Neurotoxicity In Primary Cultures Research Articles

Extracellular tau induces microglial phagocytosis of living neurons in cell cultures.

Tau is a microtubule-associated protein, found at high levels in neurons, and itsaggregation is associated with neurodegeneration. Recently, it was found that tau can be actively secreted from neurons, but the effects of extracellular tau on neuronal viability are unclear. In this study, we investigated whether extracellular tau2N4R can cause neurotoxicity in primary cultures of rat brain neurons and glial cells. Cell cultures were examined for neuronal loss, death, and phosphatidylserine exposure, as well as for microglial phagocytosis by fluorescence microscopy. Aggregation of tau2N4R was assessed by atomic force microscopy. We found that extracellular addition of tau induced a gradual loss of neurons over 1-2days, without neuronal necrosis or apoptosis, but accompanied by proliferation of microglia in the neuronal-glial co-cultures. Tau addition caused exposure of the 'eat-me' signal phosphatidylserine on the surface of living neurons, and this was prevented by elimination of the microglia or by inhibition of neutral sphingomyelinase. Tau also increased the phagocytic activity of pure microglia, and this was blocked by inhibitors of neutral sphingomyelinase or protein kinase C. The neuronal loss induced by tau was prevented by inhibitors of neutral sphingomyelinase, protein kinase C or the phagocytic receptor MerTK, or by eliminating microglia from the cultures. The data suggest that extracellular tau induces primary phagocytosis of stressed neurons by activated microglia, and identifies multiple ways in which the neuronal loss induced by tau can be prevented.

Deletion of the Na/HCO3 Transporter NBCn1 Protects Hippocampal Neurons from NMDA-induced Seizures and Neurotoxicity in Mice

The Na/HCO3 cotransporter NBCn1/SLC4A7 can affect glutamate neurotoxicity in primary cultures of rat hippocampal neurons. Here, we examined NMDA-induced neurotoxicity in NBCn1 knockout mice to determine whether a similar effect also occurs in the mouse brain. In primary cultures of hippocampal neurons from knockouts, NMDA had no neurotoxic effects, determined by lactate dehydrogenase release and nitric oxide synthase (NOS)-dependent cGMP production. Male knockouts and wildtypes (6–8 weeks old) were then injected with NMDA (75 mg/kg; ip) and hippocampal neuronal damages were assessed. Wildtypes developed severe tonic-clonic seizures, whereas knockouts had mild seizure activity (motionless). In knockouts, the NOS activity, caspase-3 expression/activity and the number of TUNEL-positive cells were significantly low. Immunochemical analysis revealed decreased expression levels of the NMDA receptor subunit GluN1 and the postsynaptic density protein PSD-95 in knockouts. Extracellular recording from hippocampal slices showed no Mg2+/NMDA-mediated epileptiform events in knockouts. In conclusion, these results show a decrease in NMDA neurotoxicity by NBCn1 deletion. Given that acid extrusion has been known to prevent pH decrease and protect neurons from acid-induced damage, our study presents novel evidence that acid extrusion by NBCn1 stimulates neurotoxicity.

Combined use of CDK4/6 and mTOR inhibitors induce synergistic growth arrest of diffuse intrinsic pontine glioma cells via mutual downregulation of mTORC1 activity.

