taurine levels. This study shows the feasibility and potential importance of examining the relationship between taurine and CB use as it may shed light on a mechanism that underlies the neuroprotective effects of CB.

Effects of pomegranate peel extract against glutamate excitotoxicity in rat primary neuron culture

Amaç: Güçlü bir antioksidan etkiye sahip olan nar kabuğunun pek çok fizyolojik özellikleri gösterilmiştir. Çalışmamızda eksitatör bir nörotransmitter olan glutamatın nörotoksik etkisine karşı, güçlü antioksidan olan nar kabuğunun etkilerini araştırmayı amaçladık. Materyal ve Metot: Çalışmamızda yeni doğan sıçan beyin korteksi kullanılmıştır. Nar kabuğu ekstresi 200, 300 ve 400 mg/ml dozunda uygulandıktan 2 saat sonra 6x10-3 ve 3x10-3 M konsantrasyonda glutamat uygulaması gerçekleştirildi. Toksisite oluşturulduktan 24 saat sonra canlılık testi, total oksidan ve antioksidan kapasite ölçümleri gerçekleştirildi. Bulgular: Glutamat uygulaması artan dozlarda hücre canlılığını önemli ölçüde azaltırken nar kabuğu ekstresi yüksek dozda en iyi nöroprotektif etkiyi ortaya koymuştur. Toksisiteye bağlı artan oksidan kapasite nar kabuğu uygulaması ile anlamlı derecede düzelmiştir. Glutamata bağlı azalan antioksidan kapasite nar kabuğu ekstresi ile düzelme göstermiştir. Nar kabuğu ekstresi tek başına yüksek doz uygulandığında proliferatif etki ortaya koymuştur. Nar kabuğu nöroprotektif etkilerini proinflamatuar sitokin olan tümör nekrozis faktör-α ve apoptotik proteinler olan caspas 3 ve 9 ekspresyonunu baskılayarak ortaya koymuştur. Sonuç: Nar kabuğu ekstresi antioksidan, antiinflamatuar ve anti-apoptotik etkisi ile glutamata bağlı gelişen nörotoksisiteyi önlemiştir. Anahtar Kelimeler: Nar, Eksitotoksisite, Glutamat, Sıçan

Protective action of the extract of blueberry leaves against glutamate excitotoxicity on neurons of the rat cerebral cortex

The aqueous extract of blueberry leaves inhibits the glutamate-induced Ca2+ influx into the synaptosomes of rat brain neurons and the IC50 value of this process is close to the IC50 for MK-801, a well-known noncompetitive antagonist of glutamate NMDA subtype receptors. The aqueous extract of blueberry leaves protected the cultured neurons of the rat cerebral cortex from the neurotoxic effect of glutamate, and the inhibition intensity depended on the incubation time with the extract.

Effect of phenytoin treatment on cell proliferation in the hippocampus and the heart and related neuroendocrine changes under non-stress and stress conditions

Objective: Phenytoin is an antiepileptic drug that has been shown to reduce some neurotoxic effects of glutamate in states when glutamate neurotransmission is increased, such as neurodegeneration and stress. In the present study, we tested the hypothesis that treatment with phenytoin prevents stress-induced alterations in the production of new cells in the hippocampus and in the heart.Methods: Adult male Wistar rats were exposed to repeated hypokinesis (restraint, 2 hours daily) and were treated with phenytoin (20 mg/kg/day, s.c.) or vehicle for 8 days. On the 7th day, all animals were treated with 5-bromo-2'-deoxyuridine (BrdU), a marker of cell proliferation.Results: The treatment with phenytoin under both control and stress conditions resulted in an inhibitory effect on cell proliferation, which was more evident in the heart than in the hippocampus. Repeated treatment with phenytoin led to adrenal gland hypertrophy as well as to a marginal increase in plasma renin activity. Basal and stress-induced concentrations of adrenal steroids were unchanged by phenytoin administration.Discussion: Treatment with the glutamate release inhibiting drug phenytoin did not prevent negative consequences of repeated stress exposure on cell proliferation in Wistar rats. Treatment with phenytoin even deepened stress-induced reduction of cell proliferation in the heart. It cannot be excluded that the phenytoin-induced increase in plasma renin activity observed in the present study may occur during therapeutic use of phenytoin and contribute to its adverse effects.

