6-cyano-7-nitroquinoxaline-2-3-dione and Brain-Ischemia

It is established that the new compound, 9-[2-(4-isopropylphenoxy)ethyl]adenine (9-IPE-adenine) in a dose of 10 mg/kg per day produces neuroprotective effect in rats with brain ischemia model. 9-IPE-adenine decreased the neurologic deficiency 1.2 times more effectively (p < 0.05) than the reference drug mexidol in analogous dose, and had equal effect with this drug at 25 mg/kg per day on the neurologic deficiency and survival of animals. Electrophysiological studies in hippocampal slices in rats showed that 9-IPE-adenine depressed orthodromic population spikes in CA1 area by 42 ± 4%. Non-competitive antagonist of NMDA receptor complex MK-801, in contrast to D-AP5 (competitive NMDA receptor antagonist) and CNQX (competitive AMPA receptor antagonist), enhanced the depressive effect of the new drug more than two times. These ese results are indicative of the ability of 9-IPE-adenine to modulate the ion channel of NMDA receptor complex.

Topics: 6-Cyano-7-nitroquinoxaline-2,3-dione; Action Potentials; Adenine; Animals; Brain Ischemia; CA1 Region, Hippocampal; Dizocilpine Maleate; Drug Administration Schedule; Excitatory Amino Acid Antagonists; Male; Neuroprotective Agents; Picolines; Rats; Receptors, AMPA; Receptors, N-Methyl-D-Aspartate; Tissue Culture Techniques

Excessive presynaptic glutamate release after cerebral ischaemia leads to neuronal death mainly through excessive calcium entry of N-methyl-D-aspartate receptors (NMDARs). Our recent study reported that cerebroside can open large-conductance Ca²⁺-activated K⁺ (BKCa) channels. The present study evaluated the effects of cerebroside-A (CS-A), a single molecule isolated from an edible mushroom, on brain injury after focal or global ischaemia in adult male mice and rats. We herein report that treatment with CS-A after 60-min middle cerebral artery occlusion dose-dependently reduced the cerebral infarction with at least a 6-h efficacious time-window, which was partially blocked by the BKCa channel blocker charybdotoxin (CTX). Treatment with CS-A after 20 min global cerebral ischaemia (four-vessel occlusion) significantly attenuated the death of pyramidal cells in hippocampal CA1 area, which was also sensitive to CTX. CS-A, by opening the BKCa channel, could prevent excessive glutamate release after oxygen-glucose deprivation (OGD). In addition, CS-A could inhibit NMDAR Ca²⁺ influx, which did not require the activation of the BKCa channel. Furthermore, CS-A blocked the OGD-induced NMDAR-dependent long-term potentiation in hippocampal CA1 region. These findings indicate that treatment with CS-A after stroke exerts potent neuroprotection through prevention of excessive glutamate release and reduction of Ca²⁺ influx through NMDARs.

Topics: 6-Cyano-7-nitroquinoxaline-2,3-dione; Analysis of Variance; Animals; Brain Ischemia; Calcium; Cerebral Infarction; Cerebrosides; Charybdotoxin; Disease Models, Animal; Dose-Response Relationship, Drug; Drug Interactions; Electric Stimulation; Excitatory Amino Acid Antagonists; Excitatory Postsynaptic Potentials; Glucose; Glutamic Acid; Hippocampus; Hypoxia; In Vitro Techniques; Infarction, Middle Cerebral Artery; Long-Term Potentiation; Male; Mice; Mice, Inbred C57BL; N-Methylaspartate; Neuroprotective Agents; Neurotoxins; Patch-Clamp Techniques; Rats; Rats, Sprague-Dawley; Receptors, N-Methyl-D-Aspartate; Tetrazolium Salts; Valine

Two types of quantal spontaneous neurotransmitter release are present in the nervous system, namely action potential (AP)-dependent release and AP-independent release. Previous studies have identified and characterized AP-independent release during hypoxia and ischemia. However, the relative contribution of AP-dependent spontaneous release to the overall glutamate released during transient ischemia has not been quantified. Furthermore, the neuronal activity that mediates such release has not been identified. Using acute brain slices, we show that AP-dependent release constitutes approximately one-third of the overall glutamate-mediated excitatory postsynaptic potentials/currents (EPSPs/EPSCs) measured onto hippocampal CA1 pyramidal neurons. However, during transient (2 mins) in vitro hypoxia-hypoglycemia, large-amplitude, AP-dependent spontaneous release is significantly enhanced and contributes to 74% of the overall glutamatergic responses. This increased AP-dependent release is due to hyper-excitability in the presynaptic CA3 neurons, which is mediated by the activity of NMDA receptors. Spontaneous glutamate release during ischemia can lead to excitotoxicity and perturbation of neural network functions.