BackgroundDiffuse intrinsic pontine glioma (DIPG) is a lethal type of pediatric brain tumor that is resistant to conventional chemotherapies. Palbociclib is a putative novel DIPG treatment that restricts the proliferation of rapidly dividing cancer cells via selective inhibition of cyclin-dependent kinase (CDK) 4 and CDK6. However, implementing palbociclib as a monotherapy for DIPG is unfeasible, as CDK4/6 inhibitor resistance is commonplace and palbociclib does not readily cross the blood–brain barrier (BBB) or persist in the central nervous system. To inhibit the growth of DIPG cells, we aimed to use palbociclib in combination with the rapamycin analog temsirolimus, which is known to ameliorate resistance to CDK4/6 inhibitors and inhibit BBB efflux.Materials and methodsWe tested palbociclib and temsirolimus in three patient-derived DIPG cell lines. The expression profiles of key proteins in the CDK4/6 and mammalian target of rapamycin (mTOR) signaling pathways were assessed, respectively, to determine feasibility against DIPG. Moreover, we investigated effects on cell viability and examined in vivo drug toxicity.ResultsImmunoblot analyses revealed palbociclib and temsirolimus inhibited CDK4/6 and mTOR signaling through canonical perturbation of phosphorylation of the retinoblastoma (RB) and mTOR proteins, respectively; however, we observed noncanonical downregulation of mTOR by palbociclib. We demonstrated that palbociclib and temsirolimus inhibited cell proliferation in all three DIPG cell lines, acting synergistically in combination to further restrict cell growth. Flow cytometric analyses revealed both drugs caused G1 cell cycle arrest, and clonogenic assays showed irreversible effects on cell proliferation. Palbociclib did not elicit neurotoxicity in primary cultures of normal rat hippocampi or when infused into rat brains.ConclusionThese data illustrate the in vitro antiproliferative effects of CDK4/6 and mTOR inhibitors in DIPG cells. Direct infusion of palbociclib into the brain, in combination with systemic delivery of temsirolimus, represents a promising new approach to developing a much-needed treatment for DIPG.

Expression of cell cycle proteins in cortical neurons-Correlation with glutamate-induced neurotoxicity.

Under physiological conditions, upon differentiation neurons become irreversibly post-mitotic by down-regulating cell cycle progression. However, recent studies have provided evidence that aberrant expression of cell cycle related proteins; especially cyclins, cyclin-dependent kinases, and their inhibitors are accompanied by programmed cell death in neurons. This abnormal phenotype has been postulated to contribute to the pathophysiology of different neurodegenerative diseases. Glutamate is the most abundant and major excitatory neurotransmitter in the central nervous system but high concentrations are reported to be involved in the pathology of many neurodegenerative diseases. The mechanisms of glutamate neurotoxicity have been intensively investigated over the past decades but still remain not fully understood. In this study, we hypothesized that aberrant regulation of cell cycle proteins may be involved in glutamate-induced neurotoxicity in primary cultures of rat cortical neurons. The results have shown that, glutamate treatment caused apoptosis by inducing active caspase-3 and p53 expression. Together with this, an increase in cyclin D1 and Cdk4 protein levels, localization of cyclin D1 to nucleus, and a decrease in the cell cycle inhibitor p27 were observed. After glutamate treatment we also detected up-regulation of protein kinase C-α (PKC-α) protein expression. Altogether, the data reported in this study show for the first time that glutamate in cortical neurons changes simultaneously the expression levels of a number of key cell cycle proteins and cell homeostasis regulators. © 2016 BioFactors, 42(4):358-367, 2016.

GET73 Prevents Ethanol-Induced Neurotoxicity in Primary Cultures of Rat Hippocampal Neurons.

N-[(4-trifluoromethyl) benzyl] 4-methoxybutyramide (GET73) may be considered a promising therapeutic agent for the treatment of alcohol use disorders. The compound displayed anti-alcohol and anxiolytic properties in rat. In the present study, an in vitro experimental model of chronic ethanol treatment was used to investigate the ability of the compound to counteract the ethanol-induced neurotoxicity. Primary cultures of rat hippocampal neurons were exposed to ethanol (75 mM; 4 days) and the neuroprotective effects of GET73 were assessed by evaluating cell viability, cell morphology, glutamate levels and reactive oxygen species production. The exposure to ethanol induced a reduction of cell viability, an alteration of cytoskeleton, a decrease in extracellular glutamate levels and an increase of reactive oxygen species production. The addiction of GET73 (1 and 10 µM) 1 h before and during chronic ethanol exposure prevented all the above ethanol-induced effects. Based on the proposed GET73 mechanism of action, the effects of mGlu5 receptor negative allosteric modulator, 2-methyl-6-(phenylethynyl)-pyridine (MPEP), on ethanol-induced reduction of cell viability were also assessed. The results indicated that the addiction of MPEP (100 µM) 1 h before and during chronic ethanol exposure prevented the ethanol-induced cell viability reduction. The present findings provide the first evidence that GET73 shows a neuroprotective role against ethanol-induced neurotoxicity in primary cultures of rat hippocampal neurons. Together with previous findings, these results suggest that GET73 possesses multifaceted properties thus lending further support to the significance of developing GET73 as a therapeutic tool for use in the treatment of alcohol use disorders.