Immunoelectron microscopic analysis of neurotoxic effect of glutamate in the vestibular end organs during ischemia

Conclusion: The excessive glutamate released from the type I and type II hair cells and the supporting cells injure the bouton-type endings and the nerve chalices in 30 min ischemia, and neuronal damage of glutamate was slight in 10 min ischemia. Objective: In the present study, we investigated by means of post-embedding immunoelectron microscopic analysis whether neuronal damage in the vestibular end organs is associated with the change of cellular glutamate concentration during ischemia. Methods: Transient local anoxia (10 min, 30 min) of guinea pig inner ear was induced by pressing the left labyrinthine artery. The right sides were used as controls. The morphological changes of the vestibular end organs and the areal gold particle densities representing glutamate were compared in the ischemia side and the control side. Results: The areal gold particle densities of the type I and type II hair cells and the supporting cells in the ischemic side were lower than those of the control side. There were no remarkable morphological changes compared to the control side in 10 min ischemia. In 30 min ischemia, the bouton-type endings were swollen and intercellular spaces between the type I hair cells and the nerve chalices were enlarged.

The Neuroprotective Effects of Oxaloacetate in Closed Head Injury in Rats is Mediated by its Blood Glutamate Scavenging Activity

Treatment with oxaloacetate after traumatic brain injury has been shown to decrease blood glutamate levels and protect against the neurotoxic effects of glutamate on the brain. A number of potential mechanisms have been suggested to explain oxaloacetate-induced neuroprotection. We hypothesize that the primary mechanism by which intravenous oxaloacetate provides neuroprotection is by activation of the blood glutamate-scavenging enzyme glutamate-oxaloacetate transaminase, increasing thereby the driving force for the efflux of excess glutamate from brain interstitial fluids into blood. If so, coadministration of maleate, a glutamate-oxaloacetate transaminase-blocker is expected to prevent the neuroprotective effects of oxaloacetate. A neurological severity score (NSS) was measured 1 hour after closed head injury (CHI) in rats. Then, rats received 30 microL/min/100 g infusion of saline, or 1 mmol/100 g solution of oxaloacetate, maleate, or a mixture of oxaloacetate and maleate. NSS was reassessed at 24 and 48 hour after CHI. Blood glutamate and glucose levels were measured at 0, 60, 90, and 120 minutes. NSS improved significantly at 24 hour (P<0.001) and 48 hour (P<0.001) only in the rats treated with oxaloacetate. Blood glutamate decreased significantly in the oxaloacetate-treated group at 90 minute (at the conclusion of oxaloacetate administration) (P<0.00001), but not in the control, maleate or oxaloacetate+maleate groups. A strong correlation r2=0.86 was found to exist between the percent decrease in blood glutamate levels and percent improvement in NSS. The results of this study demonstrate that the primary mechanism by which oxaloacetate provides neuroprotective activity after CHI is related to its blood glutamate scavenging activity. Management of blood glutamate concentration may have important implications in the treatment of acute brain conditions, including CHI and stroke.

Concomitant degradation of β‐catenin and GSK‐3β potently contributes to glutamate‐induced neurotoxicity in rat hippocampal slice cultures

Increasingly, published evidence links glutamate with the pathogenesis of Alzheimer's disease. We investigated the molecular mechanism underlying glutamate-induced neurotoxicity in hippocampus, which is primarily linked to cognitive dysfunction in Alzheimer's disease. Acute exposure of rat hippocampal slices to glutamate significantly induced cell death, as determined by media lactate dehydrogenase levels and PI staining. Moreover, this was accompanied by Ca2+ influx and calpain-1 activation, as confirmed by the proteolytic pattern of spectrin. Notably, glutamate-induced calpain-1 activation decreased the level of beta-catenin, and this process appeared to be independent of glycogen synthase kinase 3beta (GSK-3beta), since glutamate also led to loss of GSK-3beta. Calpeptin, a calpain inhibitor, attenuated the glutamate-mediated degradations of spectrin, synaptophysin, and beta-catenin except GSK-3beta and modestly increased cell survival. In contrast, the NMDA receptor antagonist 2-amino-5-phosphonopentanoic acid (APV) effectively reduced all glutamate-evoked responses, i.e., the breakdowns of spectrin, synaptophysin, beta-catenin and GSK-3beta, and cell death. Pharmacological studies and in vitro calpain-1 proteolysis confirmed that in the glutamate-treated hippocampus, calpain-1-mediated decrease of beta-catenin could occur independently of GSK-3beta and of proteasome, and that GSK-3beta degradation is independent of calpain-1. These findings together provide the first direct evidence that glutamate promotes the down-regulations of beta-catenin and GSK-3beta, which potently contribute to neurotoxicity in hippocampus during excitotoxic cell death, and a molecular basis for the protection afforded by calpeptin and APV from the neurotoxic effect of glutamate.