Topics: 2-Amino-5-phosphonovalerate; 6-Cyano-7-nitroquinoxaline-2,3-dione; Action Potentials; Animals; Brain Ischemia; CA1 Region, Hippocampal; Electrophysiology; Excitatory Amino Acid Antagonists; Excitatory Postsynaptic Potentials; Glutamates; Ischemic Attack, Transient; Mice; Neurons; Patch-Clamp Techniques; Pyramidal Cells; Receptors, AMPA; Receptors, N-Methyl-D-Aspartate; Transcription Factor AP-1

Dark neurons have plagued the interpretation of brain tissue sections, experimentally and clinically. Seen only when perturbed but living tissue is fixed in aldehydes, their mechanism of production is unknown. Since dark neurons are seen in cortical biopsies, experimental ischemia, hypoglycemia, and epilepsy, we surmised that glutamate release and neuronal transmembrane ion fluxes could be the perturbation leading to dark neuron formation while the fixation process is underway. Accordingly, we excised biopsies of rat cortex to simulate neurosurgical production of dark neurons. To ascertain the role of glutamate, blockade of N-methyl-D-aspartate (NMDA) and non-NMDA receptors was done prior to formaldehyde fixation. To assess the role of transmembrane sodium ion (and implicitly, water) fluxes, tetraethylammonium (TEA) was used. Blockade of NMDA receptors with MK-801 and non-NMDA receptors with the quinoxalinediones (CNQX and NBQX) abolished dark neuron formation. More delayed exposure of the tissue to the antagonist, CNQX, by admixing it with the fixative directly, allowed for some production of dark neurons. Aminophosphonoheptanoate (APH), perhaps due to its polarity, and TEA, did not prevent dark neurons, which were abundant in control formaldehyde fixed material unexposed to either receptor or ion channel antagonists. The results demonstrate a role for the pharmacologic subtypes of glutamate receptors in the pathogenetic mechanism of dark neuron formation. Our results are consistent with the appearance of dark neurons in biopsy where the cerebral cortex has been undercut, and rendered locally ischemic and hypoglycemic, as well as in epilepsy, hypoglycemia, and ischemia, all of which lead to glutamate release. Rather than a pressure-derived mechanical origin, we suggest that depolarization, glutamate release or receptor activation are more likely mechanisms of dark neuron production.

Topics: 6-Cyano-7-nitroquinoxaline-2,3-dione; Animals; Artifacts; Biopsy; Brain Ischemia; Cerebral Cortex; Dizocilpine Maleate; Epilepsy; Excitatory Amino Acid Antagonists; Glutamic Acid; Hypoglycemia; Male; Neurons; Potassium Channel Blockers; Quinoxalines; Rats; Rats, Wistar; Receptors, Glutamate; Receptors, N-Methyl-D-Aspartate; Tetraethylammonium

Neural excitotoxicity is a typical factor in the early phase pathogenesis of cerebral ischemia. Its cellular and molecular mechanisms are still unclear and clinical approaches are still lacking of promising therapies. We have examined the vulnerability of cortical neurons to short-term ischemia in rats by simultaneously analyzing the activities of inhibitory and principal neurons in brain slices. Our results demonstrate that short-term in vitro ischemia permanently impairs the excitability of inhibitory neurons (IN) and synaptic transmission mediated by gamma-aminobutyric acid (GABA). However, principal neurons appear to be more exciting during the reperfusion. The vulnerability of inhibitory neurons to ischemia acquires during postnatal development. Our findings signify a major contribution of the ischemic dysfunction of inhibitory neurons to neural excitotoxicity as well as a strategy to prevent the progress of ischemic stroke by protecting inhibitory neurons.