The role of the glutamatergic NMDA receptor in nanosilver-evoked neurotoxicity in primary cultures of cerebellar granule cells

Nanoparticles are known to enter the vertebrate brain, but little is known about their neurotoxicity. The aim of this study is to investigate mechanisms of the contribution of AgNPs to neuronal cell death using primary cultures of rat cerebellar granule cells (CGCs). We tested the role of glutamatergic N-methyl-d-aspartate receptors (NMDA) in AgNP-evoked neurotoxicity using MK-801, a noncompetitive inhibitor of NMDAR. We used commercially available 0.2% PVP-coated AgNPs <100nm in a concentration range of 2.5–75μg/ml sonicated with fetal calf serum. After a 10min incubation period, a dose-dependent increase in the uptake of 45Ca2+ into neurons was observed in the presence of 25–75μg/mL AgNPs which was completely abolished by addition of MK-801. Using the fluorescent dye fluo3 AM we observed an increase in the intracellular calcium level by 87% compared to control. ROS production was found to increase by about 30% over control after a 30-min incubation with 75μg/mL AgNPs. Further, we observed a significant decrease in the mitochondrial potential during a 30-min incubation with AgNPs. Administration of MK-801 was found to provide a protective effect. Our results show that excitotoxicity via activation of NMDA receptor, followed by calcium imbalance, destabilization of mitochondrial function and ROS production, indicate an important mechanism involved in neurotoxicity evoked by AgNPs in cultured neurons.

Validated LC-ESI-MS/MS Method for the Quantitation of Neopanaxadiol: a Novel Neuroprotective Agent from Panax ginseng and Its Application to a Pharmacokinetic Study in Rat Plasma

Neopanaxadiol (NPD), a new panaxadiol first obtained from the acid hydrolysate of the total ginsenosides of Panax ginseng C. A. Meyer (Araliaceae) in our laboratory, showed neuroprotective effect against glutamate-induced neurotoxicity in primary cultures of rat cortical cells (Tao et al. Chin Chem Lett 20:687-689, 2009 ). In this research, a sensitive and specific method of ultra-performance liquid chromatography-electrospray mass spectrometry (UPLC-ESI-MS/MS) for the quantitative analysis of NPD in rat plasma was developed. The NPD and internal standard of panaxadiol were extracted from 200 µL of rat plasma samples with methanol, then the samples were injected in an UPLC (Waters BEH) with C18 column (50 mm × 2.1 mm i.d., 1.7 µm) and eluted with the mobile phase consisting of methanol-water-formic acid (80:20:0.2, v/v/v). Excellent linearity was found between 5 and 160 ng mL–1 with a limit of quantitation of 4 ng mL–1. Intra- and inter-day precision values (RSD) of QC samples were both below 7 %. This study was successfully utilized for the pharmacokinetic profiles of NPD in rats after oral administration and provided an experimental basis for the further pharmacology and clinical application of ginseng products.

New neuroprotective dibenzylbutane lignans isolated from Machilus thunbergii

Two new dibenzylbutane lignans, namely rel-methyl (2S)-2-hydroxy-2-2′-((2″R,3″R)-4″-guaiacyl-2″,3″dimethylbutyl)-5′-oxo-2′,5′-dihydro-2-furanylethnoate (1) and rel-methyl (2S)-2-hydroxy-2-2′-((2″R,3″S)-4″-guaiacyl-2″,3″dimethylbutyl)-5′-oxo-2′,5′-dihydro-2-furanylethnoate (2), were isolated from the bark of Machilus thunbergii. Their structures were elucidated by spectral analysis. These two lignans exerted significant neuroprotective activities against glutamate-induced neurotoxicity in primary cultures of rat cortical cells. Compounds 1 and 2 represent a new type of lignan skeleton that has not been reported so far from nature.

Dammarane derivatives protect cultured rat cortical cells from glutamate-induced neurotoxicity.

