6-cyano-7-nitroquinoxaline-2-3-dione and Seizures

The effects of a single and multiple doses of ginkgolide A, B, C, and bilobalide, active components of Ginkgo biloba extract (EGb 761), on absence seizures were investigated in male WAG/Rij rats, a genetic animal model of absence epilepsy. Furthermore, the interactions of ginkgolide A together with NMDA receptor antagonist MK-801, AMPA/kainate receptor antagonist 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX), or L-type calcium channel blocker nicardipine were studied to figure out how ginkgolide A affects spike-wave discharges (SWDs) in the brain. The experiments were done using 6-8-month-old male WAG/Rij rats with infusion cannula and EEG electrode implanted. Ginkgolide A, B, C, and bilobalide were administered intraperitoneally for 7 days at a dose of 6 mg/kg. In interaction groups, 6 μg ginkgolide A was injected intracerebroventricularly in combination with MK-801 (10 μg), CNQX (1 μg), and nicardipine (50 μg) for 7 days. EEG was recorded from animals at the baseline, first dose, and seventh dose periods for 4 h. Ginkgolide A (p = .028), C (p = .046), and bilobalide (p = .043) significantly increased the frequency of SWDs in WAG/Rij rats. Ginkgolide A injected into the lateral ventricle with MK-801 (p = .046), CNQX (p = .043), and nicardipine (p = .046) significantly increased the number of SWDs after seventh dose. Finally, the EGb 761-related increase in absence epilepsy was determined to be caused by ginkgolide A, C, and bilobalide. All three receptor antagonists/channel blockers do not inhibit the pro-absence effect of ginkgolide A. The findings revealed that ginkgolide A's pro-absence effect is mediated by brain circuits other than ionotropic glutamate receptors or L-type calcium channels.

Topics: 6-Cyano-7-nitroquinoxaline-2,3-dione; Animals; Bilobalides; Disease Models, Animal; Dizocilpine Maleate; Electroencephalography; Epilepsy, Absence; Excitatory Amino Acid Antagonists; Ginkgolides; Male; Nicardipine; Rats; Seizures

Topics: 6-Cyano-7-nitroquinoxaline-2,3-dione; Animals; Electroencephalography; Hippocampus; Kainic Acid; Male; Pyridines; Rats, Wistar; Receptor, Metabotropic Glutamate 5; Receptors, AMPA; Receptors, Kainic Acid; Seizures

We obtained field, K(+) selective and "sharp" intracellular recordings from the rat entorhinal (EC) and perirhinal (PC) cortices in an in vitro brain slice preparation to identify the events occurring at interictal-to-ictal transition during 4-aminopyridine application. Field recordings revealed interictal- (duration: 1.1 to 2.2s) and ictal-like (duration: 31 to 103s) activity occurring synchronously in EC and PC; in addition, interictal spiking in PC increased in frequency shortly before the onset of ictal oscillatory activity thus resembling the hypersynchronous seizure onset seen in epileptic patients and in in vivo animal models. Intracellular recordings with K-acetate+QX314-filled pipettes in PC principal cells showed that spikes at ictal onset had post-burst hyperpolarizations (presumably mediated by postsynaptic GABAA receptors), which gradually decreased in amplitude. This trend was associated with a progressive positive shift of the post-burst hyperpolarization reversal potential. Finally, the transient elevations in [K(+)]o (up to 4.4mM from a base line of 3.2mM) - which occurred with the interictal events in PC - progressively increased (up to 7.3mM) with the spike immediately preceding ictal onset. Our findings indicate that hypersynchronous seizure onset in rat PC is caused by dynamic weakening of GABAA receptor signaling presumably resulting from [K(+)]o accumulation.

Topics: 4-Aminopyridine; 6-Cyano-7-nitroquinoxaline-2,3-dione; Action Potentials; Animals; Cations, Monovalent; Cerebral Cortex; Excitatory Amino Acid Antagonists; GABA Antagonists; Neurons; Periodicity; Phosphinic Acids; Picrotoxin; Piperazines; Potassium; Propanolamines; Rats, Sprague-Dawley; Receptors, GABA-A; Receptors, GABA-B; Receptors, N-Methyl-D-Aspartate; Seizures; Tissue Culture Techniques

Seizure-like activities generated in anterior cingulate cortex (ACC) are usually classified as simple partial and are associated with changes in autonomic function, motivation, and thought. Previous studies have shown that thalamic inputs can modulate ACC seizure, but the exact mechanisms have not been studied thoroughly. Therefore, we investigated the role of thalamic inputs in modulating ACC seizure-like activities. In addition, seizure onset and propagation are difficult to determine in vivo in ACC. We studied the spatiotemporal changes in epileptiform activity in this cortex in a thalamic-ACC slice to clearly determine seizure onset.. We used multielectrode array (MEA) recording and calcium imaging to investigate the modulatory effect of thalamic inputs in a thalamic-ACC slice preparation.. Seizure-like activities induced with 4-aminopyridine (4-AP; 250 μm) and bicuculline (5-50 μm) in ACC were attenuated by glutamate receptor antagonists, and the degree of disinhibition varied with the dose of bicuculline. Seizure-like activities were decreased with 1 Hz thalamic stimulation, whereas corpus callosum stimulation could increase ictal discharges. Amplitude and duration of cingulate seizure-like activities were augmented after removing thalamic inputs, and this effect was not observed with those induced with elevated bicuculline (50 μm). Seizure-like activities were initiated in layers II/III and, after thalamic lesions, they occurred mainly in layers V/VI. Two-dimensional current-source density analyses revealed sink signals more frequently in layers V/VI after thalamic lesions, indicating that these layers produce larger excitatory synchronization. Calcium transients were synchronized after thalamic lesions suggesting that ACC seizure-like activities are subjected to desynchronizing modulation by thalamic inputs. Therefore, ACC seizure-like activities are subject to desynchronizing modulation from medial thalamic inputs to deep layer pyramidal neurons.. Cingulate seizure-like activities were modulated significantly by thalamic inputs. Repeated stimulation of the thalamus efficiently inhibited epileptiform activity, demonstrating that the desynchronization was pathway-specific. The clinical implications of deep thalamic stimulation in the modulation of cingulate epileptic activity require further investigation.

Topics: 4-Aminopyridine; 6-Cyano-7-nitroquinoxaline-2,3-dione; Action Potentials; Animals; Bicuculline; Biological Clocks; Calcium; Corpus Callosum; Disease Models, Animal; Dose-Response Relationship, Drug; Electric Stimulation; Electrodes; Gyrus Cinguli; In Vitro Techniques; Male; Mice; Mice, Inbred C57BL; Muscimol; Neural Pathways; Seizures; Thalamus

Exposure to organophosphorus nerve agents induces brain seizures, which can cause profound brain damage resulting in death or long-term cognitive deficits. The amygdala and the hippocampus are two of the most seizure-prone brain structures, but their relative contribution to the generation of seizures after nerve agent exposure is unclear. Here, we report that application of 1 muM soman for 30 min, in rat coronal brain slices containing both the hippocampus and the amygdala, produces prolonged synchronous neuronal discharges (10-40 s duration, 1.5-5 min interval of occurrence) resembling ictal activity in the basolateral nucleus of the amygdala (BLA), but only interictal-like activity ("spikes" of 100-250 ms duration; 2-5 s interval) in the pyramidal cell layer of the CA1 hippocampal area. BLA ictal- and CA1 interictal-like activity were synaptically driven, as they were blocked by the AMPA/kainate receptor antagonist 6-cyano-7-nitroquinoxaline-2,3-dione. As the expression of the GluR5 subunit of kainate receptors is high in the amygdala, and kainate receptors containing this subunit (GluR5KRs) play an important role in the regulation of neuronal excitability in both the amygdala and the hippocampus, we tested the efficacy of a GluR5KR antagonist against the epileptiform activity induced by soman. The GluR5KR antagonist UBP302 reduced the amplitude of the hippocampal interictal-like spikes, and eliminated the seizure-like discharges in the BLA, or reduced their duration and frequency, with no significant effect on the evoked field potentials. This is the first study reporting in vitro ictal-like activity in response to a nerve agent. Our findings, along with previous literature, suggest that the amygdala may play a more important role than the hippocampus in the generation of seizures following soman exposure, and provide the first evidence that GluR5KR antagonists may be an effective treatment against nerve agent-induced seizures.

