Apoptosis Induced via AMPA-Selective Glutamate Receptors in Cultured Murine Cortical Neurons

Abstract: We have investigated the mechanisms of cell death induced by long-term exposure to the glutamate receptor agonist ( S )-α-amino-3-hydroxy-5-methyl-4-isoxazolepropionate [( S )-AMPA]. Using primary cultures of pure neurons (95%) grown in serum-free conditions, we found that 24-h exposure to ( S )-AMPA (0.01–1,000 µ M ) induced concentration-dependent neuronal cell death (EC₅₀ = 3 ± 0.5 µ M ) with cellular changes including neurite blebbing, chromatin condensation, and DNA fragmentation, indicative of apoptosis. ( S )-AMPA induced a delayed cell death with DNA fragmentation occurring in ∼50% of cells at concentrations between 100 and 300 µ M detected using terminal transferase-mediated dUTP nick end-labeling (TUNEL) and agarose gel electrophoresis. Apoptotic chromatin condensation was detected using 4,6-diamidino-2-phenylindole, a fluorescent DNA binding dye. Cell death induced by ( S )-AMPA was attenuated by the AMPA receptor-selective antagonist LY293558 (10 µ M ) and the non-NMDA receptor antagonist 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX; 50 µ M ), yielding EC₅₀ values of 73 ± 5 and 265 ± 8 µ M , respectively, and was unaffected by the NMDA receptor antagonist MK-801 (10 µ M ). The number of apoptotic nuclei induced by 300 µ M ( S )-AMPA (57%) was also reduced substantially by the antagonists LY293558 and CNQX, with only 20% and 18% of neurons, respectively, staining TUNEL-positive at 24 h. In addition, cycloheximide (0.5 µg/ml) also inhibited ( S )-AMPA-induced DNA fragmentation and cell death. Our results show that long-term exposure to AMPA can induce substantial neuronal death involving apoptosis in cultured cortical neurons, suggesting a wide involvement of AMPA-sensitive glutamate receptors in excitotoxic injury and neurodegenerative pathologies.     

The Desglycinyl Metabolite of Remacemide Hydrochloride Is Neuroprotective in Cultured Rat Cortical Neurons

Abstract: The neuroprotective actions of remacemide and its anticonvulsant metabolite 1,2-diphenyl-2-propylamine monohydrochloride (desglycinylremacemide; DGR), a low-affinity NMDA receptor antagonist, were investigated using primary rat cortical neuronal cultures. Exposure of cortical cultures to NMDA (100 µ M ) for 15 min killed 85% of the neurons during the next 24 h. This neurotoxicity was blocked in a concentration-dependent manner by adding DGR (5–20 µ M ), but not its remacemide precursor (10–100 µ M ), to the cultures during the time of NMDA exposure. This suggests that the neuroprotective, as well as the anticonvulsant, activity of remacemide is mediated by DGR. Neuroprotective concentrations of DGR also inhibited two of the principal acute effects of NMDA. DGR (5–20 µ M ) prevented the loss of membrane-associated protein kinase C (PKC) activity that developed by 4 h after transient exposure to 100 µ M NMDA and reduced the NMDA-triggered increases in intracellular free Ca²⁺ concentration ([Ca²⁺]_i) by up to 70%. By contrast, remacemide (50 and 100 µ M ) did not prevent the NMDA-induced loss of PKC activity or reduce the [Ca²⁺]_i responses. These data suggest that DGR protection against NMDA-mediated toxicity in cultured cortical neurons is associated with a reduction of NMDA-triggered [Ca²⁺]_i surges and a prevention of the loss of membrane-associated PKC activity. In addition, the inhibition of NMDA-triggered [Ca²⁺]_i responses by DGR was qualitatively different from the inhibition of these responses by the high-affinity NMDA-receptor antagonists MK-801 and phencyclidine. This may be a consequence of DGR's lower affinity for the NMDA receptor.     