We previously reported that ginsenosides Rb1 and Rg3, dammarane glycosides, of Panax ginseng C. A. Meyer (Araliaceae), significantly attenuated glutamate-induced neurotoxicity in primary cultures of rat cortical cells. To seek more potent neuroprotective compounds, we attempted to modify the chemical structure of dammarane glycosides and obtained six derivatives, MA-11, PT-11, PT-111, POA-101, POA-111 and N-001. The neuroprotective activity of these dammarane derivatives were evaluated employing primary cultures of rat corticoid cells. The glutamate-induced neuronal cell damage was significantly reduced by a pre-treatment with protopanaxadiol, MA-11 or PT-11 at concentrations ranging from 100 nM to 10 microM. Both MA-11 and PT-11, preserved the levels of catalase and inhibited decreases in glutathione reductase in glutamate-injured cells. Furthermore, the dammarane derivatives reduced the content of intracellular peroxide in glutamate-intoxicated cells. Finally, they inhibited the formation of malondialdehyde, a compound produced during lipid peroxidation, in glutamate-insulted cells. These results show that the dammarane derivatives, MA-11 and PT-11, exert significant neuroprotective effects on cultured cortical cells by a mechanism seemingly distinct from that afforded by ginsenosides Rb1 and Rg3. As such, the dammarane derivatives may be efficacious in protecting neurons from oxidative damage caused by exposure to excess glutamate.

Compounds with neuroprotective activity from the medicinal plant Machilus thunbergii

The dichloromethane fraction of the bark of Machilus thunbergii Sieb. et Zucc. (Lauraceae) significantly protected primary cultures of rat cortical cells exposed to the excitotoxic amino acid, L-glutamate. Through the activity-guided isolation from the CH2Cl2 fraction, (+)-9′-hydroxygalbelgin (1), isogalcatin B (2), (7S,8S,8′R)-3′,4′-dimethoxy-3,4,-methylenedioxylignan-7-ol (3), 1-hydroxy-7-hydroxymethyl-6-methoxyxanthone (4), 5,7-dimethoxy-3′,4′-methylenedioxyflavan-3-ol (5), (+)-(3S,4S,6R)-3,6-dihydroxypiperitone (6), protocatechuic acid methyl ester (7) and tyrosol (8) were obtained. All of them had significant neuroprotective activities against glutamate-induced neurotoxicity in primary cultures of rat cortical cells at concentrations ranging from 0.1 μM to 10.0 μM and were comparable to MK-801, a well-known inhibitor of glutamate receptor.

Achyranthes bidentata Blume extract protects cultured hippocampal neurons against glutamate-induced neurotoxicity

We have prepared an aqueous extract of Achyranthes bidentata Blume, a Chinese medicinal herb commonly prescribed for arthritis treatment or immnopotentiation, and have found that Achyranthes bidentata extract promotes nerve growth and prevents neuronal apoptosis. Aim of the study To investigate the protective effect of Achyranthes bidentata extract against glutamate-induced neurotoxicity in primary culture of rat hippocampal neurons. Materials and methods We accomplished MTT assay for cell viability, Hoechst 33342 staining, and flow cytometry for cell apoptosis analysis to examine the effects of Achyranthes bidentata extract on glutamate-induced neurotoxicity, and also used Fluo 4-AM measurement, RT-PCR and Western blot analysis to determine the changes in intracellular calcium concentration [Ca 2+] I, and mRNA and protein levels of Bcl-2, respectively, concurrently accompanied with the influences of Achyranthes bidentata extract. Results Achyranthes bidentata extract was found to inhibit glutamate-induced neuronal damage in a dose- and time-dependent manner. On the other hand, Achyranthes bidentata extract depressed glutamate-induced elevation of intracellular calcium concentration [Ca 2+] i , and also antagonized glutamate-evoked decreases in Bcl-2 expression at mRNA and protein levels. Conclusion The results suggest that Achyranthes bidentata extract prevents glutamate-induced cell damage in primarily cultured hippocampal neurons by inhibiting an increase in [Ca 2+] i , and reversing the down-regulation of Bcl-2.

Nicotinamide and 1-methylnicotinamide reduce homocysteine neurotoxicity in primary cultures of rat cerebellar granule cells.