Topics: 6-Cyano-7-nitroquinoxaline-2,3-dione; Alanine; Amygdala; Animals; Calcium; Evoked Potentials; Hippocampus; In Vitro Techniques; Male; Rats; Rats, Sprague-Dawley; Receptors, Kainic Acid; Seizures; Soman; Thymine

Excitatory GABA action induced by high [Cl(-)](i) is thought to contribute to seizure generation in neonatal neurons although the mechanism of this effect remains unclear. We report that bumetanide, a NKCC1 antagonist, reduces driving force of GABA-mediated currents (DF(GABA)) in neonatal hippocampal neurons and blocks the giant depolarizing potentials (GDPs), a spontaneous pattern of network activity. In the preparation composed of two intact interconnected hippocampi, bumetanide did not prevent generation of kainate-induced seizures, their propagation to the contralateral hippocampus, and formation of an epileptogenic mirror focus. However, in the isolated mirror focus, bumetanide effectively blocked spontaneous epileptiform activity transforming it to the GDP-like activity pattern. Bumetanide partially reduced DF(GABA) and therefore the excitatory action of GABA in epileptic neurons. Therefore bumetanide is a potent anticonvulsive agent although it cannot prevent formation of the epileptogenic mirror focus. We suggest that an additional mechanism other than NKCC1-mediated contributes to the persistent increase of DF(GABA) in epileptic neurons.

Topics: 6-Cyano-7-nitroquinoxaline-2,3-dione; Action Potentials; Animals; Animals, Newborn; Bicuculline; Biophysics; Bumetanide; Electric Stimulation; Epilepsy; Excitatory Amino Acid Agents; Functional Laterality; GABA Antagonists; gamma-Aminobutyric Acid; Hippocampus; In Vitro Techniques; Kainic Acid; Neurons; Patch-Clamp Techniques; Rats; Rats, Wistar; Seizures; Sodium Potassium Chloride Symporter Inhibitors; Spectrum Analysis

The neonatal period is critical for seizure susceptibility, and neocortical networks are central in infantile epilepsies. We report that application of 4-aminopyridine (4-AP) to immature (P6-P9) neocortical slices generates layer-specific interictal seizures (IISs) that transform after recurrent seizures to ictal seizures (ISs). During IISs, cell-attached recordings show action potentials in interneurons and pyramidal cells in L5/6 and interneurons but not pyramidal neurons in L2/3. However, L2/3 pyramidal neurons also fire during ISs. Using single N-methyl-d-aspartate (NMDA) channel recordings for measuring the cell resting potential (Em), we show that transition from IISs to ISs is associated with a gradual Em depolarization of L2/3 and L5/6 pyramidal neurons that enhances their excitability. Bumetanide, a NKCC1 co-transporter antagonist, inhibits generation of IISs and prevents their transformation to ISs, indicating the role excitatory GABA in epilepsies. Therefore deep layer neurons are more susceptible to seizures than superficial ones. The initiating phase of seizures is characterized by IISs generated in L5/6 and supported by activation of both L5/6 interneurons and pyramidal cells. IISs propagate to L2/3 via activation of L2/3 interneurons but not pyramidal cells, which are mostly quiescent at this phase. In superficial layers, a persistent increase in excitability of pyramidal neurons caused by Em depolarization is associated with a transition from largely confined GABAergic IIS to ictal events that entrain the entire neocortex.

Topics: 4-Aminopyridine; 6-Cyano-7-nitroquinoxaline-2,3-dione; Afferent Pathways; Animals; Animals, Newborn; Bumetanide; Dose-Response Relationship, Radiation; Electric Stimulation; Excitatory Amino Acid Antagonists; Female; gamma-Aminobutyric Acid; In Vitro Techniques; Male; Membrane Potentials; Mice; Neocortex; Neurons; Neurotransmitter Agents; Patch-Clamp Techniques; Potassium Channel Blockers; Reaction Time; Seizures; Sodium Potassium Chloride Symporter Inhibitors; Synapses; Synaptic Transmission

Reactive glial cells, for example, from patients with temporal lope epilepsy have a reduced density of inward rectifying K(+) (Kir) channels and thus a reduced K(+) buffering capacity. Evidence is accumulating that this downregulation of Kir channels could be implicated in epileptogenesis. In rat hippocampal brain slices, prolonged exposure to the nonselective Kir channel antagonist, Cs(+) (5 mM), gives rise to an epileptiform field potential (Cs-FP) in area CA1 composed of an initial positive (interictal-like) phase followed by a prolonged negative (ictal-like) phase. We have previously shown that the interictal-like phase depends on synaptic activation. The present study extends these findings by showing that the ictal-like phase of the Cs-FP is (i) sensitive to osmotic expansion of the extracellular space, (ii) reversed very quickly during wash out of Cs(+), and (iii) re-established in the presence of Ba(2+) (30-200 microM) or isosmotic low extracellular concentration of Na(+) ([Na(+)](o), 51.25 mM). The interictal-like phase showed less or no sensitivity to these treatments. In the complete absence of Cs(+), the Cs-FP could be fully reconstructed by the combined application of 4-aminopyridine (0.5 mM), an isosmotic high extracellular concentration of K(+) ([K(+)](o), 7 mM), and low [Na(+)](o) (51.25 mM). These results suggest that the interictal-like phase is initiated through synaptic activation and results from an unspecific increase in neuronal excitability, whereas the ictal-like phase is entirely dependent on blockade of Kir channels in CA1. We propose that glial dysfunction-related loss of Kir channels may not alone be sufficient for starting the induction process, but will likely increase the tendency of an epileptogenic process to proceed into seizure activity.

Topics: 4-Aminopyridine; 6-Cyano-7-nitroquinoxaline-2,3-dione; Animals; Barium; Cesium; Dizocilpine Maleate; Electric Stimulation; Excitatory Amino Acid Antagonists; Hippocampus; In Vitro Techniques; Male; Membrane Potentials; Neuroglia; Patch-Clamp Techniques; Potassium Channel Blockers; Potassium Channels, Inwardly Rectifying; Rats; Rats, Wistar; Seizures; Synaptic Transmission

In response to harmful stresses, cells induce programmed cell death (PCD) or apoptosis. Seizures can induce neural damage and activate biochemical pathways associated with PCD. Since seizures trigger intracellular calcium overload, it has been presumed that the intrinsic cell death pathway mediated by mitochondrial dysfunction would modulate cell death following seizures. However, previous work suggests that the extrinsic cell death pathway may initiate the damage program. Here we investigate intrinsic versus extrinsic cell death pathway activation using caspase cleavage as a marker for activation of these pathways in a rat in vitro model of seizures. Hippocampal cells, chronically treated with kynurenic acid, had kynurenic acid withdrawn to induce seizure-like activity for 40 min. Subjecting rat hippocampal cultures to seizures increased cell death and apoptosis-like DNA fragmentation using TUNEL staining. Seizure-induced cell death was blocked by both MK801 (10 microM) and CNQX (40 microM), which suggests multiple glutamate receptors regulate seizure-induced cell death. Cleavage of the initiator caspases, caspase 8 and 12 were increased 4h following seizure, and cleavage of the quintessential executioner caspase, caspase 3 was increased 4h following seizure. In contrast, caspase 9 cleavage only increased 24h following seizure. Using an affinity labeling approach to trap activated caspases in situ, we show that caspase 8 is the apical caspase activated following seizures. Finally, we show that the caspase 8 inhibitor Ac-IETD-CHO was more effective at blocking seizure-induced cell death than the caspase 9 inhibitor Ac-LEHD-CHO. Taken together, our data suggests the extrinsic cell death pathway-associated caspase 8 is activated following seizures in vitro.

Topics: 6-Cyano-7-nitroquinoxaline-2,3-dione; Animals; Caspase 8; Caspase 9; Caspases; Cell Death; Cells, Cultured; Dizocilpine Maleate; DNA Damage; Excitatory Amino Acid Antagonists; Hippocampus; Kynurenic Acid; L-Lactate Dehydrogenase; Neurons; Rats; Rats, Sprague-Dawley; Seizures

The temporal lobe seems particularly susceptible to seizure activity. Mesial temporal lobe structures, including the hippocampus, have the lowest seizure thresholds in the brain. Conversely, thresholds in the frontal neocortex are significantly higher. The development of intact, isolated preparations of hippocampus and neocortex in vitro allows for study into mechanisms governing seizure threshold.. Epileptiform discharges in isolated mouse neocortical blocks were compared with the contralateral intact hippocampus, isolated from the same brain, by using the low-Mg2+, 4 aminopyridine (4-AP), and low-Ca2+ in vitro seizure models. The pharmacology of low Mg(2+)-induced ictal-like events (ILEs) generated in the hippocampus and neocortex was then compared by using glutamatergic antagonists DL-2-amino-5-phosphonovaleric acid (APV) and 6-cyano-7-nitro-quinoxaline-2,3-dione (CNQX), and the Ca2+ channel antagonist, nifedipine.. Neocortical blocks generated both recurrent, spontaneous ILEs and interictal-like events under low-Mg2+ artificial CSF (aCSF) perfusion, distinct from those generated in the hippocampus. ILEs from the hippocampus displayed lower thresholds and longer durations as compared with isolated neocortical blocks. Similar results were obtained during 4-AP perfusion. Perfusion with low-Ca2+ ACSF did not produce stereotypical ILEs in the neocortical block, producing instead recurrent, slow depolarizations. Both ILEs and recurrent, slow depolarizations were produced in the hippocampus. Application of APV and nifedipine exacerbated low Mg(2+)-induced ILEs in the hippocampus but not the neocortex, indicating a distinct pharmacology for partial seizures of different brain regions.. The developing mouse hippocampus demonstrates increased ictogenesis compared with the developing neocortex in vitro, consistent with clinical observations and in vivo experimental models.

Topics: 2-Amino-5-phosphonovalerate; 6-Cyano-7-nitroquinoxaline-2,3-dione; Animals; Calcium Channel Blockers; Convulsants; Disease Models, Animal; Dose-Response Relationship, Drug; Epilepsy, Temporal Lobe; Evoked Potentials; Excitatory Amino Acid Antagonists; Frontal Lobe; Functional Laterality; Hippocampus; Hypocalcemia; In Vitro Techniques; Magnesium Deficiency; Mice; Mice, Inbred C57BL; Neocortex; Nifedipine; Seizures; Synaptic Transmission

Hypoxia is the most common cause of perinatal seizures and can be refractory to conventional anticonvulsant drugs, suggesting an age-specific form of epileptogenesis. A model of hypoxia-induced seizures in immature rats reveals that seizures result in immediate activation of the phosphatase calcineurin (CaN) in area CA1 of hippocampus. After seizures, CA1 pyramidal neurons exhibit a downregulation of GABA(A) receptor (GABA(A)R)-mediated inhibition that was reversed by CaN inhibitors. CaN activation appears to be dependent on seizure-induced activation of Ca2+-permeable AMPA receptors (AMPARs), because the upregulation of CaN activation and GABA(A)R inhibition were attenuated by GYKI 52466 [1-(4-aminophenyl)-4-methyl-7,8-methylenedioxy-5H-2,3-benzodiazepine hydrochloride] or Joro spider toxin. GABA(A)R beta2/3 subunit protein was dephosphorylated at 1 h after seizures, suggesting this subunit as a possible substrate of CaN in this model. Finally, in vivo administration of the CaN inhibitor FK-506 significantly suppressed hypoxic seizures, and posttreatment with NBQX (2,3-dihydroxy-6-nitro-7-sulfonyl-benzo[f]quinoxaline) or FK-506 blocked the hypoxic seizure-induced increase in CaN expression. These data suggest that Ca2+-permeable AMPARs and CaN regulate inhibitory synaptic transmission in a novel plasticity pathway that may play a role in epileptogenesis in the immature brain.

Topics: 6-Cyano-7-nitroquinoxaline-2,3-dione; Animals; Animals, Newborn; Blotting, Western; Calcineurin; Dose-Response Relationship, Radiation; Electric Stimulation; Excitatory Amino Acid Antagonists; Excitatory Postsynaptic Potentials; gamma-Aminobutyric Acid; Gene Expression Regulation, Developmental; Hippocampus; Hypoxia; Immunoprecipitation; In Vitro Techniques; Neural Inhibition; Patch-Clamp Techniques; Rats; Receptors, AMPA; Receptors, GABA-A; Seizures; Synapses; Synaptic Transmission; Tacrolimus; Time Factors

Previous developmental studies in vitro suggested that the inhibitory neurotransmitter GABA exerts depolarizing and excitatory actions on the immature neurons and that depolarizing GABA is causally linked to ictal activity during the first weeks of postnatal life. However, remarkably little is known on the role of GABA in the generation of neonatal seizures in vivo. Here, using extracellular recordings from CA3 hippocampus, we studied the effects of GABA(A)-acting drugs on electrographic seizures induced by local intrahippocampal injection of the epileptogenic agents (high K(+)/low Mg(2+)) in the nonanesthetized rats in vivo and in the hippocampal slices in vitro during the second postnatal week (postnatal days P8-12). We found that in vivo, the induction of ictal-like events was facilitated by co-infusion of high-K(+)/low Mg(2+) together with the GABA(A) antagonist bicuculline or gabazine. Moreover, the infusion of bicuculline alone caused ictal-like activity in approximately 30% of cases. Co-infusion of the GABA(A) receptor agonist isoguvacine or the GABA(A)-positive allosteric modulator diazepam completely prevented high-K(+)/low Mg(2+)-induced seizures. In in vitro studies using hippocampal slices, we also found that high-K(+)/low Mg(2+) produced ictal activity that was exacerbated by bicuculline and gabazine and reduced by isoguvacine. Thus in the model of high-K(+)/low Mg(2+)-induced seizures both in in vivo and in vitro conditions, GABA, acting via GABA(A) receptors, has an anticonvulsant effect during the critical developmental period of enhanced excitability.

Topics: 2-Amino-5-phosphonovalerate; 6-Cyano-7-nitroquinoxaline-2,3-dione; Animals; Animals, Newborn; Bicuculline; Disease Models, Animal; Drug Interactions; Excitatory Amino Acid Antagonists; GABA Agonists; GABA Antagonists; gamma-Aminobutyric Acid; Hippocampus; In Vitro Techniques; Isonicotinic Acids; Lipoproteins; Magnesium; Membrane Potentials; Neurons; Patch-Clamp Techniques; Potassium; Pyridazines; Rats; Seizures

We have recently reported that excitatory GABAergic and glutamatergic mechanisms may be involved in the generation of seizure-like (ictal) rhythmic synchronization (afterdischarge), induced by a strong synaptic stimulation of the CA1 pyramidal cells in the mature rat hippocampus in vitro. To clarify the network mechanism of this neuronal synchronization, dual whole-cell patch-clamp recordings of the afterdischarge responses were performed simultaneously from a variety of interneurones and their neighbouring pyramidal cells in hippocampal CA1-isolated slice preparations. According to morphological and electrophysiological criteria, the recorded interneurones were then classified into distinct subtypes. The non-fast-spiking interneurones located in the strata lacunosum-moleculare and radiatum hardly discharged during the afterdischarge, whereas most of the fast-spiking and non-fast-spiking interneurones in the strata oriens and pyramidale, including the basket, chandelier and bistratified cells, exhibited prominent firings that were precisely synchronous with oscillatory responses in the pyramidal cells. Field potential recordings showed that excitatory synaptic transmissions might take place primarily in the strata oriens and pyramidale during the afterdischarge. Restricted lesions in the strata oriens and pyramidale, but not in the other layers, resulted in the complete desynchronization of afterdischarge activity, and also, local application of glutamate receptor antagonists to these layers blocked the expression of afterdischarge. The present findings indicate that the neuronal synchronization of epileptic afterdischarge may be accomplished in a 'positive feedback circuit' formed by the excitatory GABAergic interneurones and the glutamatergic pyramidal cells within the strata oriens and/or pyramidale of the hippocampal CA1 region.

