Comparison of the Potency of Competitive NMDA Antagonists Against the Neurotoxicity of Glutamate and NMDA

Abstract: The object of this investigation was to determine whether glutamate uptake affects the apparent potency of the competitive antagonists dl -2-amino-5-phosphonovalerate and CGS-19755 in blocking NMDA receptor-mediated neurotoxicity. In astrocyte-rich rat cortical cultures we observed that dl -2-amino-5-phosphonovalerate and CGS-19755 were 24 and 16 times more potent against NMDA than against glutamate-induced toxicity. In contrast, dl -2-amino-5-phosphonovalerate was equipotent against the two agonists in astrocyte-poor cultures, in which dendrites are directly exposed to the extracellular medium. With the noncompetitive NMDA antagonist MK-801, similar potencies were observed against glutamate (212 ± 16 n M ) and against NMDA (155 ± 9 n M ) neurotoxicity. These results may be explained if we assume that the neuronal cell body is less susceptible than the dendrites to NMDA receptor-mediated toxicity, and that the action of glutamate in astrocyte-rich cultures is confined to the cell body. In this case, one would expect that higher concentrations of glutamate would be needed to produce toxicity in astrocyte-rich cultures, and that higher concentrations of competitive antagonists would be needed to overcome this toxicity. Our observations help explain the pharmacology of the competitive NMDA antagonists against NMDA receptor-mediated neurotoxicity but also suggest the possibility that, because the cell body and dendrites may be distinct sites for neurotoxicity, they might also involve different mechanisms of toxicity.     

Increase in glutamate-induced neurotoxicity by activated astrocytes involves stimulation of protein kinase C

Activation of astrocytes is a common feature of neurological disorders, but the importance of this phenomenon for neuronal outcome is not fully understood. Treatment of mixed hippocampal cultures of neurones and astrocytes from day 2-4 in vitro (DIV 2-4) with 1 micro m cytosine arabinofuranoside (AraC) caused an activation of astrocytes as detected by a stellate morphology and a 10-fold increase in glial fibrillary acidic protein (GFAP) level compared with vehicle-treated cultures. After DIV 12, we determined 43% and 97% damaged neurones 18 h after the exposure to glutamate (1 mm, 1 h) in cultures treated with vehicle and AraC, respectively. Dose-response curves were different with a higher sensitivity to glutamate in cultures treated with AraC (EC50 = 0.01 mm) than with vehicle (EC50 = 0.12 mm). The susceptibility of neurones to 1 mm glutamate did not correlate with the percentage of astrocytes and was insensitive to an inhibition of glutamate uptake. In cultures treated with vehicle and AraC, glutamate-induced neurotoxicity was mediated through stimulation of the NR1-NR2B subtype of NMDA receptors, because it was blocked by the NMDA receptor antagonist MK-801 and the NR1-NR2B selective receptor antagonist ifenprodil. Protein levels of the NR2A and NR2B subunits of NMDA receptor were similar in cultures treated with vehicle or AraC. AraC-induced changes in glutamate-induced neurotoxicity were mimicked by activation of protein kinase C (PKC), whereas neuronal susceptibility to glutamate was reduced in cultures depleted of PKC and treated with AraC suggesting that the increase in glutamate toxicity by activated astrocytes involves activation of PKC.     

β-Amyloid Neurotoxicity in Human Cortical Culture Is Not Mediated by Excitotoxins

Abstract: β-Amyloid is a metabolic product of the amyloid precursor protein, which accumulates abnormally in senile plaques in the brains of patients with Alzheimer's disease. The neurotoxicity of 0-amyloid has been observed in cell culture and in vivo, but the mechanism of this effect is unclear. In this report, we describe the direct neurotoxicity of β-amyloid in high-density primary cultures of human fetal cortex. In 36-day-old cortical cultures, β-amyloid neurotoxicity was not inhibited by the broad-spectrum excitatory amino acid receptor antagonist kynurenate or the NMDA receptor antagonist D-2-amino-5-phosphonovaleric acid under conditions that inhibited glutamate and NMDA neurotoxicity. In 8-day-old cortical cultures, neurons were resistant to glutamate and NMDA toxicity but were still susceptible to β-amyloid neurotoxicity, which was unaffected by excitatory amino acid receptor antagonists. Treatment with β-amyloid caused chronic neurodegenera-tive changes, including neuronal clumping and dystrophic neurites, whereas glutamate treatment caused rapid neuronal swelling and neurite fragmentation. These results suggest that β-amyloid is directly neurotoxic to primary human cortical neurons by a mechanism that does not involve excitatory amino acid receptors.     