Neuroprotective Effects of NMDA Receptor Glycine Recognition Site Antagonism: Dependence on Glycine Concentration

Abstract: High-affinity NMDA receptor glycine recognition site antagonists protect brain tissue from ischemic damage. The neuroprotective effect of 5-nitro-6,7-dichloro-2,3-quinoxalinedione (ACEA 1021), a selective NMDA receptor antagonist with nanomolar affinity for the glycine binding site, was examined in rat cortical mixed neuronal/glial cultures. ACEA 1021 alone did not alter spontaneous lactate dehydrogenase (LDH) release. Treatment with ACEA 1021 (0.1–10 µ M ) before 500 µ M glutamate, 30 µ M NMDA, or 300 µ M kainate exposure was found to reduce LDH release in a concentration-dependent fashion. These effects were altered by adding glycine to the medium. Glycine (1 m M ) partially reversed the effect of ACEA 1021 on kainate cytotoxicity. Glycine (100 µ M –1 m M ) completely blocked the effects of ACEA 1021 on glutamate and NMDA cytotoxicity. The glycine concentration that produced a half-maximal potentiation of excitotoxin-induced LDH release in the presence of 1.0 µ M ACEA 1021 was similar for glutamate and NMDA (18 ± 3 and 29 ± 9 µ M , respectively). ACEA 1021 also reduced kainate toxicity in cultures treated with MK-801. The effects of glycine and ACEA 1021 on glutamate-induced LDH release were consistent with a model of simple competitive interaction for the strychnine-insensitive NMDA receptor glycine recognition site, although nonspecific effects at the kainate receptor may be of lesser importance.     

NMDA Receptor Involvement in Toxicity to Dopamine Neurons In Vitro Caused by the Succinate Dehydrogenase Inhibitor 3-Nitropropionic Acid

Abstract: Exposure of mesencephalic dopamine neurons to an irreversible inhibitor of succinate dehydrogenase (SDH), 3-nitropropionic acid (3-NPA), for 24 h on day 12 in vitro, produced a dose-dependent loss of high-affinity dopamine uptake when measured 48 h following 3-NPA removal. ATP concentrations in the cultures were reduced by 57% after 3 h of treatment with the highest concentration of 3-NPA tested (500 µ M ). To determine whether glutamate receptors mediated the dopamine toxicity by 3-NPA, cultures were examined for their sensitivity to excitatory amino acid-induced toxicity. Mesencephalic cultures exposed to either 100 µ M NMDA or kainate, on day 12 for 24 h, showed complete loss of dopamine uptake following 48 h of recovery. The NMDA and non-NMDA antagonists, MK-801 (1 µ M ) or 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX; 15 µ M ), completely prevented the effects of NMDA or kainate, respectively, when present at the time of toxin exposure. In cultures treated with 3-NPA, MK-801, but not CNQX, significantly attenuated the loss of dopamine uptake. Direct measurement of the effect of 3-NPA on SDH activity showed that 3-NPA dose-dependently inhibited SDH in vitro in a manner commensurate with the loss of dopamine uptake by 3-NPA. MK-801 had no effect on basal SDH activity or on 3-NPA inhibition of SDH. These data are consistent with the interpretation that metabolic inhibition in dopamine neurons can trigger a secondary excitotoxicity that is mediated by NMDA receptors.     

NMDA Receptor-Mediated Neurotoxicity: A Paradoxical Requirement for Extracellular Mg2+ in Na+/Ca2+-Free Solutions in Rat Cortical Neurons In Vitro