Topics: 2-Amino-5-phosphonovalerate; 6-Cyano-7-nitroquinoxaline-2,3-dione; Animals; Electric Stimulation; Electrophysiology; Excitatory Amino Acid Antagonists; gamma-Aminobutyric Acid; Glutamic Acid; Hippocampus; In Vitro Techniques; Interneurons; Nerve Net; Patch-Clamp Techniques; Pyramidal Cells; Rats; Rats, Wistar; Seizures; Synapses

Renal failure has been viewed as a state of cellular calcium toxicity due to the retention of small fast-acting molecules. We have tested this hypothesis and identified potentially neuroexcitatory compounds among a number of putative uremic neurotoxins by examining the acute in vitro effects of these compounds on cultured central neurons. The in vitro neuroexcitatory and synergistic effects of guanidinosuccinate and spermine were also examined in vivo.. The acute effects of 17 candidate uremic neurotoxins on murine spinal cord neurons in primary dissociated cell culture were investigated using the tight-seal whole-cell recording technique. The compounds studied comprised low-molecular-weight solutes like urea, indoles, guanidino compounds, polyamines, purines and phenoles, homocysteine, orotate, and myoinositol. Currents evoked by these compounds were further examined using various ligand- and voltage-gated ion channel blockers. The acute in vivo effects of guanidinosuccinate and spermine were behaviorally assessed following their injection in mice.. It was shown that 3-indoxyl sulfate, guanidinosuccinate, spermine, and phenol evoked significant whole-cell currents. Inward whole-cell current evoked by 3-indoxyl sulfate was not blocked by any of the applied ligand- or voltage-gated ion channel blockers, and the compound appeared to influence miscellaneous membrane ionic conductances, probably involving voltage-gated Ca2+ channels as well. Phenol-evoked outward whole-cell currents were at least partly due to the activation of voltage-gated K+ channels, but may also involve a variety of other ionic conductances. On the other hand, inward whole-cell currents evoked by guanidinosuccinate and spermine were shown to be due to specific interaction with voltage- and ligand-gated Ca2+ channels. Guanidinosuccinate-evoked current was caused by activation of N-methyl-d-aspartate (NMDA) receptor-associated ion channels. Low (micromol/L) concentrations of spermine potentiated guanidinosuccinate-evoked current through the action of spermine on the polyamine binding site of the NMDA receptor complex, whereas current evoked by high (mmol/L) concentrations of spermine alone involved direct activation of voltage-gated Ca2+ channels. Finally, intracerebroventricular administration of 0.25 micromol/L spermine potentiated clonic convulsions induced by guanidinosuccinate. These neuroexcitatory and synergistic effects of guanidinosuccinate and spermine could take place at pathophysiologic concentrations.. The observed in vitro and in vivo effects of uremic retention solutes suggest that the identified compounds could play a significant role in uremic pathophysiology. Some of the compounds tested displayed in vitro and in vivo neuroexcitatory effects that were mediated by ligand- and voltage-gated Ca2+ channels. The findings suggest a mechanism for the involvement of calcium toxicity in the central nervous system complications in renal failure with particular reference to guanidinosuccinate and spermine.

Topics: 2-Amino-5-phosphonovalerate; 6-Cyano-7-nitroquinoxaline-2,3-dione; Animals; Bicuculline; Calcium Channels; Cells, Cultured; Drug Synergism; Excitatory Amino Acid Antagonists; GABA Antagonists; Guanidines; Membrane Potentials; Mice; Neurons; Nickel; Piperidines; Potassium Channel Blockers; Seizures; Spermine; Spinal Cord; Succinates; Synapses; Tetraethylammonium; Tetrodotoxin; Uremia

GABA, which generally mediates inhibitory synaptic transmissions, occasionally acts as an excitatory transmitter through intense GABA(A) receptor activation even in adult animals. The excitatory effect results from alterations in the gradients of chloride, bicarbonate, and potassium ions, but its functional role still remains a mystery. Here we show that such GABAergic excitation participates in the expression of seizure-like rhythmic synchronization (afterdischarge) in the mature hippocampal CA1 region. Seizure-like afterdischarge was induced by high-frequency synaptic stimulation in the rat hippocampal CA1-isolated slice preparations. The hippocampal afterdischarge was completely blocked by selective antagonists of ionotropic glutamate receptors or of GABA(A) receptor, and also by gap-junction inhibitors. In the CA1 pyramidal cells, oscillatory depolarizing responses during the afterdischarge were largely dependent on chloride conductance, and their reversal potentials (average -38 mV) were very close to those of exogenously applied GABAergic responses. Moreover, intracellular loading of the GABA(A) receptor blocker fluoride abolished the oscillatory responses in the pyramidal cells. Finally, the GABAergic excitation-driven afterdischarge has not been inducible until the second postnatal week. Thus, excitatory GABAergic transmission seems to play an active functional role in the generation of adult hippocampal afterdischarge, in cooperation with glutamatergic transmissions and possible gap junctional communications. Our findings may elucidate the cellular mechanism of neuronal synchronization during seizure activity in temporal lobe epilepsy.

Topics: 6-Cyano-7-nitroquinoxaline-2,3-dione; Acetazolamide; Animals; Animals, Newborn; Anti-Ulcer Agents; Atropine; Bicuculline; Carbenoxolone; Carbonic Anhydrase Inhibitors; Chlorides; Drug Interactions; Electric Conductivity; Electric Stimulation; Evoked Potentials; Excitatory Amino Acid Antagonists; Fluorides; GABA Antagonists; gamma-Aminobutyric Acid; Hippocampus; In Vitro Techniques; Membrane Potentials; Muscarinic Antagonists; Patch-Clamp Techniques; Phosphinic Acids; Propanolamines; Pyramidal Cells; Rats; Rats, Wistar; Seizures; Valine

Intracerebral injection of kainic acid in cerebral cortex, hippocampus or amygdala in cats chronically implanted showed that: 1) Hippocampus and amygdala presented a greater sensitivity than the cerebral cortex, while hippocampus presented a greater sensitivity than the amygdala to the generation of an epileptic focus. 2) Comparison of latency, mean duration of afterdischarges, and the mean time period to obtain the peak intensity of the afterdischarge in the three cited structures, showed that mean latency of the first afterdischarge was significantly shorter in hippocampus and amygdala compared with the cerebral cortex. Moreover the mean time period to reach the peak intensity of the afterdischarge was again shorter in the subcortical structures. 3) The epileptic foci both in hippocampus and amygdala were blocked by CNQX and muscimol. 4) The behavioral changes depended on the intensity of the epileptic process. Tonic-clonic convulsions appeared only when the motor cerebral cortex was involved. Finally, 5) kainic acid injections in hippocampus and amygdala elicited an intense neuronal destruction and gliosis of these structures. We conclude that intracerebral injection of low doses of kainic acid in cats represent a good model to study focal epileptic thresholds in the CNS.

Topics: 6-Cyano-7-nitroquinoxaline-2,3-dione; Amygdala; Animals; Cats; Cerebral Cortex; Electroencephalography; Electrophysiology; Excitatory Amino Acid Antagonists; Female; GABA Agonists; Hippocampus; Kainic Acid; Male; Microinjections; Muscimol; Seizures

The aim of the present work was to test in adult cats the capability of three glutamatergic agonists, NMDA, AMPA and ACDP as epileptogenic agents. Drugs were microinjected in amygdala or hippocampus, and once generated an epileptic focus three selective glutamatergic antagonists NMDA, CNQX and MCPG, were tested. Before and after injection both the EEG and the behavior were continuously monitored. The results were as follows: 1) AMPA showed a greater capability than NMDA or ACPD to generate a chronic epileptic focus; 2) AMPA elicited a greater epileptogenic effect in hippocampus than in amygdala; NMDA had similar epileptogenic effect in both cited structures, and ACPD had not effect; 3) of the three glutamatergic antagonists used to block a long lasting focus, the most effective one was CNQX, which showed a greater effect in hippocampus than in amygdala; 4) comparison between the epileptogenic effect of AMPA and kainic acid (first paper) in the same structure, showed that kainic acid is about 15 fold more epileptogenic. A discussion of the probable mechanisms of these results was undertaken.

Topics: 6-Cyano-7-nitroquinoxaline-2,3-dione; alpha-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic Acid; Amygdala; Animals; Cats; Dizocilpine Maleate; Electrophysiology; Excitatory Amino Acid Agonists; Excitatory Amino Acid Antagonists; Female; Hippocampus; Male; Microinjections; N-Methylaspartate; Neuroprotective Agents; Seizures

2-Methyl-4-oxo-3H-quinazoline-3-acetyl piperidine (Q5), a selective inhibitor of the fast-desensitising component of transmembrane Ca2+ ion influx to (S)-alpha-amino-3-hydroxy-5-methyliso-xazole-4-propionate ((S)-AMPA) was tested for possible anticonvulsant effects in the low-[Mg2+] model of experimental epilepsy. Evolutionary analysis of burst parameters such as half-width, decay time constant, burst multiplicity, instantaneous frequency and amplitude disclosed an approximate doubling of half-width within periods of interictal activity, being predictive for the onset of seizure-like events (SLEs). We found that SLEs observed in the CA3 region of rat hippocampal slices were suppressed by the application of 50 microM Q5. These results suggest an AMPA receptor function shaping the dynamics of spontaneous epileptiform activity.