Topics: 2-Amino-5-phosphonovalerate; 6-Cyano-7-nitroquinoxaline-2,3-dione; Action Potentials; Animals; Animals, Newborn; Bicuculline; Brain Ischemia; Cerebral Cortex; Excitatory Amino Acid Antagonists; GABA Antagonists; In Vitro Techniques; Interneurons; Neural Inhibition; Patch-Clamp Techniques; Pyramidal Cells; Rats; Rats, Sprague-Dawley

An important but poorly understood event associated with ischemia is anoxic depolarization (AD), a sudden and profound depolarization of neurons and glia in cortical and subcortical gray matter. Leao first measured the AD as a wave of electrical silence moving across the cerebral cortex in 1947 and noted its similarity to spreading depression (SD). SD is harmless when coursing through normoxic cortical tissue as during migraine aura. However for 3-4 h following focal ischemia, the additional metabolic stress arising from recurring SD in the penumbra expands the ischemic core, so SD blockade is potentially beneficial therapeutically. In the present study, we measured intrinsic optical signals (IOSs) to monitor anoxic depolarization in submerged rat neocortical slices during O2/glucose deprivation (OGD). After approximately 6 min of OGD, the AD was imaged as a focal increase in light transmittance which then propagated across neocortical gray at approximately 2 mm/min. Although the slice was globally stressed, the AD always initiated focally, sometimes at multiple sites. Its propagation was coincident with a transient negative shift in the extracellular potential, the electrical signature of AD. Acute damage to neocortex (measured as a delayed decrease in LT and as a loss of the evoked field potential) followed only where the AD had propagated, so it is the combined metabolic demands of AD and OGD that acutely damages all layers of the neocortex. Glutamate receptor antagonists (2 mM kynurenate or 25 microM AP-5/10 microM CNQX) did not block AD initiation, slow its propagation or prevent post-AD damage. This study shows that acute ischemic damage is greatly exacerberated by AD during metabolic stress and that glutamate receptor antagonists are not protective. Using this slice model, therapeutically tolerable drugs that block the AD and SD can be investigated.

Topics: 6-Cyano-7-nitroquinoxaline-2,3-dione; Action Potentials; Animals; Brain Ischemia; Cortical Spreading Depression; Excitatory Amino Acid Agonists; Excitatory Amino Acid Antagonists; Glutamic Acid; Hypoxia; Kynurenic Acid; Male; N-Methylaspartate; Neocortex; Neurons; Rats; Rats, Sprague-Dawley; Receptors, Glutamate

Ischemic injury of immature oligodendrocytes is a major component of the brain injury associated with cerebral palsy, the most common human birth disorder. We now report that cultured immature oligodendrocytes [O4(+)/galactoceramide (GC)(-)] are exquisitely sensitive to ischemic injury (80% of cells were dead after 25.5 min of oxygen and glucose withdrawal). This rapid ischemic cell death was mediated by Ca(2+) influx via non-NMDA glutamate receptors. The receptors were gated by the release of glutamate from the immature oligodendrocytes themselves via reverse glutamate transport and included a significant element of autologous feedback of glutamate from cells onto their own receptors. High (> or = 100 microM) extracellular glutamate was protective against ischemic injury as a result of non-NMDA glutamate receptor desensitization. Other potential pathways of Ca(2+) influx, such as voltage-gated Ca(2+) channels, NMDA receptors, or the Na(+)-Ca(2+) exchanger, did not significantly contribute to ischemic Ca(2+) influx or cell injury. Release of Ca(2+) from intracellular stores was also not an important factor. In agreement with previous studies, more mature oligodendrocytes (O4(-)/GC(+)) were found to be less sensitive to ischemic injury than were the immature cells studied here.

Topics: 6-Cyano-7-nitroquinoxaline-2,3-dione; Animals; Animals, Newborn; Brain Ischemia; Calcium; Cell Death; Cell Size; Cells, Cultured; Cellular Senescence; Dizocilpine Maleate; Excitatory Amino Acid Antagonists; Extracellular Space; Feedback; Glutamic Acid; Necrosis; Oligodendroglia; Rats; Rats, Long-Evans; Receptors, Glutamate