Abstract: Accumulation of intracellular Ca²⁺ is known to be critically important for the expression of NMDA receptor-mediated glutamate neurotoxicity. We have observed, however, that glutamate can also increase the neuronal intracellular Mg²⁺ concentration on activation of NMDA receptors. Here, we used conditions that elevate intracellular Mg²⁺ content independently of Ca²⁺ to investigate the potential role of Mg²⁺ in excitotoxicity in rat cortical neurons in vitro. In Ca²⁺-free solutions in which the Na⁺ was replaced by N -methyl- d -glucamine or Tris (but not choline), which also contained 9 m M Mg²⁺, exposure to 100 µ M glutamate or 200 µ M NMDA for 20 min produced delayed neuronal cell death. Neurotoxicity was correlated to the extracellular Mg²⁺ concentration and could be blocked by addition of NMDA receptor antagonists during, but not immediately following, agonist exposure. Finally, we observed that rat cortical neurons grown under different serum conditions develop an altered sensitivity to Mg²⁺-dependent NMDA receptor-mediated toxicity. Thus, the increase in intracellular Mg²⁺ concentration following NMDA receptor stimulation may be an underestimated component critical for the expression of certain forms of excitotoxic injury.     

l-Deprenyl Protects Mesencephalic Dopamine Neurons from Glutamate Receptor-Mediated Toxicity In Vitro

Abstract: l -Deprenyl is a relatively selective inhibitor of monoamine oxidase (MAO)-B that delays the emergence of disability and the progression of signs and symptoms of Parkinson's disease. Experimentally, deprenyl has also been shown to prevent neuronal cell death in various models through a mechanism that is independent of MAO-B inhibition. We examined the effect of deprenyl on cultured mesencephalic dopamine neurons subjected to daily changes of feeding medium, an experimental paradigm that causes neuronal death associated with activation of the NMDA subtype of glutamate receptors. Both deprenyl (0.5–50 µ M ) and the NMDA receptor blocker MK-801 (10 µ M ) protected dopamine neurons from damage caused by medium changes. The nonselective MAO inhibitor pargyline (0.5–50 µ M ) was not protective, indicating that protection by deprenyl was not due to MAO inhibition. Deprenyl (50 µ M ) also protected dopamine neurons from delayed neurotoxicity caused by exposure to NMDA. Because deprenyl had no inhibitory effect on NMDA receptor binding, it is likely that deprenyl protects from events occurring downstream from activation of glutamate receptors. As excitotoxic injury has been implicated in neurodegeneration, it is possible that deprenyl exerts its beneficial effects in Parkinson's disease by suppressing excitotoxic damage.     

Ca2+ and Reactive Oxygen Species in Staurosporine-Induced Neuronal Apoptosis

Abstract: Staurosporine (0.03–0.5 µ M ) induced a dose-dependent, apoptotic degeneration in cultured rat hippocampal neurons that was sensitive to 24-h pretreatments with the protein synthesis inhibitor cycloheximide (1 µ M ) or the cell cycle inhibitor mimosine (100 µ M ). To investigate the role of Ca²⁺ and reactive oxygen species in staurosporine-induced neuronal apoptosis, we overexpressed calbindin D_28K, a Ca²⁺ binding protein, and Cu/Zn superoxide dismutase, an antioxidative enzyme, in the hippocampal neurons using adenovirus-mediated gene transfer. Infection of the cultures with the recombinant adenoviruses (100 multiplicity of infection) resulted in a stable expression of the respective proteins assessed 48 h later. Overexpression of both calbindin D_28K and Cu/Zn superoxide dismutase significantly reduced staurosporine neurotoxicity compared with control cultures infected with a β-galactosidase overexpressing adenovirus. Staurosporine-induced neuronal apoptosis was also significantly reduced when the culture medium was supplemented with 10 or 30 m M K⁺, suggesting that Ca²⁺ influx via voltage-sensitive Ca²⁺ channels reduces this apoptotic cell death. In contrast, neither the glutamate receptor agonist NMDA (1–10 µ M ) nor the NMDA receptor antagonist dizocilpine (MK-801; 1 µ M ) was able to reduce staurosporine neurotoxicity. Cultures treated with the antioxidants U-74500A (1–10 µ M ) and N -acetylcysteine (100 µ M ) also demonstrated reduced staurosporine neurotoxicity. These results suggest a fundamental role for both Ca²⁺ and reactive oxygen species in staurosporine-induced neuronal apoptosis.     

Chronic Ethanol Exposure Potentiates NMDA Excitotoxicity in Cerebral Cortical Neurons

Abstract: The effect of acute and chronic ethanol exposure on excitotoxicity in cultured rat cerebral cortical neurons was examined. Neuronal death was quantitated by measuring the accumulation of lactate dehydrogenase (LDH) in the culture media 20 h after exposure to NMDA. Addition of NMDA (25–100 μ M ) to the culture dishes for 25 min in Mg²⁺-free buffer resulted in a dose-dependent increase in LDH accumulation. Phase-contrast microscopy revealed obvious signs of cellular injury as evidenced by granulation and disintegration of cell bodies and neuritic processes. Chronic exposure of neuronal cultures to ethanol (100 m M ) for 96 h followed by its removal before NMDA exposure, significantly increased NMDA-stimulated LDH release by 36 and 22% in response to 25 μ M and 50 μ M NMDA, respectively. Neither basal LDH release nor that in response to maximal NMDA (100 μ M ) stimulation was altered by chronic alcohol exposure. In contrast to the effects of chronic ethanol on NMDA neurotoxicity, inclusion of ethanol (100 m M ) only during the NMDA exposure period significantly reduced LDH release by ∼ 50% in both control and chronically treated dishes. This reduction by acute ethanol was also observed under phase-contrast microscopy as a lack of development of granulation and a sparing of disintegration of neuritic processes. These results indicate that chronic exposure of ethanol to cerebral cortical neurons in culture can sensitize neurons to excitotoxic NMDA receptor activation.     

Characterization of the Glutamate Receptors Mediating Release of Somatostatin from Cultured Hippocampal Neurons

Abstract: l -Glutamate, NMDA, dl -α-amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA), and kainate (KA) increased the release of somatostatin-like immunoreactivity (SRIF-LI) from primary cultures of rat hippocampal neurons. In Mg²⁺-containing medium, the maximal effects (reached at ∼100 µ M ) amounted to 737% (KA), 722% (glutamate), 488% (NMDA), and 374% (AMPA); the apparent affinities were 22 µ M (AMPA), 39 µ M (glutamate), 41 µ M (KA), and 70 µ M (NMDA). The metabotropic receptor agonist trans -1-aminocyclopentane-1,3-dicarboxylate did not affect SRIF-LI release. The release evoked by glutamate (100 µ M ) was abolished by 10 µ M dizocilpine (MK-801) plus 30 µ M 1-aminophenyl-4-methyl-7,8-methylenedioxy-5 H -2,3-benzodiazepine (GYKI 52466). Moreover, the maximal effect of glutamate was mimicked by a mixture of NMDA + AMPA. The release elicited by NMDA was sensitive to MK-801 but insensitive to GYKI 52466. The AMPA- and KA-evoked releases were blocked by 6,7-dinitroquinoxaline-2,3-dione (DNQX) or by GYKI 52466 but were insensitive to MK-801. The release of SRIF-LI elicited by all four agonists was Ca²⁺ dependent, whereas only the NMDA-evoked release was prevented by tetrodotoxin. Removal of Mg²⁺ caused increase of basal SRIF-LI release, an effect abolished by MK-801. Thus, glutamate can stimulate somatostatin release through ionotropic NMDA and AMPA/KA receptors. Receptors of the KA type (AMPA insensitive) or metabotropic receptors appear not to be involved.     