Astrocytes regulate N-methyl-D-aspartate receptor subunit composition increasing neuronal sensitivity to excitotoxicity

We have examined the dependence of rat cerebellar granule neurons (CGNs) for protection against glutamate toxicity. Under co-culture conditions, rat CGNs require astrocytes to protect against glutamate. The CGNs become more sensitive to glutamate toxicity in co-culture than when grown in cultures with only low numbers of astrocytes. If the protection of the astrocytes was withdrawn or blocked, this sensitivity led to neuronal death. Differing changes in NMDA receptor subunit subtype composition were noted depending on the conditions in which the CGNs were grown. Suppression of individual NMDA subunit subtypes by oligonucleotide knockdown resulted in inhibition of toxicity. This result implies that astrocytes regulate the expression of NMDA receptor subunit subtypes which influence neuronal sensitivity to glutamate toxicity.     

Induction of Ornithine Decarboxylase by N-Methyl-D-Aspartate Receptor Activation Is Unrelated to Potentiation of Glutamate Excitotoxicity by Polyamines in Cerebellar Granule Neurons

Abstract: Polyamines positively modulate the activity of the N -methyl- d -aspartate (NMDA)-sensitive glutamate receptors. The concentration of polyamines in the brain increases in certain pathological conditions, such as ischemia and brain trauma, and these compounds have been postulated to play a role in excitotoxic neuronal death. In primary cultures of rat cerebellar granule neurons, exogenous application of the polyamines spermidine and spermine (but not putrescine) potentiated the delayed neurotoxicity elicited by NMDA receptor stimulation with glutamate. Furthermore, both toxic and nontoxic concentrations of glutamate stimulated the activity of ornithine decarboxylase (ODC)—the key regulatory enzyme in polyamine synthesis—and increased the concentration of ODC mRNA in cerebellar granule neurons but not in glial cells. Glutamate-induced ODC activation but not neurotoxicity was blocked by the ODC inhibitor difluoromethylornithine. Thus, high extracellular polyamine concentrations potentiate glutamate-triggered neuronal death, but the glutamate-induced increase in neuronal ODC activity may not play a determinant role in the cascade of intracellular events responsible for delayed excitotoxicity.     

Role of group I metabotropic glutamate receptors and NMDA receptors in homocysteine-evoked acute neurodegeneration of cultured cerebellar granule neurones

Hyperhomocysteinemia is a risk factor in neurodegeneration. It has been suggested that apart from disturbances in methylation processes, the mechanisms of this effect may include excitotoxicity mediated by the N-methyl-D-aspartate (NMDA) receptors. In this study we demonstrate that apart from NMDA receptors, also group I metabotropic glutamate receptors participate in acute homocysteine (Hcy)-induced neurotoxicity in cultured rat cerebellar granule neurones. Primary neuronal cultures were incubated for 30 min in the Mg(2+)-free ionic medium containing homocysteine and other ligands, and neurodegenerative changes were assessed 24h later using propidium iodide staining. D,L-Homocysteine given alone appeared to be a weak neurotoxin, with EC(50) of 17.4mM, whereas EC(50) for L-glutamate was 0.17 mM. Addition of 50 microM glycine enhanced homocysteine neurotoxicity, and only that portion of neurotoxicity was abolished by 0.5 microM MK-801, an uncompetitive NMDA receptor antagonist. The net stimulation of 45Ca uptake by granule cells incubated in the presence of 25 mM D,L-homocysteine with 50 microM glycine was only 3% of the net uptake evoked by 1mM glutamate. Application of an antagonist of group I metabotropic glutamate receptors (mGluRs) LY367385 at 25 and 250 microM concentrations, induced a dose-dependent partial neuroprotection, whereas given together with MK-801 completely prevented neurotoxicity. In the absence of glycine, LY367385 and MK-801 given alone failed to induce neuroprotection, while applied together completely prevented homocysteine neurotoxicity. Agonist of group I mGluRs, 10 trans-azetidine-2,3-dicarboxylic acid (t-ADA) induced significant neurotoxicity. This study shows for the first time that acute homocysteine-induced neurotoxicity is mediated both by group I mGluRs and NMDA receptors, and is not accompanied by massive influx of extracellular Ca(2+) to neurones.     