Glutamate-Independent Calcium Toxicity

It is widely accepted that a critical factor in determining neuronal death during cerebral ischemia is the progressive accumulation of intracellular Na+ ([Na+]i) and Ca2+ ([Ca2+]i) ions, which can precipitate necrosis and apoptosis of vulnerable neurons. Whereas the detrimental action of [Na+]i increase is attributable to both cell swelling and microtubular disorganization—2 phenomena that lead to cell necrosis1—a change in [Ca2+]i has been shown to be a key factor in ischemic brain damage, for it modulates several death pathways, including oxidative and nitrosative stress, mitochondrial dysfunction, and protease activation. Since Olney’s seminal work firstly suggested that excitatory aminoacids could elicit neurotoxicity,2 a large amount of work has been accumulated showing that glutamate extracellular concentrations briskly rise during acute brain injury, thus triggering an influx of Ca2+ and Na+ ions into neurons through ionotropic glutamate receptor subtypes. This evidence has led to the elaboration of the paradigm of glutamate excitotoxicity that explained ischemic neuronal cell death as a mere consequence of Na+ and Ca2+ influx through glutamate receptors.3 Although this theory has been guiding basic research in the field of neurodegeneration for almost 3 decades, more recently it has become the object of serious criticism and reassessment. What has aroused such skepticism among researchers has been the fact that although first, second, and third generation glutamate receptor antagonists have long yielded promising results in animal models of brain ischemia, they have failed to elicit a neuroprotective action in stroke and traumatic brain injury in humans.4 Therefore, the theory of excitotoxicity, though a fascinating paradigm, can only explain some of the events occurring in the acute phase of anoxic insult but cannot be seen as a major target for …

A new anti-obesity drug treatment: First clinical evidence that, antagonising glutamate-gated Ca 2+ ion channels with memantine normalises binge-eating disorders

The regulation of appetite relies on complex hypothalamic neurocircuitry of which the arcuate nucleus, and the hormone leptin play important roles. Arcuate nucleus neurones are essential for the regulation of eating behaviour, but they can be intoxicated by elevated serum levels of the amino acid glutamate (GLU). Neurotoxic effects of GLU are mediated by the N-methyl- d-aspartate receptor (NMDA-R). But the neurotoxic effects of GLU can be prevented. Concurrent administration of dizocilpine maleate (MK-801), a selective and highly potent non-competitive NMDA-R antagonist, antagonises GLU-gated Ca 2+ ion channels and completely prevents the adverse effects of GLU. Also the non-competitive NMDA-R antagonist memantine displays neuroprotective properties. In view of a previously published hypothesis that human obesity results from chronic over-consumption of GLU, we performed a therapeutic trial in five obese, but otherwise healthy women. Memantine treatment markedly decreased appetite within few hours and complete suppressed the binge-eating disorder within 24 h. Body weight decreased markedly within a few days. The findings strongly support the hypothesis that elevated levels of nutritional GLU play an important role in the pathomechanism of human obesity. We suggest to treat human obesity by protecting the hypothalamic signalling cascade of leptin action with low to moderate affinity, non-competitive NMDA-R antagonists that selectively block the GLU-gated Ca 2+ ion channels.

Effect of Glutamate and Inhibitors of Various Pathways of Free Radical Production on Na+,K+-ATPase Activity and Accumulation of Lipids Peroxidation Products in Rat Brain Cortex Synaptosomes

It has been shown that treatment of the rat brain cortex synaptosomes with glutamate produced both a significant reduction in Na+,K+-ATPase activity and accumulation of products of lipid peroxidation (LPO) like malone dialdehyde, dienoic conjugates, and Schiff bases. A suppression of different routes of free radical production in cytosol by quinacrine, indomethacin, and allopurinol (blockers of phospholipase A2, cyclooxygenases, and xanthine oxidases, respectively) as well as by MK-801 (a antagonist of MDA-receptors) prevented or lowered significantly the effect of glutamate on Na+,K+-ATPase activity. No significant effect of glutamate on the Na+,K+-ATPase activity was also observed in the presence of L-NAME (inhibitor of NO-synthase). Inhibitors of the arachidonate and NO-synthase pathway of free radical production also prevented accumulation of LPO end products in the rat brain cortex under the effect of glutamate. In the presence of rotenone and olygomycin (blockers of mitochondrial electron transport and ATP synthase, respectively), glutamate led to even a greater inactivation of Na+,K+-ATPase and accumulation of malone dialdehyde. The data obtained suggest that at early stages of ischemia the neurotoxic effect of glutamate is due to an inflow of calcium ions through NMDA receptors and activation of different pathways of free radical production in cytosol of nerve cells. At these stages, protective functions of mitochondria appear to predominate due to their ability to accumulate calcium ions and to prevent an excessive increase of the cytosol calcium concentration under the effect of excitatory amino acids.