Evidence that Brain-Derived Neurotrophic Factor Neuroprotection Is Linked to Its Ability to Reverse the NMDA-Induced Inactivation of Protein Kinase C in Cortical Neurons

Abstract : Several lines of evidence indicate that a rapid loss of neuronal protein kinase C (PKC) activity is a characteristic feature of cerebral ischemia and is a necessary step in the NMDA-induced death of cultured neurons. Exposing embryonic day 18 primary rat cortical neurons to 50 μ M NMDA or 50 μ M glutamate for 10 min caused ~80% cell death over the next 24 h, but excitotoxic death was largely averted, i.e., by 70-80%, in cells pretreated with brain-derived neurotrophic factor (BDNF). An 8-h preexposure to BDNF (50-100 ng/ml) maximally protected cortical cells from the effects of NMDA and glutamate, although the transient application of BDNF between 8 and 4 h before NMDA was equally protective. These effects of BDNF were abolished at supralethal, i.e., >100 μ M , NMDA concentrations. It is significant that BDNF pretreatment prevented the inactivation of PKC in cortical cells normally seen 30 min to 2 h following lethal NMDA or glutamate exposure. This BDNF effect did not arise from changes in NMDA channel activity because neither whole-cell NMDA current amplitudes nor increases in intracellular free Ca²⁺ concentration were altered by the 8-h BDNF pretreatment. Furthermore, BDNF offered no neuroprotection to cells treated with the PKC inhibitors staurosporine (10-20 n M ), calphostin C (1-2.5 μ M ), or GF-109203X (100 n M ) at the time of NMDA addition. These results underscore the importance of PKC inactivation in glutamate-induced neuronal death. They also suggest that BDNF neuroprotection arises, at least in part, via its ability to block the mechanism by which pathophysiological Ca²⁺ influx through the NMDA receptor causes membrane PKC inactivation.     

M3 Muscarinic Receptor-Mediated Enhancement of NMDA-Evoked Adenosine Release in Rat Cortical Slices In Vitro

Abstract: Acetylcholine plays an important role in cortical arousal. Adenosine is released during increased metabolism and has been suggested to be a sleep-promoting factor. To understand the interaction of acetylcholine and adenosine in regulating cortical excitability, we examined the effect of carbachol on NMDA-evoked adenosine release and identified the muscarinic receptor subtype that mediated this effect in adult rat cortical slices in vitro. Carbachol (to 300 µ M ) alone did not affect the basal release of adenosine. However, carbachol (100 µ M ) induced a 253% increase in NMDA (20 µ M )-evoked adenosine release in the presence of Mg²⁺. In the absence of Mg²⁺, carbachol's potentiating effect was less (60% increase). The nonselective muscarinic antagonist atropine (1.5 µ M ) blocked the facilitatory effect of carbachol on NMDA-evoked adenosine release, and this was mimicked by the M3-selective antagonist 4-diphenylacetoxy- N -methylpiperidine (1 µ M ). Neither an M1-selective dose of pirenzepine (50 n M ) nor the M2-selective antagonist methoctramine (1 µ M ) affected carbachol's action on NMDA-evoked adenosine release. Carbachol had no effect on adenosine release evoked by α-amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA). These results suggest that acetylcholine does not affect basal adenosine release but enhances NMDA receptor-mediated evoked adenosine release by acting at M3 receptors in the cortex. This interaction may have a role in regulating cortical neuronal excitability on a long-term basis.     

Neurotrophic Effects of l-DOPA in Postnatal Midbrain Dopamine Neuron/Cortical Astrocyte Cocultures

Abstract: l -DOPA is toxic to catecholamine neurons in culture, but the toxicity is reduced by exposure to astrocytes. We tested the effect of l -DOPA on dopamine neurons using postnatal ventral midbrain neuron/cortical astrocyte cocultures in serum-free, glia-conditioned medium. l -DOPA (50 µ M ) protected against dopamine neuronal cell death and increased the number and branching of dopamine processes. In contrast to embryonically derived glia-free cultures, where l -DOPA is toxic, postnatal midbrain cultures did not show toxicity at 200 µ M l -DOPA. The stereoisomer d -DOPA (50–400 µ M ) was not neurotrophic. The aromatic amino acid decarboxylase inhibitor carbidopa (25 µ M ) did not block the neurotrophic effect. These data suggest that the neurotrophic effect of l -DOPA is stereospecific but independent of the production of dopamine. However, l -DOPA increased the level of glutathione. Inhibition of glutathione peroxidase by l -buthionine sulfoximine (3 µ M for 24 h) blocked the neurotrophic action of L-DOPA. N -Acetyl- l -cysteine (250 µ M for 48 h), which promotes glutathione synthesis, had a neurotrophic effect similar to that of l -DOPA. These data suggest that the neurotrophic effect of l -DOPA may be mediated, at least in part, by elevation of glutathione content.     

