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

GABAA receptor-mediated inhibition is active and may contribute to epileptiform synchronization. The efficacy of inhibition relies on low levels of intracellular Cl(-), which are controlled by KCC2 activity. This evidence has led us to analyze with field potential recordings the effects induced by the KCC2 blockers VU0240551 (10 μM) or bumetanide (50 μM) and by the KCC2 enhancer CLP257 (100 μM) on the epileptiform discharges generated by piriform and entorhinal cortices (PC and EC, respectively) in an in vitro brain slice preparation. Ictal- and interictal-like discharges along with high-frequency oscillations (HFOs, ripples: 80-200 Hz, fast ripples: 250-500 Hz) were recorded from these two regions during application of 4-aminopyridine (4AP, 50 μM). Blocking KCC2 activity with either VU024055 or high doses of bumetanide abolished ictal discharge in both PC and EC; in addition, these experimental procedures decreased the interval of occurrence and duration of interictal discharges. In contrast, enhancing KCC2 activity with CLP257 increased ictal discharge duration in both regions. Finally, blocking KCC2 activity decreased the duration and amplitude of pharmacologically isolated synchronous GABAergic events whereas enhancing KCC2 activity led to an increase in their duration. Our data demonstrate that in vitro ictogenesis is abolished or facilitated by inhibiting or enhancing KCC2 activity, respectively. We propose that these effects may result from the reduction of GABAA receptor-dependent increases in extracellular K(+) that are known to rest on KCC2 function.

Topics: 4-Aminopyridine; 6-Cyano-7-nitroquinoxaline-2,3-dione; Animals; Bumetanide; Cerebral Cortex; