自由基介导兴奋性氨基酸对培养皮层神经细胞毒性的研究

本文在体外对新生大鼠（０～１ｄ）大脑皮层神经细胞进行原代培养的基础上，建立谷氨酸（Ｇｌｕ）对培养的皮层神经细胞兴奋毒损伤模型。通过检测乳酸脱氢酶释出率和形态学观察，证实了Ｇｌｕ对神经细胞存在兴奋毒作用。通过测定不同条件下Ｇｌｕ兴奋毒对培养的神经细胞造成损伤时细胞膜脂质过氧化物（ＬＰＯ）浓度，发现膜ＬＰＯ浓度与Ｇｌｕ剂量及作用时间紧密相关，表明Ｇｌｕ对神经细胞兴奋毒作用有自由基产生并介导了神经细胞的损伤。     

Pharmacology of glutamate neurotoxicity in cortical cell culture: attenuation by NMDA antagonists

The antagonist pharmacology of glutamate neurotoxicity was quantitatively examined in murine cortical cell cultures. Addition of 1-3 mM DL-2-amino-5-phosphonovalerate (APV), or its active isomer D-APV, acutely to the exposure solution selectively blocked the neuroexcitation and neuronal cell selectively blocked the neuroexcitation and neuronal cell loss produced by N-methyl-D-aspartate (NMDA), with relatively little effect on that produced by either kainate or quisqualate. As expected, this selective NMDA receptor blockade only partially reduced the neuroexcitation or acute neuronal swelling produced by the broad-spectrum agonist glutamate; surprisingly, however, this blockade was sufficient to reduce glutamate-induced neuronal cell loss markedly. Lower concentrations of APV or D-APV had much less protective effect, suggesting that the blockade of a large number of NMDA receptors was required to acutely antagonize glutamate neurotoxicity. This requirement may be caused by the amplification of small amounts of acute glutamate-induced injury by subsequent release of endogenous NMDA agonists from injured neurons, as the "late" addition of 10-1000 microM APV or D-APV (after termination of glutamate exposure) also reduced resultant neuronal damage. If APV or D-APV were present both during and after glutamate exposure, a summation dose-protection relationship was obtained, showing substantial protective efficacy at low micromolar antagonist concentrations. Screening of several other excitatory amino acid antagonists confirmed that the ability to antagonize glutamate neurotoxicity might correlate with ability to block NMDA-induced neuroexcitation: The reported NMDA antagonists ketamine and DL-2-amino-7-phosphono-heptanoate, as well as the broad-spectrum antagonist kynurenate, were all found to attenuate glutamate neurotoxicity substantially; whereas gamma-D-glutamylaminomethyl sulfonate and L-glutamate diethyl ester, compounds reported to block predominantly quisqualate or kainate receptors, did not affect glutamate neurotoxicity. The present study suggests that glutamate neurotoxicity may be predominantly mediated by the activation of the NMDA subclass of glutamate receptors--occurring both directly, during exposure to exogenous compound, and indirectly, due to the subsequent release of endogenous NMDA agonists. Given other studies linking NMDA receptors to channels with unusually high calcium permeability, this suggestion is consistent with previous data showing that glutamate neurotoxicity depends heavily on extracellular calcium.     

Direct observation of the agonist-specific regional vulnerability to glutamate, NMDA, and kainate neurotoxicity in organotypic hippocampal cultures.

Excessive activation of excitatory amino acid receptors has been implicated in the neuronal degeneration caused by ischemia, hypoglycemia, and prolonged seizures. We have observed directly the time course and regional vulnerability of hippocampal neurons to glutamate receptor-mediated injury in organotypic hippocampal cultures, a preparation which combines accessibility and long-term survival with preservation of regional differentiation and neuroanatomic organization. Cultures were incubated with the fluorescent dye propidium iodide which selectively enters and stains cells only after membrane damage. After 5 to 10 min of a 30-min exposure to kainate (100 microM), large neurons in the hilus of the dentate were first to become brightly fluorescent. Propidium staining subsequently appeared in the other regions of the hippocampus and increased to a maximum over the first 6 h of recovery. NMDA (10 microM) caused propidium staining that was limited to CA1 and the dentate gyrus of the cultures, sparing CA3, consistent with the regions of highest NMDA receptor density in vivo. Glutamate (1 mM) caused a delayed, progressive pattern of staining that began in CA1 (2 to 4 h after exposure), then extended to include CA3 and finally the dentate gyrus over the next 24 h. Release of LDH activity into the media was slower and less sensitive than propidium staining. Histologic degeneration was limited to neurons 24 h after agonist exposure and was consistent with the propidium staining. NMDA, kainate, and glutamate each produced a unique pattern of neuronal injury. Most notably, glutamate had low potency as a toxin and its pattern of neuronal injury was not reproduced by NMDA.     