Enhanced expression of RNase L as a novel intracellular signal generated by NMDA receptors in mouse cortical neurons

Recently we showed that the level of mitochondrial mRNA was decreased prior to neuronal death induced by glutamate. As the level of mRNA is regulated by ribonuclease (RNase), we examined RNase activity and its expression in the primary cultures of cortical neurons after glutamate treatment in order to evaluate the involvement of RNase in glutamate-induced neuronal death. A 15-min exposure of the cultures to glutamate at the concentration of 100muM produced marked neuronal damage (more than 70% of total cells) at 24-h post-exposure. Under the experimental conditions used, RNA degradation was definitely observed at a period of 4-12-h post-exposure, a time when no damage was seen in the neurons. Glutamate-induced RNA degradation was completely prevented by the N-methyl-d-aspartic acid (NMDA) receptor channel blocker MK-801 or the NR2B-containing NMDA receptor antagonist ifenprodil. Glutamate exposure produced enhanced expression of RNase L at least 2-12h later, which was absolutely abolished by MK-801. However, no significant change was seen in the level of RNase H1 mRNA at any time point post-glutamate treatment. Immunocytochemical studies revealed that RNase L expressed in response to glutamate was localized within the nucleus, mitochondria, and cytoplasm in the neurons. Taken together, our data suggest that expression of RNase L is a signal generated by NMDA receptor in cortical neurons. RNase L expression and RNA degradation may be events that cause neuronal damage induced by NMDA receptor activation.     

Terfenadine induces toxicity in cultured cerebellar neurons: A role for glutamate receptors

Summary Exposure of cultured cerebellar neurons to the histamine H1 receptor antagonist terfenadine resulted in neuronal degeneration and death. Terfenadine neurotoxicity was dependent upon concentration and time of exposure. After 2h exposure, 20µM terfenadine reduced the number of surviving neurons by 75%, and as low as 10nM terfenadine induced significant neurotoxicity after 5 days of exposure. Neuronal sensitivity to terfenadine changed with age in culture, and at 25 days in culture neurons appeared to be much less sensitive than at 5 or 9–17 days in culture. Neurotoxicity by terfenadine could not be prevented by high concentrations of histamine (5 mM), but it was significantly delayed by blocking NMDA or non-NMDA glutamate receptors with MK-801 or CNQX respectively, suggesting the involvement of excitatory transmission mediated by glutamate in the neurotoxicity induced by terfenadine in these neurons. We also found that the presence of terfenadine (5,µM) unveiled the potential excitotoxicity of the non-NMDA receptor agonist AMPA (100µM), and reduced the concentration of glutamate necessary to induce excitotoxicity, compared to untreated cultures. These results suggest a role for terfenadine in the modulation of the excitotoxic response mediated in cerebellar neurons through ionotropic glutamate receptors.     