Nicotinamide is an important cofactor in many metabolic pathways and a known neuroprotective substance, while its methylated product, 1-methylnicotinamide, is a suspected neurotoxin. Homocysteine is a risk factor in Alzheimer's disease and neurodegeneration, causing inhibition of methylation processes and inducing excitotoxicity. In this study, using primary cultures of rat cerebellar granule cells and propidium iodide staining, we investigated the neurotoxicity of nicotinamide and 1-methylnicotinamide, and their neuroprotective potential in acute and sub-acute homocysteine neurotoxicity. Our results demonstrated that nicotinamide and 1-methylnicotinamide applied for 24 h to cultures at concentrations of up to 25 mM had no effect on neuronal viability. Moreover, nicotinamide at concentrations of 5-20 mM and 1-methylnicotinamide at 1-10 mM applied to cells 24 h before, and for 24 h after an acute 30 min application of 25 mM D,L homocysteine, reduced neuronal damage. 1-Methylnicotinamide at concentrations of 250 and 500 muM showed neuroprotective activity during a sub-acute 24-h exposure to 2.5 mM D,L-homocysteine, while 5 and 25 mM nicotinamide also evoked neuroprotection. These findings do not support suggestions that 1-methylnicotinamide may act as an endogenous neurotoxic agent; rather, they indicate the neuroprotective ability of nicotinamide and 1-methylnicotinamide in homocysteine neurotoxicity. The exact mechanisms of this neuroprotection are unclear and require further investigation.

Neuroprotective Limonoids of Root Bark of Dictamnus dasycarpus

A methanolic extract of Dictamnus dasycarpus root bark afforded four new degraded limonoids, 9alpha-hydroxyfraxinellone-9- O-beta- d-glucoside ( 1), dictamnusine ( 2), dictamdiol A ( 3), and dictamdiol B ( 4), together with eight known compounds, dictamdiol ( 5), fraxinellone ( 6), fraxinellonone ( 7), 9beta-hydroxyfraxinellone ( 8), calodendrolide ( 9), obacunone ( 10), limonin ( 11), and rutaevin ( 12). Compounds, 2, 3, 6, 9, 10, and 11 showed significant neuroprotective activity against glutamate-induced neurotoxicity in primary cultures of rat cortical cells at a concentration of 0.1 microM.

Isolation and identification of neuroprotective compounds from Machilus thunbergii

The CH2Cl2 fraction of the bark of Machilus thunbergii Sieb. et Zucc. (Lauraceae) significantly protected primary cultures of rat cortical cells exposed to the excitotoxic amino acid, L-glutamate. Through the activity-guided isolation of the CH2Cl2 fraction, (+)-9'-hydroxygalbelgin (1), isogalcatin B (2), (7S,8S,8'R)-3',4'-dimethoxy-3,4,-methylenedioxylignan-7-ol (3), 1-hydroxy-7-hydroxymethyl-6-methoxyxanthone (4), 5,7-dimethoxy-3',4'-methylenedioxyflavan-3-ol (5), (+)-(3S,4S,6R)-3,6-dihydroxypiperitone (6), protocatechuic acid methyl ester (7) and tyrosol (8) were obtained. Among them, compound 3, 5, 7, 8 had significant neuroprotective activities against glutamate-induced neurotoxicity in primary cultures of rat cortical cells at concentrations ranging from 0.1µM to 10.0µM. At a concentration of 10.0µM, neuroprotection against glutamate-induced toxicity of compounds 3, 5, 7 or 8 were 25.1, 31.1, 37.9 or 40.1% (p<0.01), respectively.

Neuroprotective 2-(2-Phenylethyl)chromones of Imperata cylindrica

Bioactivity-guided fractionation of the methanolic extract of the rhizomes of Imperata cylindrica afforded a new compound, 5-hydroxy-2-(2-phenylethyl)chromone (1), together with three known compounds, 5-hydroxy-2-[2-(2-hydroxyphenyl)ethyl]chromone (2), flidersiachromone (3), and 5-hydroxy-2-styrylchromone (4). Among these four compounds, 1 and 2 showed significant neuroprotective activity against glutamate-induced neurotoxicity in primary cultures of rat cortical cells.