Glutamate Neurotoxicity Is Independent of Calpain I Inhibition in Primary Cultures of Cerebellar Granule Cells

Glutamate-induced neurotoxicity and calpain activity were studied in primary cultures of rat cerebellar granule neurons and glial cells. Calpain activation, as monitored by quantitative immunoblotting of spectrin, required micromolar concentrations of Ca2+ in neuronal homogenates (calpain I) and millimolar Ca2+ concentrations in glial homogenates (calpain II). Glutamate-induced toxicity and calpain activation were observed in neuronal, but not in glial, cultures. In neurons, calpain I activation by glutamate was dose-dependent and persisted after withdrawal of neurotoxic doses of glutamate. Natural (GM1) and semisynthetic (LIGA4) gangliosides or the glutamate receptor blocker MK-801 prevented calpain I activation and delayed neuronal death elicited by glutamate. GM1 and LIGA4 had no effect on calpain I activity in neuronal homogenates, however. Furthermore, two calpain I inhibitors (leupeptin and N-acetyl-Leu-Leu-norleucinal) prevented glutamate-induced spectrin degradation, but failed to affect glutamate neurotoxicity. These results thus suggest that glutamate-induced neurotoxicity is independent of calpain I activation.     

N-Methyl-d-Aspartate- or Glutamate-Mediated Toxicity in Cultured Rat Cortical Neurons Is Antagonized by FPL 15896AR

Abstract: The neuroprotective action of ( S )-α-phenyl-2-pyridineethanamine dihydrochloride (FPL 15896AR), a novel noncompetitive N -methyl- d -aspartate (NMDA) receptor antagonist, was examined in primary rat cortical neuronal cultures. Exposure of cortical cultures to NMDA (50 µ M ) or glutamate (50 µ M ) for 15 min resulted in the death of 85–95% of the neurons during the next 24 h. This neurotoxicity was completely eliminated by adding FPL 15896AR (50 µ M ) to the cultures during the time of NMDA or glutamate exposure. Neuroprotective concentrations of FPL 15896AR also inhibited other acute effects of NMDA. FPL 15896AR (50 µ M ) prevented the loss of membrane-associated protein kinase C activity that developed by 4 h after transient exposure to 50 µ M NMDA or 50 µ M glutamate. FPL 15896AR also reduced by ∼35% the magnitude of NMDA-triggered increases in intracellular free Ca²⁺ concentration in the cortical cultures. These data indicate that NMDA-mediated toxicity in cultured cortical neurons can be blocked by the NMDA antagonist FPL 15896AR.     

Characterization of glutamate toxicity in cultured rat cerebellar granule neurons at reduced temperature