Topics: 6-Cyano-7-nitroquinoxaline-2,3-dione; Animals; Anticonvulsants; Electrophysiology; Excitatory Amino Acid Antagonists; In Vitro Techniques; Magnesium; Male; Membrane Potentials; Piperidines; Quinazolines; Rats; Rats, Wistar; Receptors, AMPA; Receptors, Glutamate; Seizures

This study examined the conditions that are required for the appearance of the long-duration seizure-like activity that can be recorded in hippocampal slices. Spontaneous interictal activity was induced in CA1 and CA3 by perfusing hippocampal slices with high potassium, cesium, 4-aminopyridine, or tetraethylammonium chloride, in normal levels of calcium. Synaptic transmission was then blocked by the addition of neurotransmitter receptor blockers (6-cyano-7-nitroquinoxaline-2,3-dione, D,L-2-amino-5-phosphonopentanoic acid, and bicuculline) or the calcium channel blocker cadmium, resulting in complete blockade of the interictal discharges and the appearance of spontaneous seizure-like events (ictal-like discharges) primarily in CA1 and the dentate gyrus. Blocking synaptic transmission in normal artificial cerebrospinal fluid did not induce ictal-like discharges in any region. The results demonstrate that ictal-like discharges can appear in normal levels of extracellular calcium when chemical synaptic transmission is blocked pharmacologically. The results suggest that an increase in neuronal excitability and absence of interictal activity promote the appearance of the longer ictal-like discharges.

Topics: 2-Amino-5-phosphonovalerate; 6-Cyano-7-nitroquinoxaline-2,3-dione; Animals; Bicuculline; Calcium; Convulsants; Electrophysiology; Excitatory Amino Acid Antagonists; Extracellular Space; Female; Hippocampus; In Vitro Techniques; Male; Rats; Rats, Sprague-Dawley; Reaction Time; Reference Values; Seizures; Synaptic Transmission

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

Postsynaptic metabotropic glutamate (mGlu) receptor-activated inward current mediated by Na(+)-Ca(2+) exchange was compared in basolateral amygdala (BLA) neurons from brain slices of control (naïve and sham-operated) and amygdala-kindled rats. In control neurons, the mGlu agonist, quisqualate (QUIS; 1-100 microM), evoked an inward current not associated with a significant change in membrane slope conductance, measured from current-voltage relationships between -110 and -60 mV, consistent with activation of the Na(+)-Ca(2+) exchanger. Application of the group I selective mGlu receptor agonist (S)-3,5-dihydroxyphenylglycine [(S)-DHPG; 10-1000 microM] or the endogenous agonist, glutamate (10-1000 microM), elicited the exchange current. QUIS was more potent than either (S)-DHPG or glutamate (apparent EC(50) = 19 microM, 57 microM, and 0.6 mM, respectively) in activating the Na(+)-Ca(2+) exchange current. The selective mGlu5 agonist, (R, S)-2-chloro-5-hydroxyphenylglycine [(R,S)-CHPG; apparent EC(50) = 2. 6 mM] also induced the exchange current. The maximum response to (R, S)-DHPG was about half of that of the other agonists suggesting partial agonist action. Concentration-response relationships of agonist-evoked inward currents were compared in control neurons and in neurons from kindled animals. The maximum value for the concentration-response relationship of the partial agonist (S)-DHPG- (but not the full agonist- [QUIS or (R,S)-CHPG]) induced inward current was shifted upward suggesting enhanced efficacy of this agonist in kindled neurons. Altogether, these data are consistent with a kindling-induced up-regulation of a group I mGlu-, possibly mGlu5-, mediated responses coupled to Na(+)-Ca(2+) exchange in BLA neurons.

Topics: 2-Amino-5-phosphonovalerate; 6-Cyano-7-nitroquinoxaline-2,3-dione; Amygdala; Animals; Calcium; Dose-Response Relationship, Drug; Epilepsy; Excitatory Amino Acid Agonists; Excitatory Amino Acid Antagonists; Glutamic Acid; Glycine; Kindling, Neurologic; Male; Membrane Potentials; Methoxyhydroxyphenylglycol; Phenylacetates; Quisqualic Acid; Rats; Rats, Sprague-Dawley; Receptors, Metabotropic Glutamate; Seizures; Sodium; Tetrodotoxin; Up-Regulation

Application of the convulsant 4-aminopyridine (4AP, 50 microM) to adult mouse combined hippocampus-entorhinal cortex (EC) slices induces interictal and ictal discharges originating from CA3 and EC respectively. In this model of limbic seizures, ictal discharges disappear over time and are reestablished after Schaffer collateral cut, a procedure that blocks interictal propagation from CA3 to EC. Here we tested whether this form of network plasticity is operant in hippocampus-EC slices obtained from young (10-25 day-old) mice. In these experiments 4AP elicited interictal (duration = 100-250 ms; interval = 0.7 +/- 0.2 s, mean +/- SD, n = 20) and ictal (duration = 267 +/- 37 s; interval = 390 +/- 37 s, n = 20) discharges in both CA3 and EC. However, in young mouse slices the ictal events occurred throughout the experiment, whereas Schaffer collateral cut abolished CA3-driven interictal discharges in EC without influencing ictal activity (n = 10). Perforant path lesion prevented the spread of EC-driven ictal events to CA3, where interictal and short ictal discharges (duration = 32 +/- 11 s; interval = 92 +/- 9.7 s, n = 8) continued to occur. Hence, two independent forms of ictal activity were seen in CA3 and in EC after separation of these structures. In intact hippocampus-EC slices, ictal discharges were reduced by an N-methyl-D-aspartate receptor antagonist (n = 10). Under these conditions, Schaffer collateral cut abolished ictal activity in EC, not in CA3 (n = 6). Thus the young mouse hippocampus-EC loop has different properties as compared with adult tissue. These differences, which include the inability of hippocampal outputs to control ictal discharge generation in EC and the ability of the loop to sustain ictal activity, may contribute to the low-seizure threshold seen in young individuals.

Topics: 4-Aminopyridine; 6-Cyano-7-nitroquinoxaline-2,3-dione; Animals; Entorhinal Cortex; Excitatory Amino Acid Antagonists; Hippocampus; In Vitro Techniques; Limbic System; Membrane Potentials; Mice; Mice, Inbred BALB C; Perforant Pathway; Piperazines; Receptors, N-Methyl-D-Aspartate; Seizures

We studied the contribution of GABAergic (gamma-aminobutyric acid) neurotransmission to epileptiform activity using the horizontal hippocampal rat brain slice. Seizure-like (ictal) activity was evoked in the CA1 area by applying high-frequency trains (80 Hz for 2 s) to the Schaffer collaterals. Whole-cell recordings from stratum oriens-alveus interneurons revealed burst firing with superimposed high-frequency spiking which was synchronous with field events and pyramidal cell firing during ictal activity. On the other hand, interictal interneuronal bursts were synchronous with large-amplitude inhibitory postsynaptic potentials (IPSPs) in pyramidal cells. Excitatory and inhibitory postsynaptic potentials were simultaneously received by pyramidal neurons during the ictal afterdischarge, and were synchronous with interneuronal bursting and field potential ictal events. The GABAA receptor antagonist bicuculline greatly reduced the duration of the ictal activity in the CA1 layer, and evoked rhythmic interictal synchronous bursting of interneurons and pyramidal cells. With intact GABAergic transmission, interictal field potential events were synchronous with large amplitude IPSPs (9.8 +/- 2.4 mV) in CA1 pyramidal cells, and with interneuronal bursting. Simultaneous dual recordings revealed synchronous IPSPs received by widely separated pyramidal neurons during ictal and interictal periods, indicative of widespread interneuronal firing synchrony throughout the hippocampus. CA3 pyramidal neurons fired in synchrony with interictal field potential events recorded in the CA1 layer, and glutamate receptor antagonists abolished interictal interneuronal firing and synchronous large amplitude IPSPs received by CA1 pyramidal cells. These observations provide evidence that the interneuronal network may be entrained in hyperexcitable states by GABAergic and glutamatergic mechanisms.