HIV-1 Tat neurotoxicity in primary cultures of rat midbrain fetal neurons: Changes in dopamine transporter binding and immunoreactivity

HIV-1 neurotoxic proteins (Tat, gp120) are believed to play a major role in pathogenesis of dementia in a significant portion of the AIDS patient population. Dopaminergic systems appear to be particularly important in HIV-associated dementia. In the current studies, we determined that primary cell cultures prepared from the midbrain of 18-day-old rat fetuses are sensitive to Tat neurotoxicity and investigated the possible effects of Tat on DAT-specific ligand binding and DAT immunoreactivity in rat fetal midbrain cultures. We found that Tat neurotoxicity was associated with a significant decrease in [3H]WIN 35428 binding. Immunostaining of cell cultures with antibodies recognizing the C-end epitope of DAT did not reveal significant changes in DAT immunoreactivity. The results of this study implicate involvement of monoamine transmission systems in HIV-associated dementia.

Protective effect of donepezil against Aβ(1-40) neurotoxicity in rat septal neurons

Donepezil, a potent acetylcholinesterase (AChE) inhibitor used for the treatment of Alzheimer's disease (AD), is thought to have a neuroprotective effect in AD patients. Because a deficit in cholinergic neurotransmission is a major feature in AD, and amyloid-beta (Aβ) accumulation has been proposed as a possible causative phenomenon, we were interested to examine the effect of donepezil on Aβ(1-40) induced neurotoxicity in primary cultures of rat septal neurons. Using immunohistochemical staining, almost all the neurons were found to be positive for vesicular acetylcholine transporter (VAChT) in these septal cultures. Septal neuronal cells were cultured for 7 days and then 15 μmol/L of Aβ(1-40) was added to the cell medium for 48 h. The cultured septal neurons were highly susceptible to Aβ toxicity, as shown by morphological examination and lactate dehydrogenase (LDH) assay. Donepezil concentration-dependently reduced the LDH efflux induced by Aβ(1-40), and the effect was significant at 100 nmol/L and above. Donepezil decreased both the negative peak at around 215 nm in the circular dichroism (CD) spectrum and the fluorescence intensity of thioflavin T in the presence of Aβ(1-40). These results suggest that donepezil exerts a neuroprotective effect by reducing the amount of the toxic form of Aβ fibrils in septal neuron cultures. These findings support the idea that the clinical efficacy of donepezil in AD is due to not only activation of cholinergic transmission, but also attenuation of neuronal damage.

Neuroprotective lignans from the bark of Machilus thunbergii.

The CH (2)Cl (2) fraction of the bark of Machilus thunbergii Sieb. et Zucc. (Lauraceae) significantly protected primary cultures of rat cortical cells exposed to the excitotoxic amino acid, L-glutamate. (-)-Isoguaiacin, meso-dihydroguaiaretic acid, machilin A, (+)-galbelgin, licarin A, (-)-sesamin, and (+)-guaiacin were isolated by bioactivity-guided fractionation from the CH (2)Cl (2) fraction and further separated using chromatographic techniques. Isoguaiacin, meso-dihydroguaiaretic acid, licarin A and (+)-guaiacin had significant neuroprotective activities against glutamate-induced neurotoxicity in primary cultures of rat cortical cells at concentrations ranging from 0.1 microM to 10.0 microM.

α 7 Nicotinic acetylcholine receptor–mediated protection against ethanol-induced neurotoxicity

The α 7-selective nicotinic partial agonist 3-[2,4-dimethoxybenzylidene]anabaseine (DMXB) was examined for its ability to modulate ethanol-induced neurotoxicity in primary cultures of rat neurons. Primary cultures of hippocampal neurons were established from Long–Evans, embryonic day (E)-18 rat fetuses and maintained for 7 days. Ethanol (0–150 mM), DMXB (0–56 μM), or both were subsequently co-applied to cultures. Ethanol was added two additional times to the cultures to compensate for evaporation. After 5 days, neuronal viability was assessed with the MTT cell proliferation assay. Results demonstrated that ethanol reduces neuronal viability in a concentration-dependent fashion and that DMXB protects against this ethanol-induced neurotoxicity, also in a concentration-dependent fashion. These results support the suggestion that nicotinic partial agonists may be useful in treating binge drinking–induced neurotoxicity and may provide clues as to why heavy drinkers are usually smokers.

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.