Cholecystokinin-induced protection of cultured cortical neurons against glutamate neurotoxicity

The effects of cholecystokinin (CCK) on glutamate-induced neurotoxicity were examined using cultured rat cortical neurons. Brief exposure of glutamate followed by an incubation with normal solution for more than 60 min reduced cell viability by 60–70%, compared with control values. Glutamate-induced neurotoxicity was significantly inhibited by MK-801 and ketamine, which are non-competitive blockers of N-methyl-d-aspartate (NMDA) receptors. Octapeptide CCK-8S and CCK-related decapeptide ceruletide at concentrations of 10⁻⁹−10⁻⁷ M dose-dependently reduced glutamate-induced neurotoxicity. A desulfated analog CCK-8NS, which acts selectively as an antagonist of CCK_B receptors, also reduced glutamate neurotoxicity. The neuroprotective effects of CCK were antagonized by L-365260, a CCK_B receptor antagonist, but not by L-364718, a CCK_A receptor antagonist. These results suggest that CCK protects cortical neurons against NMDA receptor-mediated glutamate neurotoxicity via CCK_B receptors.     

Characterization and mechanism of glutamate neurotoxicity in primary striatal cultures

Excitatory amino acids may play a role in the pathogenesis of cell death in neurodegenerative diseases, including Huntington's disease (HD). In an attempt to develop a tissue culture model for HD, the toxicity of glutamate was examined in primary striatal cultures derived from newborn rats. Morphological criteria were used to determine the toxic effects of glutamate in 6-, 12-, and 18-day-old cultures which were examined before and after 1-3 h of exposure to glutamate. Although younger cultures demonstrated little susceptibility to glutamate relative to controls, the number of neurons in older cultures was significantly depleted in the presence of glutamate. Glutamate toxicity was dose-dependent, with an ED50 of approximately 300 microns glutamate, and a maximal effect was observed within 3 h of initial exposure. Affected neurons demonstrated somal swelling within 1 h of glutamate exposure and disruption of neuritic processes and somal integrity within 3 h. Cell death was significantly increased by raising the extracellular calcium concentration and could be decreased by the addition of magnesium to the incubation medium. Moreover, the N-methyl-D-aspartate (NMDA) receptor agonist, quinolinic acid, showed a toxicity profile similar to that of glutamate. The NMDA receptor competitive antagonist, 2-amino-5-phosphonovalerate (APV) significantly reduced toxicity, albeit incompletely. An additional component of glutamate mediated toxicity in striatal cultures could be explained by activation of non-NMDA receptor subtypes. These in vitro studies indicate that glutamate is toxic to a subset of mature striatal neurons in the absence of a glutamatergic afferent input, and that this toxicity is mediated partially by the NMDA receptor, with an additional component due to non-NMDA receptors.     

TNF alpha potentiates glutamate neurotoxicity by inhibiting glutamate uptake in organotypic brain slice cultures: neuroprotection by NF kappa B inhibition

Glutamate and the proinflammatory cytokine, tumor necrosis factor alpha (TNF alpha), have been suggested to contribute to neurodegenerative diseases. We investigated the interaction of TNF alpha and glutamate on neuronal cell death using fluorescence propidium iodide uptake in rat organotypic hippocampal-entorhinal cortex (HEC) brain slice culture that maintains the cytoarchitecture of the intact brain. Time course and concentration studies indicate that glutamate produced significant neuronal cell death in all four brain areas examined, for example, entorhinal cortex, hippocampal CA1 and CA3 fields, and dentate gyrus. TNF alpha alone at concentration of 20 ng/ml caused little or no detectable neuronal cell death, however, when combined with submaximal glutamate (3.3 mM), TNF alpha significantly increased and accelerated glutamate neurotoxicity. TNF alpha potentiation of glutamate neurotoxicity is blocked by NMDA receptor antagonists but not by AMPA antagonists CNQX and NBQX. Studies directly measuring [14C]-glutamate uptake in HEC slices indicate that TNF alpha dose-dependently inhibited glutamate uptake. Further, inhibitors of glial glutamate transporters potentiated glutamate neurotoxicity similar to TNF alpha. The antioxidant butylated hydroxytoluene (BHT) and the NF kappa B inhibitor PTD-p65 peptide inhibit NF kappa B activation and TNF alpha potentiation of glutamate neurotoxicity. BHT prevented the inhibition of TNFalpha on glutamate transport in HEC slices and also blocked nuclear translocation of NF kappa B subunit p65. These data indicate that TNF alpha and glutamate can act synergistically to induce neuronal cell death. TNF alpha potentiation of glutamate neurotoxicity through the blockade of glutamate transporter activity may represent an important mechanism of neurodegeneration associated with neuroinflammation.     