We have defined conditions whereby glutamate becomes toxic to isolated cerebellar granule neurons in a physiologic salt solution (pH 7.4). In the presence of a physiologic Mg⁺⁺ concentration, acute glutamate excitotoxicity manifests only when the temperature was reduced from 37°C to 22°C. In contrast to glutamate, N-methyl-D-aspartate (NMDA) was non-toxic at either temperature at concentrations as high as 1 mM. Glycine strongly potentiated both the potency and efficacy of glutamate but revealed only a modest NMDA response. The non-NMDA receptor antagonist, 6-cyano-7-nitroquinoxalinedione (CNQX), potently protected against glutamate challenge, although the contribution of antagonism at strychnine-insensitive glycine sites could not be excluded. To further characterize the non-NMDA receptor contribution to the excitotoxic response, the promiscuity of glutamate interaction with ionotropic receptors was simulated by exposing neurons to NMDA in the presence of non-NMDA receptor agonists. NMDA toxicity was potentiated four- to sevenfold when non-NMDA receptors were coactivated by a subtoxic concentration of AMPA, kainate, or domoate. These results suggest that non-NMDA receptor activation participates in the mechanism of acute glutamate toxicity by producing neuronal depolarization (via sodium influx), which in turn promotes the release of the voltage-dependent magnesium blockade of NMDA receptor ion channels. © 1997 John Wiley & Sons, Inc.     

Ethanol Differentially Inhibits Homoquinolinic Acid- and NMDA-Induced Neurotoxicity in Primary Cultures of Cerebellar Granule Cells

The potency of ethanol to inhibit N-methyl-D-aspartate (NMDA) receptor functions may depend on the subunit composition of the NMDA receptors. We used a NR2A-B subunit-selective NMDA receptor agonist, homoquinolinic acid (HQ), and a subunit-unselective agonist, NMDA, to induce neurotoxicity in cerebellar granule cells and examined the neuroprotective actions of ethanol, as well as NR2A- and NR2B-subunit selective antagonists, respectively. HQ was a more potent neurotoxic agent than NMDA, as measured by the MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) assay. NR2A- and NR2B-selective NMDA receptor antagonists displayed quite similar neuroprotective potencies against the NMDA- and HQ-produced cell death, indicating that the higher potency of HQ to induce neurotoxicity cannot be simply explained by NR2A- or NR2B-subunit selectivity. As expected, ethanol (25 and 50 mM) attenuated the NMDA-induced neurotoxicity in a non-competitive manner by significantly reducing the maximum neurotoxicity produced by NMDA. By contrast, ethanol inhibited the HQ-induced neurotoxicity in a manner resembling a competitive-like interaction significantly increasing the EC₅₀ value for HQ, without reducing the maximum neurotoxicity produced by HQ. These results suggest that HQ reveals either a novel site or a not previously observed mechanism of interaction between ethanol and NMDA receptors in rat cerebellar granule cell cultures.     

Responses of Bergmann Glia and Granule Neurons In Situ to N-Methyl-d-Aspartate, Norepinephrine, and High Potassium

Abstract: To gain insight into neuronal-glial signaling in brain, cerebellar Bergmann glia and granule neurons were studied in acutely isolated slices with the aid of laser scanning confocal microscopy. Both Bergmann glia and granule neurons responded to N -methyl- d -aspartate (NMDA) with a rise in [Ca²⁺]_i. However, the glial NMDA response was frequently inhibited by tetrodotoxin, suggesting that the response depended on neuronal action potentials, rather than on direct activation of NMDA receptors on the Bergmann glia. Further experiments demonstrated that the NMDA response in Bergmann glia was not inhibited by a combination of non-NMDA glutamate receptor blockers 6-cyano-7-nitroquinoxaline-2,3-dione and α-methyl-4-carboxyphenylglycine. Bergmann glia also responded to norepinephrine and high K⁺, and the responses were not inhibited by tetrodotoxin. The glial norepinephrine response was blocked by phentolamine but not by the removal of external Ca²⁺, indicating a direct activation of α₁-adrenergic receptors that mediated release of Ca²⁺ from intracellular stores. The KCI-induced response in both neurons and glia was dependent on external Ca²⁺ and was blocked by verapamil or nifedipine. In summary, our data indicate that Bergmann glia in situ recognize a signal(s) released from neurons during neuronal activity.     