This study argues that, in contrast to accepted excitotoxicity theory, O2/glucose deprivation damages neurons acutely by eliciting ischemic spreading depression (SD), a process not blocked by glutamate antagonists. In live rat hippocampal slices, the initiation, propagation, and resolution of SD can be imaged by monitoring wide-band changes in light transmittance (i.e., intrinsic optical signals). Oxygen/glucose deprivation for 10 minutes at 37.5 degrees C evokes a propagating wave of elevated light transmittance across the slice, representing the SD front. Within minutes, CA1 neurons in regions undergoing SD display irreversible damage in the form of field potential inactivation, swollen cell bodies, and extensively beaded dendrites, the latter revealed by single-cell injection of lucifer yellow. Importantly, glutamate receptor antagonists do not block SD induced by O2/glucose deprivation, nor do they prevent the resultant dendritic beading of CA1 neurons. However, CA1 neurons are spared if SD is suppressed by reducing the temperature to 35 degrees C during O2/glucose deprivation. This supports previous electrophysiologic evidence in vivo that SD during ischemia promotes acute neuronal damage and that glutamate antagonists are not protective of the metabolically stressed tissue. The authors propose that the inhibition of ischemic SD should be targeted as an important therapeutic strategy against stroke damage.

Topics: 2-Amino-5-phosphonovalerate; 6-Cyano-7-nitroquinoxaline-2,3-dione; Animals; Brain Ischemia; Cortical Spreading Depression; Evoked Potentials; Excitatory Amino Acid Antagonists; Glucose; Glutamic Acid; Hippocampus; In Vitro Techniques; Kynurenic Acid; Male; Neurotoxins; Oxygen; Pyramidal Cells; Rats; Rats, Sprague-Dawley; Temperature

The effects of ischemia were examined on CA3 pyramidal neurons recorded in hippocampal slices 2-4 mo after a global forebrain insult. With intracellular recordings, CA3 post-ischemic neurons had a more depolarized resting membrane potential but no change of the input resistance, spike threshold and amplitude, fast and slow afterhyperpolarization (AHP) or ADP, and firing properties in response to depolarizing pulses. With both field and whole-cell recordings, synaptic responses were similar in control and post-ischemic neurons. Although there were no spontaneous network-driven discharges, the post-ischemic synaptic network had a smaller threshold to generate evoked and spontaneous synchronized burst discharges. Thus lower concentrations of convulsive agents (kainate, high K(+)) triggered all-or-none network-driven synaptic events in post-ischemic neurons more readily than in control ones. Also, paired-pulse protocol generates, in post-ischemics but not controls, synchronized field burst discharges when interpulse intervals ranged from 60 to 100 ms. In conclusion, 2-4 mo after the insult, the post-ischemic CA3 pyramidal cells are permanently depolarized and have a reduced threshold to generate synchronized bursts. This may explain some neuropathological and behavioral consequences of ischemia as epileptic syndromes observed several months to several years after the ischemic insult.

Topics: 6-Cyano-7-nitroquinoxaline-2,3-dione; Action Potentials; Animals; Brain Ischemia; Epilepsy; Excitatory Amino Acid Agonists; Excitatory Amino Acid Antagonists; Excitatory Postsynaptic Potentials; Hippocampus; In Vitro Techniques; Kainic Acid; Male; Patch-Clamp Techniques; Periodicity; Potassium; Pyramidal Cells; Rats; Rats, Wistar; Seizures; Stimulation, Chemical; Synapses; Time Factors

Whole cell recordings of fura-2 dialyzed vagal neurons of brain stem slices were used to monitor interstitial glutamate accumulation within the dorsal vagal complex. Anoxia produced a sustained outward current (60 pA) and a moderate [Ca(2+)](i) rise (40 nM). These responses were neither mimicked by [1S,3R]-1-aminocyclo-pentane-1, 3-dicarboxylic acid nor affected by Ca(2+)-free solution, 6-cyano-7-nitroquino-xaline-2,3-dione (CNQX), 2-amino-5-phosphonovalerate (APV), or tetrodotoxin. Anoxia or cyanide in glucose-free saline (in vitro ischemia) as well as ouabain or iodoacetate elicited an initial anoxia-like [Ca(2+)](i) increase that turned after several minutes into a prominent Ca(2+) transient (0.9 microM) and inward current (-1.8 nA). APV plus CNQX (plus methoxyverapamil) inhibited this inward current as well as accompanying spontaneous synaptic activity, and reduced the secondary [Ca(2+)](i) rise to values similar to those during anoxia. Each of the latter drugs delayed onset of both ischemic current and prominent [Ca(2+)](i) rise by several minutes and attenuated their magnitudes by up to 40%. Ca(2+)-free solution induced a twofold delay of the ischemic inward current and suppressed the prominent Ca(2+) increase but not the initial moderate [Ca(2+)](i) rise. Cyclopiazonic acid or arachidonic acid in Ca(2+)-free saline delayed further the ischemic current, whereas neither inhibitors of glutamate uptake (dihydrokainate, D,L-threo-beta-hydroxyaspartate, L-transpyrrolidone-2,4-dicarboxylate) nor the Cl(-) channel blocker 5-nitro-2-(3-phenylpropyl-amino) benzoic acid had any effect. In summary, the response to metabolic arrest is due to activation of ionotropic glutamate receptors causing Ca(2+) entry via N-methyl-D-aspartate receptors and voltage-activated Ca(2+) channels. An early Ca(2+)-dependent exocytotic phase of ischemic glutamate release is followed by nonvesicular release, not mediated by reversed glutamate uptake or Cl(-) channels. The results also show that glycolysis prevents glutamate release during anoxia.