Glial activation modulates glutamate neurotoxicity in cerebellar granule cell cultures

We studied the influence of glial cells on the neuronal response to glutamate toxicity in cerebellar granule cell cultures. We compared the effect of glutamate on neuronal viability in neuronal vs. neuronal-glial cultures and determined this effect after pretreating the cultures with the lipopolysaccharide (LPS) of Escherichia coli, agent widely used to induce glial activation. Morphological changes in glial cells and nitric oxide (NO) production were evaluated as indicators of glial activation. We observed that glutamate neurotoxicity in neuronal-glial cultures was attenuated in a certain range of glutamate concentration when compared to neuronal cultures, but it was enhanced at higher glutamate concentrations. This enhanced neurotoxicity was associated with morphological changes in astrocytes and microglial cells in the absence of NO production. LPS treatment induced morphological changes in glial cells in neuronal-glial cultures as well as NO production. These effects occurred in the absence of significant neuronal death. However, when LPS-pretreated cultures were treated with glutamate, the sensitivity of neuronal-glial cultures to glutamate neurotoxicity was increased. This was accompanied by additional morphological changes in glial cells in the absence of a further increase in NO production. These results suggest that quiescent glial cells protect neuronal cells from glutamate neurotoxicity, but reactive glial cells increase glutamate neurotoxicity. Therefore, glial cells play a key role in the neuronal response to a negative stimulus, suggesting that this response can be modified through an action on glial cells.     

Effect of anticonvulsant drugs on glutamate neurotoxicity in cortical cell culture

Pretreatment with anticonvulsants partially protects animals against the brain damage induced by intraparenchymal injection of kainate, an analogue of the neurotransmitter glutamate. In murine cortical cell culture, high concentrations of phenobarbital, diazepam, phenytoin, or GABA itself did not prevent glutamate-induced neuronal loss. Addition of a glutamate receptor antagonist (gamma-D-glutamyl glycine) did reduce glutamate neurotoxicity. The in vivo protective effect of anticonvulsant drugs against the toxicity of excitatory amino acids must be indirect.     

Dual effect of glycine on NMDA-induced neurotoxicity in rat cortical cultures

To examine the roles of glycine in neurotoxicity caused by NMDA, primary rat cortical cultures were exposed to 100-300 microM NMDA plus glycine (0-3000 microM) or other glycine analogs in a simple saline solution, and toxicity was assessed by the amount of lactate dehydrogenase (LDH) released from the cultures. NMDA-induced neurotoxicity was abolished by 100 microM D-2-amino-5-phosphonovaleric acid (D-APV), phencyclidine (IC50, 4.1 microM), and Mg (IC50, 7.5 mM), or by reducing [Ca]0 to 0.1 mM. NMDA-induced neurotoxicity could also be abolished by 7-chlorokynurenic acid (IC50, 8.6 microM), suggesting the presence of residual glycine in the culture medium (confirmed by high-performance liquid chromatography measurement). Moreover, in the presence of 30 microM 7-chlorokynurenic acid, glycine, D-serine, D-alanine, beta-fluoro-D-alanine, and 1-aminocyclopropanecarboxylic acid could restore the neurotoxic action of NMDA, and their relative potencies and relative efficacies were the same as measured in electrophysiological assays in Xenopus oocytes or cultured neurons. The addition of greater than 100 microM glycine doubled the excitotoxic effect of NMDA. The potency of glycine was low (EC50, 27 microM), and this effect was not due to a direct action on the NMDA receptor. The above-mentioned agonists were unable to substitute for glycine, even at high concentrations (1 mM). On the other hand, beta-alanine, taurine, and GABA (1 mM) did potentiate NMDA neurotoxicity, and strychnine (IC50, 550 nM) could greatly reduce neurotoxicity in the presence of 1 mM glycine plus 300 microM NMDA.(ABSTRACT TRUNCATED AT 250 WORDS)     

Extracellular pH modulates N-methyl-D-aspartate receptor-mediated neurotoxicity and calcium accumulation in rat cortical cultures.