Topics: 2-Amino-5-phosphonovalerate; 6-Cyano-7-nitroquinoxaline-2,3-dione; Action Potentials; Animals; Electric Stimulation; Evoked Potentials; gamma-Aminobutyric Acid; Hippocampus; In Vitro Techniques; Interneurons; Male; Nerve Net; Patch-Clamp Techniques; Pyramidal Cells; Rats; Rats, Wistar; Receptors, GABA-A; Seizures

Synaptic physiology was studied in horizontal slices of rat somatosensory neocortex. Intrinsic properties of pyramidal neurons from horizontal slices resembled those recorded in cells from coronal slices, but cells in superficial horizontal slices displayed more prominent fast and slow inhibition, while cells from deeper slices showed disinhibition. This disinhibition in deeper horizontal slices resulted in epileptogenesis in 81% of middle and 35% of deep layer horizontal slices. Brief exposure to glutamate antagonists and dantrolene was ineffective in preventing epileptic activity, but limited pentobarbital exposure reduced the proportion of deep slices manifesting epileptiform activity by 75%. Thus, within cortex inhibition dominates superficially and excitation predominates in deeper layers. While the cortex is vulnerable to hyperexcitability when superficial cortex is compromised, enhancing fast inhibition can reset the excitation-inhibition balance, and prevent epileptogenesis.

Topics: 6-Cyano-7-nitroquinoxaline-2,3-dione; Animals; Dantrolene; Electric Stimulation; Excitatory Amino Acid Antagonists; Excitatory Postsynaptic Potentials; GABA Modulators; In Vitro Techniques; Muscle Relaxants, Central; Neocortex; Neural Inhibition; Pentobarbital; Piperazines; Pyramidal Cells; Rats; Seizures; Somatosensory Cortex; Synapses

Alpha-dendrotoxin (alpha-DTx), a snake venom toxin which blocks several types of fast-activating voltage-dependent potassium channels, induces limbic seizures and neuronal damage when injected into the brain. The mechanisms underlying these convulsant and neuropathological actions are not fully understood. We have studied the effects of alpha-DTx on neurotransmitter release and electrical activity in rat hippocampal brain slices and the role of excitatory amino acid receptors in mediating these actions of the toxin. alpha-DTx increased the basal release of acetylcholine, glutamate, aspartate, and GABA in a concentration-dependent manner and induced epileptiform bursting in the CA1 and CA3 regions of the slice. The increase in neurotransmitter release was evident during the first 4 min after toxin addition, whereas the bursting appeared after a concentration-dependent delay (20-40 min with 250 nM toxin). The N-methyl-D-aspartate (NMDA) receptor antagonists AP5 and MK-801 had no effect on the frequency or amplitude of dendrotoxin-induced epileptiform bursts, but the non-NMDA antagonists CNQX and DNQX abolished bursting in both CA1 and CA3 within 4-6 min. In contrast, the toxin-induced increases in neurotransmitter release were not blocked by DNQX. This study has demonstrated that, following exposure to alpha-DTx, there is a rapid increase in the release of neurotransmitters which precedes the onset of epileptiform bursting in the hippocampus. Since DNQX abolished the bursting but had no effect on the increase in neurotransmitter release, these results suggest that DNQX blocks alpha-DTx-induced epileptiform activity by antagonism of postsynaptic non-NMDA receptors.

Topics: 2-Amino-5-phosphonovalerate; 6-Cyano-7-nitroquinoxaline-2,3-dione; Acetylcholine; Action Potentials; Animals; Aspartic Acid; Dizocilpine Maleate; Elapid Venoms; Excitatory Amino Acid Antagonists; gamma-Aminobutyric Acid; Glutamic Acid; Hippocampus; Male; Neurotoxins; Neurotransmitter Agents; Potassium Channels; Quinoxalines; Rats; Rats, Wistar; Receptors, N-Methyl-D-Aspartate; Seizures

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

Cortical structures such as the hippocampus and cerebral cortex are considered to be particularly susceptible to seizure and epileptiform electrical activity and, as such, are the focus of intense investigation relative to hyperexcitability. To determine whether parallel glutamate-mediated hyperexcitability and seizure-like activity in the rat can be generated by neurons irrespective of their origin within the CNS, we maintained cells from the spinal cord,hippocampus, olfactory bulb, striatum, hypothalamus, and cortex in the long-term presence of glutamate receptor antagonists 2-amino-5-phosphonovalerate and 6-cyano-7-nitroquinoxaline-2-3-dione. After removal of chronic (three to 11 weeks) glutamate receptor block, whole-cell patch-clamp recordings from current-clamped neurons (n = 94) revealed an immediate increase in large excitatory postsynaptic potentials and a depolarization of 20-35 mV that was often sustained for recording periods lasting 5 min (54% of 66 neurons from all six areas). The intense activity was not seen in age-matched control neurons not subjected to chronic glutamate receptor block. Selective blockade of ionotropic glutamate receptors showed that the hyperexcitability was due to an enhanced response through both AMPA/kainate and N-methyl-D-aspartate receptors. Relief from chronic glutamate receptor block also increased inhibitory activity, as revealed by an increase in inhibitory postsynaptic currents while neurons were voltage-clamped at -25 mV. These inhibitory postsynaptic currents could be blocked with bicuculline, indicating that they were mediated by an enhanced GABA release. This enhanced GABA activity reduced, but did not eliminate, the glutamate-mediated hyperactivity, shown by an increase in both intracellular Ca2+ and excitatory electrical activity when bicuculline was added. When the glutamate receptor block was removed, cells (n > 1000) from all six regions showed exaggerated Ca2+ activity, characterized by abnormally high increases in intracellular Ca2+, rising from basal levels of 50-100 nM up to 150-1600 nM. Cd2+ eliminated the hyperexcitability by blocking Ca2+ channels, and reducing excitatory transmitter release and response. Fura-2 digital imaging revealed Ca2+ oscillations with periods ranging from 4 to 60 s. Ca2+ peaks in oscillations in oscillations were synchronized among most neurons recorded simultaneously. That synchronization was dependent on a mechanism involving voltage-dependent Na+ channels was demonst

Topics: 2-Amino-5-phosphonovalerate; 6-Cyano-7-nitroquinoxaline-2,3-dione; Animals; Brain; Cadmium; Calcium; Cell Survival; Cells, Cultured; Cerebral Cortex; Corpus Striatum; Embryo, Mammalian; Excitatory Amino Acid Antagonists; Glutamic Acid; Hippocampus; Hypothalamus; Membrane Potentials; Neurons; Olfactory Bulb; Organ Specificity; Patch-Clamp Techniques; Rats; Rats, Sprague-Dawley; Receptors, Glutamate; Seizures; Spinal Cord; Tetrodotoxin

Excitatory amino acid (EAA)-like and excitotoxic properties of the secondary metabolite of cyanide, 2-iminothiazolidine-4-carboxylic acid, (2-ICA) were evaluated because of its possible role in cyanide-induced neurotoxicity. Intracerebroventricular (i.c.v.) injections of 2-ICA in mice produced wild-running seizures that were qualitatively and quantitatively similar to seizures observed with glutamate. 2-ICA, kainate and proline seizures were prevented by both the NMDA and non-NMDA antagonists, MK-801 and CNQX, respectively. In contrast, NMDA-induced seizures were prevented by MK-801, but not CNQX. When infused i.c.v. in rats over a seven day period, 2-ICA produced extensive and selective loss of CA-1 pyramidal neurons of the hippocampus. In hippocampal slices preloaded with D-[3H]aspartate, 2-ICA superfusion did not evoke release nor significantly augment potassium stimulated release of the radiolabeled transmitter. These findings indicate 2-ICA has excitotoxic properties and its role in cyanide neurotoxicity deserves further study.

Topics: 6-Cyano-7-nitroquinoxaline-2,3-dione; Animals; Carboxylic Acids; Cyanides; Dizocilpine Maleate; Dose-Response Relationship, Drug; Glutamic Acid; Hippocampus; Proline; Rats; Rats, Sprague-Dawley; Seizures

Non-NMDA receptor antagonists CNQX, DNQX, and NBQX (10-40 mg/kg IP) were tested against pentylenetetrazol-induced (100 mg/kg SC) seizures in 7 to 90-day-old rats. All three drugs significantly decreased the incidence of tonic hindlimb component of tonic-clonic pentylenetetrazol seizures, often in favor of increased incidence of forelimb tonus throughout development. In addition, in 7 to 25-day-old rats, DNQX and NBQX decreased the severity of seizures due to a decrease in total incidence of the tonic component of tonic-clonic seizures compared to age-matched controls. However, neither drug was able to consistently suppress the incidence or increase latency to onset of clonic and tonic-clonic pentylenetetrazol seizures. The data suggest that, during development, non-NMDA receptor transmission may play a role in the generation of the tonic component, but not in the generation of other components of pentylenetetrazol-induced seizures.