Protective effects of 1α,25-(OH) 2D 3 against the neurotoxicity of glutamate and reactive oxygen species in mesencephalic culture

This study was undertaken to determine whether 1α,25-dihydroxyvitamin D3 [1α,25-(OH) 2D 3], an active metabolite of vitamin D, protects dopaminergic neurons against the neurotoxic effects of glutamate and dopaminergic toxins using rat mesecephalic culture. Brief glutamate exposure elicited cytotoxicity in both dopaminergic and non-dopaminergic neurons. Pretreatment, but not co-administration, of 1α,25-(OH) 2D 3 protected both types of neurons against the cytotoxicity of glutamate in a concentration- and time-dependent manner. The neuroprotective effect of 1α,25-(OH) 2D 3 was inhibited by the protein synthesis inhibitor, cycloheximide. To investigate the mechanisms of these neuroprotective effects, we examined the effects of 1α,25-(OH) 2D 3 on neurotoxicity induced by calcium ionophore and reactive oxygen species (ROS). Pretreatment with 1α,25-(OH) 2D 3 protected both types of neurons against the cytotoxicity induced by A23187 in a concentration-dependent manner. Furthermore, 24-h pretreatment with 1α,25-(OH) 2D 3 concentration-dependently protected both types of neurons from ROS-induced cytotoxicity. A 24-h incubation with 1α,25-(OH) 2D 3 inhibited the increase in intracellular ROS level following H 2O 2 exposure. A 24-h exposure to 1-methyl-4-phenylpyridium ion (MPP +) or 6-hydroxydopamine (6-OHDA) exerted selective neurotoxicity on dopaminergic neurons, and these neurotoxic effects were ameliorated by 1α,25-(OH) 2D 3. These results suggest that 1α,25-(OH) 2D 3 provides protection of dopaminergic neurons against cytotoxicity induced by glutamate and dopaminergic toxins by facilitating cellular functions that reduce oxidative stress.

A microdialysis study investigating the mechanisms of hydroxyl radical formation in rat striatum exposed to glutamate

Considerable evidence has linked hydroxyl radicals ( · OH ) to excitotoxicity. Glutamate infused through a microdialysis probe into rat striatum induced a massive · OH production, which was completely blocked by PBN and attenuated by dizocilpine, 2-amino-5-phosphonopentanoic acid (AP-5), N G-nitro- l-arginine methyl ester ( l-NAME) and mepacrine. Thus, we suggest that the neurotoxic effects of glutamate in vivo may derive from an increased formation of · OH resulting from excessive activation of NMDA receptors and downstream enzymes such as NOS and PLA 2.

P-368 - Effect of SA4503, a novel σ1 receptor agonist, against glutamate neurotoxicity in cultured rat retinal neurons

Abstract We examined the effects of σ1 receptor agonists against glutamate-induced neurotoxicity in cultured retinal neurons. Primary cultures obtained from fetal rat retinas (16–19 d gestation) were used. The neurotoxic effect of glutamate was quantitatively assessed using the trypan blue exclusion method. A brief exposure of retinal cultures to glutamate (500 μM) led to delayed neuronal cell death. The glutamate-induced neurotoxicity was inhibited by (5R,10S)-(+)-5-methyl-10,11-dihydro-5H-dibenzo-[a,b]-cyclohepten-5,10-imine hydrogen maleate (MK-801). The σ1 receptor agonists, 1-(3,4-dimethoxyphenethyl)-4-(3-phenylpropyl)-piperazine dihydrochloride (SA4503) and (+)-pentazocine at a concentration range of 0.1~100 μM reduced the glutamate-induced neurotoxicity in a dose-dependent manner. In addition, the neuroprotective effects of both SA4503 and (+)-pentazocine were antagonized by co-treatment with N,N-dipropyl-2-[4-methoxy-3-(2-phenylethoxy)phenyl]ethylamine monohydrochloride (NE-100), a putative σ1 receptor antagonist. These findings suggest that σ1 receptor agonists protect retinal cells against glutamate-induced neurotoxicity.