The Effect of Magnesium on Oxidative Neuronal Injury In Vitro

Abstract: The effect of magnesium on the oxidative neuronal injury induced by hemoglobin was assessed in murine cortical cell cultures. Exposure to 5 µ M hemoglobin in physiologic (1 m M ) magnesium for 26 h resulted in the death of about one-half the neurons and a sixfold increase in malondialdehyde production; glia were not injured. Increasing medium magnesium to 3 m M reduced neuronal death by about one-half and malondialdehyde production by about two-thirds; neuronal death and lipid peroxidation were approximately doubled in 0.3 m M magnesium. Comparable results were observed in spinal cord cultures. The NMDA antagonist MK-801 weakly attenuated hemoglobin neurotoxicity in low-magnesium medium, but tended to potentiate injury in physiologic magnesium. Incubation in low-magnesium medium alone for 24 h reduced cellular glutathione by ∼50% in mixed neuronal and glial cultures but by only 10% in pure glial cultures. The iron-dependent oxidation of phosphatidylethanolamine liposomes was attenuated in a concentration-dependent fashion by 2.5–10 m M magnesium; a similar effect was provided by 0.01–0.1 m M cobalt. However, oxidation was weakly enhanced by 0.5–1 m M magnesium. These results suggest that the vulnerability of neurons to iron-dependent oxidative injury is an inverse function of the extracellular magnesium concentration. At high concentrations, magnesium inhibits lipid peroxidation directly, perhaps by competing with iron for phospholipid binding sites. At low concentrations, enhancement of cell death may be due to the combined effect of increased NMDA receptor activity, glutathione depletion, and direct potentiation of lipid peroxidation.     

Delayed Neuronal Death After Brief Histotoxic Hypoxia In Vitro

Abstract: The effect of three metabolic inhibitors—iodoacetate, potassium cyanide, and potassium arsenate—on neuronal viability was studied in primary rat cortical and hippocampal CA1 neuronal cultures. Iodoacetate (0.1 m M ) applied for 5 min to 8-day-old cultures resulted in delayed neuronal death within 3–24 h in cortical and hippocampal CA1 neurons. Neuronal degeneration was preceded by transient inhibition of energy metabolism to ∼40% and a permanent inhibition of protein synthesis to ∼50%. The inhibition of protein synthesis and the neuronal death were prevented by the free radical scavenger vitamin E but not by the glutamate antagonist MK-801. Removal of calcium during iodoacetate exposure could not protect against toxicity, and there was no increase of intracellular calcium concentration during and shortly after iodoacetate treatment. Cyanide and arsenate produced only partial neuronal degeneration, even at a dose of 10 m M . These observations demonstrate that brief exposure of neurons to low concentrations of iodoacetate produces a delayed type of neuronal death that is not mediated by either calcium or glutamate. The therapeutic effect of vitamin E points to a free-radical mediated injury and suggests that this type of pathology may also be involved in delayed neuronal death after transient energy depletion in vivo.     

Glutamate Receptor-Mediated Calcium Surges in Neurons Derived from P19 Cells

Abstract: Retinoic acid-treated murine P19 embryonal carcinoma cells differentiate into cells with neuronal morphology that display typical neuronal markers. In this study, the presence of glutamate receptors linked to Ca²⁺-signaling mechanisms on these neurons was demonstrated by testing the effects of glutamate agonists and antagonists on the intracellular calcium ion concentration ([Ca²⁺]_i). Glutamate (1 m M ) induced either sustained or transient increases in [Ca²⁺]_i. The sustained glutamate-induced increase in [Ca²⁺]_i was mimicked by NMDA (40 µ M ). The NMDA-triggered [Ca²⁺]_i response was abolished by incubating the cells in Ca²⁺-free medium or by pretreating them with Mg²⁺ (2 m M ) or MK-801 (0.1 µ M ). These responses were unaffected by the non-NMDA antagonist CNQX (10 µ M ), but they required glycine (3–30 µ M ). Kainate (40 µ M ) and AMPA (40 µ M ) did not affect [Ca²⁺]_i. Without external Ca²⁺, glutamate triggered transient, sometimes oscillating, increases in [Ca²⁺]_i. These responses were mimicked by the metabotropic agonist trans -(1 S ,3 R )-1-amino-1,3-cyclopentanedicarboxylic acid (300 µ M ). These results suggest that neurons derived from P19 embryonal carcinoma cells have NMDA and metabotropic, but not AMPA/kainate receptors, which are linked to Ca²⁺-signaling mechanisms. These cells could provide a consistent and reproducible model with which to study neuronal differentiation, neurotoxicity, and glutamate receptor-signaling mechanisms.     