Topics: 6-Cyano-7-nitroquinoxaline-2,3-dione; Aging; Animals; Anticonvulsants; Behavior, Animal; Epilepsy, Tonic-Clonic; Hindlimb; Male; Muscle Tonus; Pentylenetetrazole; Quinoxalines; Rats; Rats, Wistar; Receptors, AMPA; Receptors, Kainic Acid; Seizures; Synaptic Transmission

Two rabbits immunized with a portion of glutamate receptor (GluR) subunit GluR3 (amino acids 245-457) exhibited seizure-like behaviors, suggesting that antibodies to GluR3 may modulate neuronal excitability. Using whole-cell recording, rabbit GluR3 antisera were tested on cultured fetal mouse cortical neurons. In a subset of kainate-responsive neurons, miniperfusion of antisera and IgG evoked currents that were blocked by CNQX. Immunoreactivity to synthetic peptides prepared to subregions GluR3A (amino acids 245-274) and GluR3B (amino acids 372-395) was present in both rabbit sera. Peptide GluR3B, but not GluR3A, specifically blocked antisera- and IgG-evoked currents. Similar receptor activation and anti-GluR3 reactivity was present in sera from patients with active Rasmussen's encephalitis, an intractable pediatric epilepsy. Thus, antibodies to GluR3 define a region involved in agonist binding and specific receptor activation. These data suggest that antibodies to neuronal receptors can function as agonists and that autoantibodies to GluRs may be highly specific neurotoxicants in some neurological diseases.

Topics: 6-Cyano-7-nitroquinoxaline-2,3-dione; Amino Acid Sequence; Animals; Antibodies; Binding Sites; Cell Line; Cells, Cultured; Electric Conductivity; Embryo, Mammalian; Humans; Immunoglobulin G; Kainic Acid; Kidney; Mice; Molecular Sequence Data; Neurons; Peptide Fragments; Receptors, Glutamate; Seizures; Transfection

The behavioural and convulsant effects of imipenem (Imi), a carbapenem derivative, were studied after intraperitoneal (i.p.) or intracerebroventricular (i.c.v.) administration in DBA/2 mice, a strain genetically susceptible to sound-induced seizures. The anticonvulsant effects of some excitatory amino acid antagonists and muscimol (Msc), a GABAA agonist, against seizures induced by i.p. or i.c.v. administration of Imi were also evaluated. The present study demonstrated that the order of anticonvulsant activity in our epileptic model, after i.p. administration, was (+)-5-methyl-10,11-dihydro-5H-dibenzo(a,d)-cyclohepten-5,10-imine maleate (MK-801) > (+/-)(E)-2-amino-4-methyl-5-phosphono-3-pentenoate ethyl ester (CGP 39551) > 3-((+/-)-2-carboxypiperazin-4-yl)propenyl-1-phosphonic acid (CPPene) > 3-((+/-)-2-carboxypiperazin-4-yl)-propyl-1-phosphonic acid (CCP) > 2,3-dihydroxy-6-nitro-7-sulfamoylbenzo(F)-quinoxaline (NBQX). Ifenprodil, a compound acting on the polyamine site of NMDA receptor complex was unable to protect against seizures induced by Imi, suggesting that the poliamine site did not exert a principal role in the genesis of seizures induced by Imi. In addition, the order of anticonvulsant potency in our epileptic model, after i.c.v. administration, was CPPene > MK-801 > Msc > (-)-2-amino-7-phosphonic acid (AP7) > gamma-D-glutamylaminomethylsulphonate (gamma-D-GAMS) > NBQX > kynurenic acid (KYNA) > 6-cyano-7-nitro-quinoxaline-2,3-dione (CNQX). The relationship between the different site of action and the anticonvulsant activity of these derivatives was discussed. Although the main mechanism of Imi induced seizures cannot be easily determined, potential interactions with the receptors of the excitatory amino acid neurotransmitters exists. In fact, antagonists of excitatory amino acids are able to increase the threshold for the seizures or to prevent the seizures induced by Imi. In addition, Imi acts on the central nervous system by inhibition of GABA neurotransmission and Msc, a selective GABAA agonist, was able to protect against seizures induced by Imi.

Topics: 2-Amino-5-phosphonovalerate; 6-Cyano-7-nitroquinoxaline-2,3-dione; Amino Acids; Animals; Anticonvulsants; Dizocilpine Maleate; Excitatory Amino Acids; Glutamine; Imipenem; Kynurenic Acid; Mice; Mice, Inbred DBA; Muscimol; Piperazines; Piperidines; Quinoxalines; Seizures

Pilocarpine injection into rodents leads to the development of chronic limbic seizures that follow an initial status epilepticus and a seizure-free interval. It has been proposed that a decreased efficacy of the mechanisms that buffer the extracellular concentration of K+ ([K+]o) leads to an increase in seizure susceptibility. Therefore, we analyzed the changes in [K+]o associated with the synchronous activity induced by 4-aminopyridine (4AP) in hippocampal slices obtained from control and pilocarpine-treated rats. At all recording sites (i.e. stratum radiatum of the CA1 and CA3 subfields, and hilus of the dentate gyrus), the amplitude of GABA-mediated synchronous field potentials induced by 4AP, as well as the associated [K+]o increases, were significantly reduced in slices obtained from the pilocarpine-treated rats. In the control group, the field-potential amplitudes reached 1 mV (i.e. 1.7 +/- 0.3 mV in CA1, 0.93 +/- 0.2 mV in CA3, and 1.03 +/- 0.12 mV in the hilus; mean +/- SEM), while the accompanying rises in [K+]o exceeded 4 mM (i.e. 4.17 +/- 0.15 mM in CA1, 4.04 +/- 0.12 mM in CA3, 4.04 +/- 0.11 mM in the hilus) from a baseline of 3.25 mM. The corresponding values in slices from the pilocarpine-treated group were rarely greater than 0.4 mV (i.e. 0.3 +/- 0.09 mV in CA1, 0.27 +/- 0.03 mV in CA3 and 0.38 +/- 0.06 mV in the hilus), and larger than 3.6 mM (i.e. 3.63 +/- 0.04 mM in CA1, 3.64 +/- 0.03 mM in CA3 and 3.60 +/- 0.04 mM in the hilus) from a similar baseline value. With pilocarpine, the rate of occurrence of the GABA-mediated potential significantly decreased from 0.035 to 0.016 s-1. Since the rises in [K+]o decreased rather than increased and their overall duration was unchanged (possibly reflecting cell loss), we conclude that a modification of [K+]o buffering capacity is unlikely to account for the appearance of in vivo seizures in the pilocarpine model of epilepsy.

Topics: 4-Aminopyridine; 6-Cyano-7-nitroquinoxaline-2,3-dione; Animals; Evoked Potentials; Excitatory Amino Acid Agonists; Excitatory Amino Acid Antagonists; Extracellular Space; Hippocampus; In Vitro Techniques; Male; Muscarinic Agonists; Pilocarpine; Piperazines; Potassium; Rats; Seizures

Nerve growth factor (NGF) and brain-derived neurotrophic factor (BDNF) are two structurally-related neurotrophins synthesized in dentate gyrus granule cells and pyramidal neurons of the hippocampal formation. These neurons receive excitatory glutamatergic afferents from the entorhinal cortex via the angular bundle/perforant path. In the present study, we tested whether electrophysiological stimulation of this glutamatergic pathway modifies NGF or BDNF messenger RNA (mRNA) expression in vivo. Within hours following brief trains of high frequency angular bundle stimulation, the levels of mRNA encoding both neurotrophins were increased exclusively in granule cells of the ipsilateral dentate gyrus. The increase in neurotrophic factor mRNA expression was found to be mediated through the N-methyl-D-aspartate (NMDA) glutamate receptor subtype, and occurred in the absence of seizure. These findings provide evidence that neurotrophic factor mRNA levels in the hippocampal formation are increased by direct activation of excitatory afferents originating in the entorhinal cortex. We suggest that the function of some neurotrophin-responsive neuronal populations may depend upon the integrity and activity of neurons in the entorhinal cortex, a population of neurons reported to be compromised in patients with Alzheimer's disease.