Astrocytes modulate thapsigargin-induced changes in calcium concentration and neuronal survival

When mature cerebellar granule neurons (CGN) grown in high K+ (25 mM K+, HK)-serum containing medium are subjected to the HK/serum deprivation, they are destined for neuronal death. In this study, we attempted to elucidate the roles of endoplasmic reticular (ER) Ca2+-store and co-cultured astrocytes in HK/serum deprivation induced neuronal death. Thapsigargin (TG), an inhibitor of ER Ca2+-ATPase was simultaneously applied with normal K+ (5 mM K+, NK) serum free medium, and its effects on neuronal death in either astrocyte-poor or astrocyterich culture were examined. By means of the fura-2 microfluorimetric technique, we monitored the changes of the intracellular Ca2+ concentration, [Ca2+]i, associated with neuronal death under various treatments. The results obtained showed that in astrocyte-poor cultures of mature CGN (10 days in vitro, DIV), the basal level of [Ca2+]i markedly decreased from 184 +/- 5 to 89.7 +/- 5 nM 24 h after HK/serum deprivation. Although treatment with TG slightly increased the [Ca2+]i to 117.6 +/- 4 nM, the survival rate of the neurons was even worse; it was reduced from 49 +/- 4% to 28 +/- 2%. In the astrocyte-rich cultures, HK/serum deprivation also caused a profound reduction of neuronal [Ca2+]i, from 166 +/- 3 to 90.2 +/- 6 nM, accompanied by even more serious neuronal death (95.5 +/- 1%). On the other hand, treatment with TG in astrocyterich cultures further lowered the [Ca2+]i to 65 +/- 2 nM but markedly improved the neuronal survival rate from 4.5 +/- 1% to 60 +/- 2% in a concentration-dependent manner. The strong implication of these findings is that ER Ca2+-store and astrocytes participate in modulating the responses of neurons to stress stimulation.     

Epidermal Growth Factor and Basic Fibroblast Growth Factor Protect Dopaminergic Neurons from Glutamate Toxicity in Culture

Abstract: In this report we characterize the toxicity of the excitatory amino acid l -glutamate with respect to dopaminergic neurons cultured from embryonic rat mesencephalon. We also demonstrate that two growth factors, epidermal growth factor (EGF) and basic fibroblast growth factor (bFGF), can protect these neurons from damage. Micromolar concentrations of l -glutamate, as well as agonists that specifically activate N -methyl- d -aspartate (NMDA) and non-NMDA receptors, are all toxic to dopamine neurons in a concentration-dependent manner, as reflected by decreases in high-affinity dopamine uptake and confirmed by decreases in numbers of tyrosine hydroxylase-immunoreactive neurons. Although the non-NMDA receptor antagonist 6-cyano-7-nitroquinoxaline-2,3-dione could attenuate the effects of quisqualate, treatment with this antagonist could not eliminate the effects of glutamate itself. Similarly, (±)-2-amino-5-phosphonopentanoic acid was effective against NMDA toxicity but could not protect cells from quisqualate toxicity. Thus, each type of receptor could mediate neurotoxicity independently of the other. The presence of EGF or bFGF in the culture medium conferred a relative resistance of dopaminergic neurons to glutamate and quisqualate neurotoxicity by increased glutamate transport. However, treatment of the cultures with l - trans -pyrrolidine-2,4-dicarboxylic acid, an inhibitor of glutamate transport, attenuated but did not eliminate the protective effects of both growth factors against glutamate toxicity. When cultures were incubated with conditioned medium from growth factor-treated cultures, neuroprotection was also achieved. These results suggest that both EGF and bFGF can protect neurons from neurotoxicity in culture by increasing the capacity of the culture for glutamate uptake as well as by the secretion of soluble factors into the medium.     

DNQX-induced toxicity in cultured rat hippocampal neurons: an apparent AMPA receptor-independent effect?