Effect of SA4503, a novel σ1 receptor agonist, against glutamate neurotoxicity in cultured rat retinal neurons

We examined the effects of sigma1 receptor agonists against glutamate-induced neurotoxicity in cultured retinal neurons. Primary cultures obtained from fetal rat retinas (16-19 d gestation) were used. The neurotoxic effect of glutamate was quantitatively assessed using the trypan blue exclusion method. A brief exposure of retinal cultures to glutamate (500 microM) led to delayed neuronal cell death. The glutamate-induced neurotoxicity was inhibited by (5R,10S)-(+)-5-methyl-10,11-dihydro-5H-dibenzo-[a,b]-cyclohepten-5 ,10-imine hydrogen maleate (MK-801). The sigma1 receptor agonists, 1-(3,4-dimethoxyphenethyl)-4-(3-phenylpropyl)-piperazine dihydrochloride (SA4503) and (+)-pentazocine at a concentration range of 0.1 approximately 100 microM reduced the glutamate-induced neurotoxicity in a dose-dependent manner. In addition, the neuroprotective effects of both SA4503 and (+)-pentazocine were antagonized by co-treatment with N,N-dipropyl-2-[4-methoxy-3-(2-phenylethoxy)phenyl]ethylamine monohydrochloride (NE-100), a putative sigma1 receptor antagonist. These findings suggest that sigma1 receptor agonists protect retinal cells against glutamate-induced neurotoxicity.

Dodecylglycerol provides partial protection against glutamate toxicity in neuronal cultures derived from different regions of embryonic rat brain.

Primary cultures enriched in neurons dissociated from embryonic rat cerebral cortex, cerebellum, or hippocampus were treated in a chemically defined serum-free media with either vehicle, dodecylglycerol (DDG, 3 microM), or glutamate (75 microM), or preincubated with DDG for 4 or 24 h, and further incubated with glutamate. Their morphological and biochemical assessments (lactate dehydrogenase [LDH] release in the culture media, neuronal viability and intracellular Ca2+ mobilization) were made. Neurotoxic effects of glutamate and glutamate-mediated increases in intracellular Ca2+ were maximal in neurons from cerebellum and minimal in neurons from cortex. Cotreatment of cells with DDG and glutamate failed to provide significant neuronal protection against glutamate in the three brain regions. Pretreatment of cells with DDG for 4 or 24 h prior to glutamate treatment provided significant neuroprotection as judged by morphological changes and a decrease in LDH activity. Neuroprotection of approximately 15-35% was observed following 4 h of DDG pretreatment, increasing to 60-85% protection after 24 h of DDG pretreatment. Although the mechanism of DDG's neuroprotective action remains to be elucidated, these results demonstrate that both glutamate and DDG have differential specificity for anatomical regions of the brain.

AMPA Neurotoxicity in Cultured Cerebellar Granule Neurons: Mode of Cell Death

Various forms of cell death induced by the glutamate receptor agonist, α-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA), were analyzed by determining the capacity of cultured cerebellar granule cells to metabolize 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) into formazan, by measuring the leakage of lactate dehydrogenase (LDH), by using confocal microscopy to visualize propidium iodide staining of apoptotic nuclei, and by using field inversion gel electrophoresis (FIGE) for the detection of AMPA-produced cleavage of DNA into high molecular-weight fragments (50 kbp). All these measures indicated that stimulation of AMPA receptors may be involved in the neurotoxic effects of glutamate, and that AMPA-induced neurotoxicity in cerebellar granule cells display morphologically distinct features of both necrotic and apoptotic modes of cell death. In agreement with previous observations, a blockade of AMPA receptor desensitization was necessary to unmask AMPA-induced functional responses in cultured cerebellar granule neurons in vitro. Microfluorimetric measurements of free cytoplasmic calcium concentrations ([Ca2+]i) in single cerebellar neurons revealed that AMPA neurotoxicity was accompanied by a pronounced elevation of [Ca2+]i. Our current results add further evidence to the notion that glutamate-induced neurotoxicity in cerebellar granule cells is mediated not only through NMDA receptors but also through a direct activation of AMPA receptor-regulated cation channels.