Topics: 2-Amino-5-phosphonovalerate; 6-Cyano-7-nitroquinoxaline-2,3-dione; Animals; Brain-Derived Neurotrophic Factor; Electric Stimulation; Female; Gene Expression Regulation; Hippocampus; Long-Term Potentiation; Nerve Growth Factors; Nerve Tissue Proteins; Quinoxalines; Rats; Receptors, N-Methyl-D-Aspartate; RNA, Messenger; Seizures; Tetany

Repetitive synchronized neuronal discharging that lasts for seconds and even minutes in in vitro brain slice preparations are important new models in experimental epilepsy. In hippocampal slices from 1-2-week-old rats, individual CA3 pyramidal cells undergo a sustained depolarization during such electrographic seizures, induced by GABAA receptor antagonists. In experiments reported here these events were produced in small isolated segments of the CA3 subfield, measuring only 400-500 microns along the cell body layer. In such minisclices local application of either kynurenic acid or 6-cyano-7-nitroquinoxaline-2-3-dione (CNQX) to the proximal basilar dendrites abolished the synchronized discharges of electrographic seizures. Interictal spikes appeared unaffected by this treatment. Application of these excitatory amino acid receptor antagonists to distal basilar dendrites or apical dendrites was ineffective. In "larger" minislices, measuring 700-1000 microns along the cell body layer, application of kynurenic acid, CNQX, or TTX to the proximal basilar dendrites did not abolish electrographic seizures but instead selectively suppressed the intracellularly recorded sustained depolarization and the coincident slow negative field potential recorded in proximal basilar dendrites. Results of several experiments suggest that electrographic seizures recorded under these conditions were produced by a remote network of "generator cells." Since the remote neurons were unaffected by local application of the drugs, it seemed likely that they continued to undergo a sustained depolarization. Simultaneous blockade of basilar dendritic synapses in the "generator" population abolished electrographic seizures throughout these larger minislices. These results suggest that the sustained depolarization plays a central role in seizure generation and that it does not have to be generated in every neuron, only in a critical number of "generator cells" for a seizure to occur. Taken together, results presented here suggest that the sustained depolarization of electrographic seizures is a separate physiological process from the more rapid repetitive depolarizations of the seizure discharges and is required if electrographic seizures are to occur. This slow depolarization appears to be synaptically mediated and generated exclusively in proximal basilar dendrites. Therefore, in addition to the excitatory synaptic potentials involved in paroxysmal depolarization shift generation, a second

Topics: 6-Cyano-7-nitroquinoxaline-2,3-dione; Animals; Dendrites; Evoked Potentials; GABA Antagonists; Hippocampus; In Vitro Techniques; Kynurenic Acid; Membrane Potentials; Pyramidal Tracts; Quinoxalines; Rats; Seizures; Synapses; Tetrodotoxin

The excitatory and excitotoxic actions of the endogenous excitatory amino acid (EAA) neurotransmitter, glutamate, are mediated by activation of three common subtypes of EAA receptors: N-methyl-D-aspartate (NMDA), alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA)/quisqualate and kainate receptors. EAA neurotransmitter systems play a number of physiological roles in the regulation and organization of neural systems during development. However, excessive activation of this neurotransmitter system is also implicated in the pathophysiology of several forms of acute and chronic brain injury. In this study, the susceptibility of the developing rat brain to AMPA/quisqualate receptor mediated injury was examined at eight postnatal ages (1-90 days). The receptor agonists, AMPA (25 nmol) or quisqualate (100 nmol), were stereotaxically microinjected unilaterally into the anterior striatum. The severity of resulting brain injury was assessed 5 days later by comparison of reductions in regional cortical and striatal cross-sectional areas. Microinjection of AMPA (25 nmol) produced widespread unilateral forebrain injury in the intermediate postnatal period (days 5-28). The severity of injury resulting from microinjection of a fixed dose of AMPA (25 nmol) transiently exceeded the severity of injury in adults between PND 5-28 with peak sensitivity occurring near PND 10. At PND 1, microinjection of AMPA produced a 24.5 +/- 1.7% reduction in striatal cross-sectional area, which is similar to the response observed in adult animals, and the lesion was confined to the injection site. Susceptibility to AMPA toxicity increased 2-fold from PND 1 to PND 5. At PND 10, the age of maximal sensitivity, the excitotoxic reaction to AMPA extended throughout the entire cerebral hemisphere and the mean striatal cross-sectional area was reduced by 81.7 +/- 3.9%. With advancing postnatal age, the severity of injury progressively diminished and the lesion became confined to the injection site. The developmental pattern of sensitivity to AMPA toxicity in other brain regions differed although peak sensitivity consistently occurred near PND 10. Microinjection of quisqualate produced a developmental pattern of striatal susceptibility similar to AMPA although quisqualate was a considerable less potent neurotoxin. In additional experiments, the in vivo pharmacology of AMPA and quisqualate mediated brain injury was evaluated in a PND 7 rat model in order to determine the neurotoxic cha

Topics: 6-Cyano-7-nitroquinoxaline-2,3-dione; Aging; alpha-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic Acid; Analysis of Variance; Animals; Brain; Brain Injuries; Diazepam; Dizocilpine Maleate; Female; Ibotenic Acid; Male; N-Methylaspartate; Neurotoxins; Organ Specificity; Phenytoin; Quinoxalines; Quisqualic Acid; Rats; Rats, Inbred Strains; Seizures

The involvement of excitatory amino acid (EAA) receptors in mediation of the toxic effects of cocaine was studied in male ICR mice. Cocaine HCl (90 mg/kg, IP) induced seizures in 95% and death within 24 h in 68% (n = 135) of the animals. There was a significant correlation (r = .54) between the time to onset of convulsions and the time to death in mice which died within 30 min of injection (n = 84). Pretreatment with selected EAA receptor antagonists 15 min prior to cocaine differentially blocked cocaine toxicity. Selective N-methyl-D-aspartic acid (NMDA) receptor antagonists (MK-801, dextrorphan, CPP) decreased both the incidence of seizures and mortality. A nonselective EAA antagonist, kynurenic acid, decreased lethality in doses which did not reduce convulsions. A similar action was observed following pretreatment with the selective kainic acid/AMPA receptor antagonist, GDEE. Antagonists at EAA receptors can provide significant protection against cocaine-induced toxicity. Moreover, the data provide evidence for the involvement of both NMDA and non-NMDA receptor subtypes in aspects of cocaine toxicity.

Topics: 6-Cyano-7-nitroquinoxaline-2,3-dione; Animals; Anticonvulsants; Cocaine; Death; Dextrorphan; Dizocilpine Maleate; Dose-Response Relationship, Drug; Glutamates; Kynurenic Acid; Male; Mice; Mice, Inbred ICR; Piperazines; Quinoxalines; Receptors, Glutamate; Receptors, N-Methyl-D-Aspartate; Receptors, Neurotransmitter; Seizures; Time Factors

CNQX and DNQX are compounds that have recently been reported to show potent non-NMDA excitatory amino acid receptor antagonist activity. Effects of these compounds on seizures induced by homocysteine thiolactone and quisqualic acid were studied in order to examine the pharmacological properties of these compounds. In a dosage of 1.16 micrograms intracerebroventricularly (ICV), CNQX prolonged the latency to the onset of quisqualate-, but not homocysteine-induced seizures. DNQX was not effective when given either ICV or systemically, although a 3.78 micrograms dose of DNQX given ICV markedly increased the variability in latency to seizure onset, suggesting a combination of pro- and anticonvulsant effects. Higher dosages of both CNQX and DNQX induced seizure-like activity after ICV injection. These data confirm that CNQX has pharmacological effects corresponding to its effects on cellular responses to quisqualate and kainate agonists, but these effects are weak and may limit its usefulness as a pharmacological tool.

Topics: 6-Cyano-7-nitroquinoxaline-2,3-dione; Animals; Anticonvulsants; Female; Homocysteine; Mice; Oxadiazoles; Quinoxalines; Quisqualic Acid; Seizures

Hippocampal neurons that were grown for prolonged periods in the continuous presence of agents that interfere with synaptic transmission, especially excitatory synaptic transmission, appeared to become seizure-prone. Washout of the synaptic blocking agents, that had been continuously present for several weeks to several months, caused the population of neurons to produce an abnormal and intense electrical activity. This consisted of two major components: spontaneously arising phasic responses that closely resembled paroxysmal depolarization shifts and, less frequently, slowly rising depolarizations similar to the sustained depolarizations observed during ictus-like episodes in intact cortex or cortical slices. We describe here observations on the role of the N-methyl-D-aspartate (NMDA) and non-NMDA types of glutamate receptors in the generation of these activities.

Topics: 2-Amino-5-phosphonovalerate; 6-Cyano-7-nitroquinoxaline-2,3-dione; Animals; Cells, Cultured; Electrophysiology; Glutamates; Hippocampus; Neurons; Quinoxalines; Receptors, Glutamate; Receptors, Neurotransmitter; Seizures; Tetrodotoxin