The effect of extracellular pH (pHo) on the excitotoxicity of N-methyl-D-aspartate (NMDA) in cultured rat cortical cells was studied. Treatment of cells with 500 microM NMDA for 15 min at various pH's in a range from 6.5 to 8.0 progressively enhanced staining with Trypan blue and release of lactate dehydrogenase with increased pH after 18 h of culture following treatment. The cytotoxic effect of high concentration of K+ (40 mM) or veratridine (10 microM) was also directly related to the increase in pHo. Free calcium accumulation in cells on addition of NMDA increased parallel to pHo. Changes in intracellular pH were estimated to be minor compared with extracellular changes. Specific NMDA antagonists could block both the NMDA- and membrane depolarization-induced neurotoxicity and calcium accumulation completely. These results suggest that the proton concentration outside of cells attenuates NMDA-induced neurotoxicity by blocking calcium accumulation.     

Ruthenium red neurotoxicity and interaction with gangliosides in primary cortical cultures

Ruthenium red (RR) is an inorganic polycationic dye able to exert several effects on the nervous system, including neurodegeneration, both in vivo and in cell cultures. Gangliosides have been shown to protect cultured neurons against several damaging conditions, and it has been postulated that RR can interact with the negative charges of the sialic acid residues of these molecules. In the present work we have tested the effect of the trisialoganglioside GT1b and the monosialoganglioside GM1 on the RR-induced neuronal damage in primary cortical cultures, as well as on the binding of RR to synaptosomes. GT1b at 100–200 μM concentrations partially protected against RR-induced neurodegeneration, as judged by light microscopy and by measurement of the reduction of a tetrazolium salt, while GM1 was ineffective. GT1b, but not GM1, also partly blocked both RR binding and its diminution in the culture medium occurring during incubation. These results suggest that the three negative charges of GT1b enable it to interact with RR and as a consequence the entrance of the dye into the cells is blocked and neurotoxicity is diminished, although other mechanisms of protection cannot be excluded. Endogenous polysialic acid–containing molecules do not seem to be involved in RR effects, since the removal of sialic acid residues by treatment with neuraminidase did not prevent the cell damage. J. Neurosci. Res. 49:72–79, 1997. © 1997 Wiley-Liss Inc.     

Iridoids from Scrophularia buergeriana attenuate glutamate-induced neurotoxicity in rat cortical cultures

In previous work, we isolated 7 neuroprotective iridoid glycosides from the 90% MeOH fraction of Scrophularia buergeriana (Scrophulariaceae). We therefore investigated the mode of action of 8-O-E-p-methoxycinnamoyl-harpagide (8-MCA-Harp), the most potent neuroprotective iridoid, and its aglycone, harpagide (Harp) using primary cultures of rat cortical cells in vitro. 8-MCA-Harp only revealed its neuroprotective activity in a pretreatment paradigm; this iridoid had more selectivity in protecting neurons against N-methyl-D-aspartate (NMDA)-induced neurotoxicity as opposed to that induced by kainic acid (KA). On the other hand, Harp exerted significant neuroprotective activity when it was administered either before or after glutamate insult and protected cultured neuronal cells from neurotoxicity induced by NMDA or KA. Furthermore, Harp significantly prevented the decrease of glutathione, an antioxidative compound in the brain, in our cultures. Finally, 8-MCA-Harp and Harp could successfully reduce the overproduction of nitric oxide and the level of cellular peroxide in cultured neurons. Collectively, these results suggested that Harp and 8-MCA-Harp protected primary cultured neurons against glutamate-induced oxidative stress primarily by acting on the antioxidative defense system and on glutamatergic receptors, respectively.     

Gypenosides protect primary cultures of rat cortical cells against oxidative neurotoxicity