To evaluate the involvement of AMPA receptor activation in neuronal cell death and survival, rat hippocampal neurons in culture were treated with AMPA receptor antagonists. A 46 h treatment with 6,7-dinitroquinoxaline-2,3-dione (DNQX), added 2 h after cell plating, induces a dose-dependent neurotoxicity. Similar effects are also observed in more mature hippocampal neurons (treatment at 14 days in vitro). DNQX toxic effect is neuron-specific since cultured hippocampal glial cells are unaffected. Attempts to characterise the site of action of DNQX suggest that ionotropic glutamate receptors would not be implicated. Indeed, (i) other AMPA receptor antagonists are either ineffective or only moderately efficient in mimicking DNQX effects; (ii) AMPA alone or in the presence of cyclothiazide, as well as, other AMPA receptor agonists, do not reverse DNQX action; (iii) DNQX neurotoxicity is not likely to involve blockade of NMDA receptor glycine site, since this effect is neither mimicked by 7-chlorokynurenate nor reversed by D-serine. Thus, DNQX toxicity in cultured hippocampal neurons is apparently mediated through an ionotropic glutamate receptor-independent way.     

Astrocytes protect neurons from nitric oxide toxicity by a glutathione-dependent mechanism

Nitric oxide (NO) contributes to neuronal death in cerebral ischemia and other conditions. Astrocytes are anatomically well positioned to shield neurons from NO because astrocyte processes surround most neurons. In this study, the capacity of astrocytes to limit NO neurotoxicity was examined using a cortical co-culture system. Astrocyte-coated dialysis membranes were placed directly on top of neuronal cultures to provide a removable astrocyte layer between the neurons and the culture medium. The utility of this system was tested by comparing neuronal death produced by glutamate, which is rapidly cleared by astrocytes, and N-methyl-D-aspartate (NMDA), which is not. The presence of an astrocyte layer increased the LD(50) for glutamate by approximately four-fold, but had no effect on NMDA toxicity. Astrocyte effects on neuronal death produced by the NO donors S-nitroso-N-acetyl penicillamine and spermine NONOate were examined by placing these compounds into the medium of co-cultures containing either a control astrocyte layer or an astrocyte layer depleted of glutathione by prior exposure to buthionine sulfoximine. Neurons in culture with the glutathione-depleted astrocytes exhibited a two-fold increase in cell death over a range of NO donor concentrations. These findings suggest that astrocytes protect neurons from NO toxicity by a glutathione-dependent mechanism.     

Contribution of extrasynaptic N-methyl-d-aspartate and adenosine A1 receptors in the generation of dendritic glutamate-mediated plateau potentials

Thin basal dendrites can strongly influence neuronal output via generation of dendritic spikes. It was recently postulated that glial processes actively support dendritic spikes by either ceasing glutamate uptake or by actively releasing glutamate and adenosine triphosphate (ATP). We used calcium imaging to study the role of NR2C/D-containing N-methyl-d-aspartate (NMDA) receptors and adenosine A1 receptors in the generation of dendritic NMDA spikes and plateau potentials in basal dendrites of layer 5 pyramidal neurons in the mouse prefrontal cortex. We found that NR2C/D glutamate receptor subunits contribute to the amplitude of synaptically evoked NMDA spikes. Dendritic calcium signals associated with glutamate-evoked dendritic plateau potentials were significantly shortened upon application of the NR2C/D receptor antagonist PPDA, suggesting that NR2C/D receptors prolong the duration of calcium influx during dendritic spiking. In contrast to NR2C/D receptors, adenosine A1 receptors act to abbreviate dendritic and somatic signals via the activation of dendritic K⁺ current. This current is characterized as a slow-activating outward-rectifying voltage- and adenosine-gated current, insensitive to 4-aminopyridine but sensitive to TEA. Our data support the hypothesis that the release of glutamate and ATP from neurons or glia contribute to initiation, maintenance and termination of local dendritic glutamate-mediated regenerative potentials.     