Topics: 2-Amino-5-phosphonovalerate; 6-Cyano-7-nitroquinoxaline-2,3-dione; Action Potentials; Animals; Brain Ischemia; Brain Stem; Calcium; Chloride Channels; Enzyme Inhibitors; Excitatory Amino Acid Antagonists; Female; Glutamic Acid; Hypoglycemic Agents; Hypoxia, Brain; In Vitro Techniques; Indoles; Male; Neurons; Patch-Clamp Techniques; Rats; Rats, Wistar; Sulfonylurea Compounds; Synaptic Transmission; Tolbutamide; Vagus Nerve

Ca2+ influx and activation of protein kinase C (PKC) and mitogen-activated protein kinase (MAPK) during nonlethal ischemic preconditioning have been implicated in the protection of the heart against subsequent lethal ischemic injury. Thus, we determined if Ca2+ influx, PKC and MAPK also mediate ischemic preconditioning-induced protection in neurons. Preconditioning by exposure of E18 rat cortical cultures to 90 min of nonlethal oxygen-glucose deprivation (OGD) 24 h prior to 180-240 min of lethal OGD was neuroprotective. Exposure to nominally free Ca2+, or blockade of the alpha-amino-hydroxy-5-methyl-isoxazolepropionate (AMPA) receptor with CNQX did not eliminate protection. MAPK activity did not change and PKC activity decreased by 50% relative to normal baseline levels at 0 and 24 h following preconditioning. The sustained decrease in PKC activity was not due to a loss of enzyme as determined from immunoblots using pan and epsilon-, beta- and zeta-specific PKC antibodies. Neuroprotection was maintained with pharmacological inhibition of PKC activity by staurosporine, chelerythrine and calphostin C and MAPK activity by PD 98059 during preconditioning, indicating that activation of these enzymes during preconditioning was not necessary for protection. Therefore, in contrast to cardiac tissue, ischemic preconditioning of neurons does not require activation of PKC and MAP kinase, and protection is maintained with substantial removal of extracellular Ca2+ or blockade of the AMPA receptor.

Topics: 6-Cyano-7-nitroquinoxaline-2,3-dione; Animals; Brain Ischemia; Calcium; Calcium-Calmodulin-Dependent Protein Kinases; Cells, Cultured; Cerebral Cortex; Enzyme Inhibitors; Excitatory Amino Acid Antagonists; Flavonoids; Glucose; Ischemic Preconditioning; Myocardial Ischemia; Myocardium; Neurons; Oxygen; Protein Kinase C; Rats

We have investigated the relative contributions of oxygen and glucose deprivation to ischaemic neurodegeneration in organotypic hippocampal slice cultures. Cultures prepared from 10-day-old rats were maintained in vitro for 14 days and then deprived of either oxygen (hypoxia), glucose (hypoglycaemia), or both oxygen and glucose (ischaemia). Hypoxia alone induced degeneration selectively in CA1 pyramidal cells and this was greatly potentiated if glucose was removed from the medium. We have also characterised the effects of both pre- and post-treatment using glutamate receptor antagonists and the sodium channel blocker tetrodotoxin (TTX). Neuronal death following either hypoxia or ischaemia was prevented by pre-incubation with CNQX, MK-801 or tetrodotoxin. MK-801 or CNQX also prevented death induced by either hypoxia or ischaemia if added immediately post-insult, however, post-insult addition of TTX prevented hypoxic but not ischaemic damage. Organotypic hippocampal slice cultures are sensitive to both NMDA and non-NMDA glutamate receptor blockade and thus represent a useful in vitro system for the study of ischaemic neurodegeneration paralleling results reported using in vivo models of ischaemia.