Gypenosides (GPs) were tested for their ability to protect primary cultures of immature cortical cells against oxidative glutamate toxicity. In immature neural cells, glutamate cytotoxicity is known to be mediated by the inhibition of cystine uptake, leading to depletion of intracellular glutathione (GSH). The depletion of GSH impairs cellular antioxidant defenses resulting in oxidative stress and cell death. We found that pretreatment with GPs (100-400 microg/ml) significantly protected cells from glutamate-induced cell death. It was therefore of interest to investigate whether GPs protect cortical cells against glutamate-induced oxidative injury through preventing GSH depletion. Results show that GPs significantly up-regulated mRNAs encoding gamma-glutamylcysteine synthetase (gamma-GCS) and glutathione reductase (GR) and enhanced their activities for GSH synthesis as well as recycle. Furthermore, GPs lowered the consumption of GSH through decreased accumulation of intracellular peroxides, leading to an increase in the intracellular GSH content. GPs were also found to prevent lipid peroxidation and reduce the influx of Ca(2+) which routinely follows glutamate oxidative challenge. GPs treatment significantly blocked glutamate-induced decrease in levels of Bcl-2 and increase in Bax, leading to a decrease in glutamate-induced apoptosis. Thus, we conclude that GPs protect cortical cells by multiple antioxidative actions via enhancing intracellular GSH, suppressing glutamate-induced cytosolic Ca(2+) elevation and blocking glutamate-induced apoptosis. The novel role of GPs implies their remarkable preventative and therapeutic potential in treatment of neurological diseases involving glutamate and oxidative stress.     

Involvement of platelet-activating factor (PAF) in glutamate neurotoxicity in rat neuronal cultures

To clarify the role of platelet-activating factor (PAF) in glutamate neurotoxicity, in vitro experiments using primary neuronal cultures were performed. The anti-PAF immunoglobulin-G (aPAF-IgG) and the three PAF receptor antagonists (BN52021, CV6209, and E5880) were tested for their neuroprotective activity in primary neuronal cultures isolated from embryonic rat cerebral cortex. The cultured neurons were exposed to glutamate (1 mM) for 60 min. Twenty-four hours after this exposure, aPAF-IgG demonstrated evidence of protective effects against neuronal damage in a dose-dependent manner. Protective effects also were observed in cultures treated with the three PAF antagonists (P<0.05 at 1 μg/ml aPAF-IgG, P<0.01 at 100 μM BN52021, P<0.05 at 10 nM CV6209 and P<0.01 at 10 nM E5880). The Fura-2 assay was used to estimate whether low dosages of exogenous PAF affect cultured neurons. The cultured neurons were loaded with Fura-2/AM. After preincubation for 120 min, the Fura-2-loaded neurons were exposed to various concentrations of PAF for 60 min. By measuring the fluorescent intensity of the medium as representing the amount of Fura-2 released from damaged neurons, we detected an increased release of Fura-2, even at low doses of PAF (P<0.01 at 10 nM PAF). We further studied PAF production by neurons in response to glutamate. The level of PAF measured in the medium exposed to glutamate was significantly higher than the level in the medium unexposed to glutamate (P<0.05). Our results suggest an important role of PAF in glutamate neurotoxicity. © 1997 Elsevier Science B.V. All rights reserved.     

Accumulation of extracellular glutamate and neuronal death in astrocyte-poor cortical cultures exposed to glutamine.

The function of astrocytes in cerebral cortex may be studied by comparing the properties of conventional, astrocyte-rich cultures with astrocyte-poor cultures in which astrocyte proliferation has been stringently suppressed. Exposure of astrocyte-poor, but not astrocyte-rich, cultures to fresh medium containing 2 mM glutamine resulted in the death of most neurons within 24 h. This study was undertaken to understand the basis for the apparent toxicity of glutamine in astrocyte-poor cultures. The toxicity of glutamine was found to be mediated by glutamate, which demonstrated an LD50 as a neurotoxin in astrocyte-poor cultures of 2 microM. Exposure of astrocyte-poor (but not astrocyte-rich) cultures to fresh medium containing glutamine for 17.5-24 h resulted in the accumulation of substantial quantities of glutamate (255 +/- 158 microM; mean +/- standard deviation) coincident with the death of neurons in the cultures. Exposure of astrocyte-poor cultures to glutamate in the absence of glutamine did not result in the accumulation of extracellular glutamate. Both the neuronal death and the extracellular glutamate accumulation in astrocyte-poor cultures exposed to glutamine could be blocked by N-methyl-D-aspartate (NMDA) antagonists. These observations suggest that astrocytes as well as glutamine may play an important role in the pathogenesis of glutamate neurotoxicity in the central nervous system.     

Ascorbate neurotoxicity in cortical cell culture.