P-19-31 - Effect of “Kangenkaryu” on cerebral blood flow and gene expression in rat

We examined the effects of σ1 receptor agonists against glutamate-induced neurotoxicity in cultured retinal neurons. Primary cultures obtained from fetal rat retinas (16–19 d gestation) were used. The neurotoxic effect of glutamate was quantitatively assessed using the trypan blue exclusion method. A brief exposure of retinal cultures to glutamate (500 μM) led to delayed neuronal cell death. The glutamate-induced neurotoxicity was inhibited by (5R,10S)-(+)-5-methyl-10,11-dihydro-5H-dibenzo-[a,b]-cyclohepten-5,10-imine hydrogen maleate (MK-801). The σ1 receptor agonists, 1-(3,4-dimethoxyphenethyl)-4-(3-phenylpropyl)-piperazine dihydrochloride (SA4503) and (+)-pentazocine at a concentration range of 0.1~100 μM reduced the glutamate-induced neurotoxicity in a dose-dependent manner. In addition, the neuroprotective effects of both SA4503 and (+)-pentazocine were antagonized by co-treatment with N,N-dipropyl-2-[4-methoxy-3-(2-phenylethoxy)phenyl]ethylamine monohydrochloride (NE-100), a putative σ1 receptor antagonist. These findings suggest that σ1 receptor agonists protect retinal cells against glutamate-induced neurotoxicity.

Transcriptional or translational inhibition blocks low dose NMDA-mediated cell death.

Glutamate toxicity in nerve cells has been well documented and may play a role in a broad spectrum of neurological and ophthalmic diseases. Recent work in several laboratories has suggested that an apoptotic-like mechanism may be implicated in glutamate toxicity under certain circumstances. We therefore studied the effects of transcriptional and translational inhibition on glutamate-mediated cell death in retinal ganglion cells. We now report that either cycloheximide or actinomycin D can, even when added 2 h after the initial excitotoxic insult, save retinal ganglion cells from low dose glutamate toxicity. However, cycloheximide or actinomycin D are unable to prevent glutamate-mediated death at higher concentrations of excitotoxin. This result indicates that at low doses, the neurotoxic effects of glutamate may develop through an apoptotic-like mechanism.

Glutamate neurotoxicity in mesencephalic dopaminergic neurons in culture.

The neurotoxic effect of glutamate in cultured mouse mesencephalic dopaminergic neurons was investigated. Neuron-rich cell cultures were prepared from 13-14-day-old fetal mouse ventral mesencephalic tissue. Cultures were exposed to glutamate for 10 min and evaluated for glutamate neurotoxicity (GNT) 18-24 hr later by tyrosine hydroxylase (TH) immunostaining, microtubule associated protein-2 (MAP2) immunostaining, and radiolabeled dopamine uptake assay. In glutamate-exposed cultures, the number of TH-positive neurons and the level of dopamine uptake were reduced to 40% (35-45%) and 50% (47-52%), respectively, of control cultures. The number of MAP2-positive neurons was also reduced to 47%, indicating that the GNT was not restricted or selective to dopaminergic neurons. It is concluded that GNT was mediated by the N-methyl-D-aspartic acid (NMDA) receptor from the following observations: 1) GNT was completely blocked by MK-801, an NMDA receptor antagonist; 2) NMDA itself was as toxic as glutamate; 3) 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX), an antagonist of the alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid/kainate (AMPA/KA) receptor, did not block GNT; 4) kainate did not show neurotoxicity at a low concentration; and 5) two modulators of the NMDA receptor, 7-chlorokynurenic acid and magnesium, were effective in blocking GNT. Protective effects of phorbol myristate acetate, a tumor promoter, and gangliosides (GM1 and GT1b) on GNT were also demonstrated. Possible interactions between GNT and several protein kinase cascades were also investigated. Forskolin, an activator of adenyl cyclase and protein kinase A, showed some protective effect on GNT. But okadaic acid, an inhibitor of phosphatases, and genistein, a tyrosine kinase inhibitor, did not show any protective effect. These results suggest that 1) glutamate is capable of causing neuronal death in the substantia nigra; 2) GNT on dopaminergic neurons is mainly mediated by the NMDA receptor under the conditions of our study; 3) protein kinase C translocation is a key mechanism of GNT; and 4) there is an interplay of a signal transduction system in the pathomechanism of GNT.

Neurotoxic effects of glutamate on mouse area postrema

Neurotoxic effects of glutamate on mouse area postrema