Topics: 6-Cyano-7-nitroquinoxaline-2,3-dione; Animals; Brain Ischemia; Cell Death; Excitatory Amino Acid Antagonists; Hippocampus; Hypoglycemia; Hypoxia, Brain; Neurons; Neuroprotective Agents; Organ Culture Techniques; Rats; Rats, Wistar; Receptors, AMPA; Receptors, N-Methyl-D-Aspartate; Sodium Channels

We previously demonstrated that 2-iminothiazolidine-4-carboxylic acid (2-ICA), formed by cyanide reacting with cysteine, caused glutamate antagonist-sensitive seizures when injected i.c.v. (intracerebroventricular) in mice and produced hippocampal CA1 damage following i.c.v. infusion in rats. In this study, the ability of either 2-ICA, glutamate, proline or NMDA (N-methyl-D-aspartate) injected i.c.v. to produce hippocampal lesions sensitive to glutamate antagonists was compared in mice. Hippocampal CA1 damage was observed 5-days following either a seizure (3.2 mumol) or subseizure (1.0 mumol) dose of 2-ICA. Glutamate (3.2 mumol) or proline (10 mumol) also produced hippocampal damage; glutamate damage was primarily to the CA1 subfield, whereas proline damaged neurons throughout the entire hippocampal formation. NMDA (3.2 nmol) caused seizure activity in all animals with a 50% lethality. No hippocampal damage was observed in surviving mice. Neither MK-801 (dizocilpine maleate) nor CNQX (6-cyano-7-nitroquinoxaline-2,3-dione) pretreatment prevented hippocampal lesions produced by 2-ICA. In contrast, MK-801 significantly reduced the frequency of mice displaying glutamate hippocampal lesions, but failed to block seizures produced by glutamate. MK-801 also protected neurons in the CA2-3 zone and the dentate gyrus, but not in the CA1 region of proline-injected mice. Finally, pretreatment with the mixed metabotropic glutamate receptor (mGluR)1/mGluR2 antagonist-agonist (S)-4-carboxy-3-hydroxyphenylglycine (CHPG) prevented hippocampal damage produced by the mGluR1 agonist (RS)-3,5-dihydroxyphenylglycine (DHPG), but did not protect against 2-ICA hippocampal lesions. These results show that 2-ICA hippocampal CA1 damage is not mediated through ionotropic or metabotropic glutamate receptors. 2-ICA hippocampal damage may represent a neurotoxicity that is distinct from excitotoxic-mediated cell death.

Topics: 6-Cyano-7-nitroquinoxaline-2,3-dione; Animals; Brain Ischemia; Dizocilpine Maleate; Excitatory Amino Acid Agonists; Glutamic Acid; Hippocampus; Injections, Intraventricular; Male; Neuroprotective Agents; Neurotoxins; Proline; Rats; Rats, Sprague-Dawley; Seizures

Ischemia-induced cell damage studies have revealed a complex mechanism that is thought to involve glutamate excitotoxicity, intracellular calcium increase, and free radical production. We provide direct evidence that free radical generation occurs in rat CA1 pyramidal neurons of organotypic slices subjected to a hypoxic-hypoglycemic insult. The production of free radicals is temporally correlated with intracellular calcium elevation, as measured by injection of fluo-3 in individual pyramidal cells, using patch electrodes. Free radical production (measured as changes in the fluorescence emission of dihydrorhodamine 123) peaked during reoxygenation and paralleled rising intracellular calcium. Electrophysiological whole-cell recordings revealed membrane potential depolarization and decreased input resistance during the ischemic insult. Glutamate receptor blockade resulted in decreased free radical production and markedly diminished intracellular calcium accumulation, and prevented neuronal depolarization and input resistance decrease during the ischemic episode. These results provide evidence for a direct involvement of glutamate in oxidative damage resulting from ischemic episodes.