D-Serine as a putative glial neurotransmitter

Abundant recent evidence favors a neurotransmitter/neuromodulator role for D-serine. D-serine is synthesized from L-serine by serine racemase in astrocytic glia that ensheath synapses, especially in regions of the brain that are enriched in NMDA-glutamate receptors. D-serine is more potent than glycine at activating the 'glycine' site of these receptors. Moreover, selective degradation of D-serine but not glycine by D-amino acid oxidase markedly reduces NMDA neurotransmission. D-serine appears to be released physiologically in response to activation by glutamate of AMPA-glutamate receptors on D-serine-containing glia. This causes glutamate-receptor-interacting protein, which binds serine racemase, to stimulate enzyme activity and D-serine release. Thus, glutamate triggers the release of D-serine so that the two amino acids can act together on postsynaptic NMDA receptors. D-serine also plays a role in neural development, being released from Bergmann glia to chemokinetically enhance the migration of granule cell cerebellar neurons from the external to the internal granular layer.     

Cerebellar Granule Neurons are More Vulnerable to Transient Transport-Mediated Glutamate Release than to Glutamate Uptake Blockade. Correlation with Excitatory Amino Acids Levels

The extracellular concentration of glutamate is highly regulated due to its excitotoxic nature. Failure of glutamate uptake or reversed activation of its transporters contributes to neurodegeneration related to some pathological conditions. We have compared the neurotoxicity of the substrate glutamate uptake inhibitor, l-trans-pyrrolidine-2,4-dicarboxylate (PDC), which promotes glutamate release by heteroexchange, with that of DL-threo-beta-benzyloxyaspartate (DL-TBOA), a non-substrate inhibitor, in cerebellar granule cell cultures. PDC substantially increases the extracellular concentration of glutamate during 30 min exposure and causes neuronal death at high concentrations, while DL-TBOA neurotoxicity is only observed after long-term exposure (8–24 h). During mitochondrial inhibition by 3-nitropropionic acid (3-NP), PDC-induced neuronal death is facilitated, but not that of DL-TBOA. In cultures containing a higher population of astrocytes DL-TBOA-induced increase in glutamate levels is more pronounced, but neuronal death is only triggered in the presence of 3-NP. Results suggest that cerebellar granule neurons are more vulnerable to acute transport-mediated glutamate release than to uptake blockade, which correlates with the extracellular excitatory amino acids levels.     

Immature cortical neurons are uniquely sensitive to glutamate toxicity by inhibition of cystine uptake

Using the N18-RE-105 neuroblastoma X retina cell line, we previously described Ca2(+)-dependent quisqualate-type glutamate toxicity caused by the inhibition of high-affinity cystine uptake, leading to glutathione depletion and accumulation of cellular oxidants. We now demonstrate that primary cultures of rat cortical neurons (E17; 24-72 h in culture), but not glia, also degenerate when exposed to culture medium with reduced cystine or containing competitive inhibitors of cystine uptake, including glutamate. At this developmental stage, neurotoxicity did not occur as a consequence of continuous exposure to glutamate receptor subtype agonists, N-methyl-D-aspartate, kainate, or 2(RS)-amino-3-hydroxy-5-methyl-4-isoxazolepropionate. However, those that inhibited neuronal cystine uptake--quisqualate, glutamate, homocysteate, beta-N-oxalyl-L-alpha,beta-diaminopropionic acid, and ibotenate--were neurotoxic. Toxicity related to quisqualate did not correlate with the development of quisqualate-stimulated phosphatidylinositol turnover. The toxic potencies of glutamate, quisqualate, and homocysteate were inversely proportional to the concentration of cystine in the medium, suggesting that they competitively inhibit cystine uptake. Autoradiographic analysis of the cellular localization of L-[35S]cystine uptake indicated that embryonic neurons have a high-affinity transport system that is sensitive to quisqualate, whereas non-neuronal cells in the same cultures have a low-affinity system that is insensitive to quisqualate but potently blocked by D-aspartate and glutamate. Exposure to glutamate or homocysteate resulted in a time-dependent depletion of the cellular antioxidant glutathione. The centrally acting antioxidant idebenone and alpha-tocopherol completely blocked the neurotoxicity resulting from glutamate exposure. We propose that competitive inhibition of cystine transport and reduction of extracellular cystine levels result in neuronal cell death due to accumulation of cellular oxidants.