Ascorbate (vitamin C) is believed to act as a neuromodulator that facilitates the release of neurotransmitters and inhibits neurotransmitter binding to receptors, including dopamine and N-methyl-D-aspartate receptors. Extracellular levels of ascorbate are known to reach the low millimolar range after ischemic brain injury. This study shows that treatment of cultured cortical neurons with micromolar to low millimolar ascorbate first inhibits total protein synthesis and then results in late neuronal death. Astrocytes are much less vulnerable to ascorbate than neurons. Ascorbate may exacerbate neuronal and glial damage after brain ischemia, and it may play a pathological role in other neurological diseases.     

3-Nitropropionic acid neurotoxicity in hippocampal slice cultures: developmental and regional vulnerability and dependency on glucose

We investigated whether neurotoxic effects of the mitochondrial toxin 3-nitropropionic acid (3-NP) in hippocampal slice cultures are dependent on glucose levels in the culture medium and whether such effects occur via apoptosis or necrosis. In addition, 3-NP toxicity was investigated at two developmental stages of the cultures, prepared from rat brain at postnatal day 5-7 and grown in Neurobasal medium for 1 or 3 weeks. Cultures were exposed to 3-NP in the presence of high (25 mM), normal (5 mM), or low (3 mM) glucose for 48 h, followed by 48 h incubation in medium without 3-NP. Cellular propidium iodide (PI) uptake and lactate dehydrogenase (LDH) efflux into the medium revealed time- and dose-dependent cell death by 3-NP, with EC(50) values of about 60 microM in high or normal glucose. Regional vulnerability, as assessed by PI uptake and MAP2 immunostaining, in 3-week-old cultures was as follows: CA1 > CA3 > fascia dentata. In low glucose much lower concentrations of 3-NP (25 microM) triggered neurotoxicity. One-week-old cultures were less susceptible to 3-NP toxicity than 3-week-old cultures, but the dentate granule cells were relatively more affected in the immature cultures. We found no evidence for apoptotic cell death by 3-NP in 3-week-old cultures, but in 1-week-old cultures the putative apoptotic marker c-JUN/AP1 and nuclear fragmentation (Hoechst) were significantly increased in the dentate granule cells.     

Modulation of glutamate neurotoxicity on mesencephalic dopaminergic neurons in primary cultures by the presence of striatal target cells

Glutamate toxicity was compared in substantia nigra (SN)/striatum (STR) and SN/cerebellum (CRB) co-cultures on both the entire neuronal population (neuron specific enolase (NSE) immunopositive cells) and dopaminergic neurons (tyrosine hydroxylase (TH) immunopositive cells). In SN/CRB co-cultures NSE- and TH-positive cells were more sensitive to glutamate-induced toxicity than in SN/STR co-cultures. Moreover, in SN/STR co-cultures as compared to SN/CRB and SN cultures, glutamate toxicity was prevented to a larger extent by TCP, a non-competitive NMDA antagonist. These results suggest that target cells induce a differential expression of the different glutamate receptor subtypes in mesencephalic dopaminergic cells. Alternatively, the presence of target cells may induce the selective development of a subpopulation of dopaminergic neurons expressing predominantly NMDA receptors.     

Involvement of enhanced sensitivity of N-methyl-D-aspartate receptors in vulnerability of developing cortical neurons to methylmercury neurotoxicity

The developing cortical neurons have been well documented to be extremely vulnerable to the toxic effect of methylmercury (MeHg). In the present study, a possible involvement of N-methyl-D-aspartate (NMDA) receptors in MeHg neurotoxicity was examined because the sensitivity of cortical neurons to NMDA neurotoxicity has a similar developmental profile. Rats on postnatal day 2 (P2), P16, and P60 were orally administered MeHg (10 mg/kg) for 7 consecutive days. The most severe neuronal damage was observed in the occipital cortex of P16 rats. When MK-801 (0.1 mg/kg), a non-competitive antagonist of NMDA, was administered intraperitoneally with MeHg, MeHg-induced neurodegeneration was markedly ameliorated. Furthermore, there was a marked accumulation of nitrotyrosine, a reaction product of peroxynitrite and L-tyrosine, after chronic treatment of MeHg in the occipital cortex of P16 rats. The accumulation of nitrotyrosine was also significantly suppressed by MK-801. In the present electrophysiological study, the amplitude of synaptic responses mediated by NMDA receptors recorded in cortical neurons of P16 rats was significantly larger than those from P2 and P60 rats. These observations strongly suggest that a generation of peroxynitrite through activation of NMDA receptors is a major causal factor for MeHg neurotoxicity in the developing cortical neurons. Furthermore, enhanced sensitivity of NMDA receptors may make the cortical neurons of P16 rats most susceptible to MeHg neurotoxicity.