Topics: 6-Cyano-7-nitroquinoxaline-2,3-dione; Animals; Brain Ischemia; Calcium; Cell Hypoxia; Energy Metabolism; Fluorescent Dyes; Free Radicals; Glucose; Glutamic Acid; Hippocampus; Ion Transport; Membrane Potentials; Oxidative Stress; Oxygen; Patch-Clamp Techniques; Pyramidal Cells; Rats; Rats, Wistar; Receptors, Glutamate; Reperfusion Injury; Rhodamine 123; Rhodamines

Glutamate antagonists mitigate hypoxic damage upon acute inhibition of energy metabolism. The goal of this study was to investigate their effect on increased hypoxic tolerance induced by preceding chemical inhibition of energy metabolism. While recovery of population spike amplitude (psap) is 30% of onset in slices prepared from control animals (15 min hypoxia, 45 min recovery), recovery exceeds 90% in slices prepared from animals that underwent mild chemical hypoxia in vivo by treatment with 20 mg/kg 3-nitropropionic acid 1 h prior to slice preparation (p-slices). In p-slices perfused for 5 min with D(-)-2-amino-5-phosphonopentanoic acid (APV) (100 microM) 45 min prior to hypoxia, recovery declines to 42 +/- 13% (mean +/- SEM). In contrast, posthypoxic recovery after similar perfusion with 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) (10 microM) is 72 +/- 15% (P < 0.05). We conclude that increased hypoxic tolerance is abolished by N-methyl-D-aspartate (NMDA)-antagonists but not non-NMDA-antagonists.

Topics: 2-Amino-5-phosphonovalerate; 6-Cyano-7-nitroquinoxaline-2,3-dione; Animals; Antihypertensive Agents; Brain Ischemia; Energy Metabolism; Excitatory Amino Acid Antagonists; Hypoxia, Brain; In Vitro Techniques; Male; Nitro Compounds; Oxygen Consumption; Propionates; Rats; Rats, Wistar; Receptors, N-Methyl-D-Aspartate

Using an in situ patch clamp in hippocampal CA1 mini-slices, we measured excitatory postsynaptic currents (EPSC) by varying the strength of the stimulus applied to the axons of CA3 neurones. The kinetics of the EPSC was initially independent of the stimulus strength. Post-ischaemic potentiation of the EPSC was observed 60-80 min after brief periods (10 min) of anoxia/aglycaemia. The decay of the EPSC slowed significantly in most of the examined neurones. In 11 of 17 cells the EPSC kinetics became dependent on stimulus strength: a slower decay corresponded to a stronger stimulus. This effect was not abolished by N-methyl-D-aspartate (NMDA) or a non-NMDA receptor blocker (D-2-amino-5-phosphonovaleric acid or 6-cyano-7-nitroquinoxaline-2,3-dione respectively) indicating the polysynaptic nature of the modified EPSC: transient ischaemia led to the long-term recruitment of previously inactive, possibly latent NMDA synapses between CA1 neurones.

Topics: 6-Cyano-7-nitroquinoxaline-2,3-dione; Animals; Brain Ischemia; Hippocampus; Rats; Rats, Wistar; Receptors, N-Methyl-D-Aspartate; Synapses

Severe forebrain ischemia induces a large increase in expression of NMDA receptor subunits in rat brain. One week after ischemia, levels of NMDA-R1 mRNAs in the CA1 pyramidal cells of hippocampus are 7 times higher than those observed in control rats. At 7 days postischemia, an enhanced immunostaining of the NMDA-R1 subunit was observed in all hippocampal structures indicating that changes in mRNA levels are accompanied by changes in receptor protein level. Riluzole, a potent inhibitor of glutamate release and CNQX, a selective AMPA/kainate antagonist, drastically reduced the ischemia-induced expression of mRNAs for the three NMDA receptor subunits while D-AP5, a selective NMDA antagonist, had essentially no effect. Therefore ischemia-induced expression of NMDA receptor subunits is associated with glutamate release and proceeds via an AMPA/kainate pathway. These results together with those of other groups concerning ischemia effects on AMPA and GABAA receptor levels, suggest an important role of the induced expression of NMDA receptor subunits in the deleterious effects of ischemia.

Topics: 6-Cyano-7-nitroquinoxaline-2,3-dione; Animals; Base Sequence; Brain Ischemia; Excitatory Amino Acid Antagonists; Glutamic Acid; Hippocampus; Male; Molecular Sequence Data; Oligonucleotide Probes; Prosencephalon; Rats; Rats, Wistar; Receptors, AMPA; Receptors, Kainic Acid; Receptors, N-Methyl-D-Aspartate; Riluzole; RNA, Messenger; Thiazoles