Neuroprotection by the N-Methyl-d-Aspartate Receptor Antagonist CGP 40116: In Vivo and In Vitro Studies

Abstract: The goal of this study was to evaluate the effects of a novel competitive N -methyl- d -aspartate (NMDA) receptor antagonist, d -( E )-2-amino-4-methyl-5-phosphono-3-pentoic acid (CGP 40116), on neuronal damage in vivo and in vitro. We studied 20 rabbits that underwent a 2-h occlusion of the left internal carotid, anterior cerebral, and middle cerebral arteries followed by 4 h of reperfusion. Ten minutes after occlusion the animals were treated with either normal saline (n = 7) or CGP 40116 at two different doses (20 mg/kg, n = 6; 40 mg/kg, n = 7) administered over a 5-min period. Somatosensory evoked potentials were used to confirm adequate ischemia and neuronal injury was assessed by histopathology and magnetic resonance imaging. CGP 40116 decreased cortical ischemic neuronal damage by 74 and 77% (control, 37.8%± 13.1%; CGP 20 mg/kg, 9.9 ± 3.6%; CGP 40 mg/kg, 8.7 ± 3.7%; p < 0.01) and reduced cortical ischemic edema by 52 and 35% (control, 42.3 ± 10.4%; CGP 20 mg/kg, 20.1 ± 6.7%; CGP 40 mg/kg, 27.5 ± 13.3%; p < 0.05) but did not protect against striatal injury. We performed a second study using primary cell cultures from mouse neocortex to determine the effects of CGP 40116 on neuronal death induced by a 10-min exposure to 500 µ M NMDA or by 45 min of oxygen-glucose deprivation (OGD). Our results demonstrate that CGP 40116 was effective at attenuating neuronal death in a concentration-dependent manner (ED₅₀ of 3.2 µ M against NMDA toxicity and 23.1 µ M against OGD) as measured by lactate dehydrogenase levels 24 h after the insult. The neuroprotective effects of CGP 40116 in vivo and in vitro suggest it may be of potential clinical therapeutic value.     

Magnesium Sulfate Protects Against the Bioenergetic Consequences of Chronic Glutamate Receptor Stimulation

Extracellular glutamate is elevated following brain ischemia or trauma and contributes to neuronal injury. We tested the hypothesis that magnesium sulfate (MgSO₄, 3 mM) protects against metabolic failure caused by excitotoxic glutamate exposure. Rat cortical neuron preparations treated in medium already containing a physiological concentration of Mg²⁺ (1 mM) could be segregated based on their response to glutamate (100 µM). Type I preparations responded with a decrease or small transient increase in oxygen consumption rate (OCR). Type II neurons responded with >50% stimulation in OCR, indicating a robust response to increased energy demand without immediate toxicity. Pre-treatment with MgSO₄ improved the initial bioenergetic response to glutamate and ameliorated subsequent loss of spare respiratory capacity, measured following addition of the uncoupler FCCP, in Type I but not Type II neurons. Spare respiratory capacity in Type I neurons was also improved by incubation with MgSO₄ or NMDA receptor antagonist MK801 in the absence of glutamate treatment. This finding indicates that the major difference between Type I and Type II preparations is the amount of endogenous glutamate receptor activity. Incubation of Type II neurons with 5 µM glutamate prior to excitotoxic (100 µM) glutamate exposure recapitulated a Type I phenotype. MgSO₄ protected against an excitotoxic glutamate-induced drop in neuronal ATP both with and without prior 5 µM glutamate exposure. Results indicate that MgSO₄ protects against chronic moderate glutamate receptor stimulation and preserves cellular ATP following treatment with excitotoxic glutamate.     

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.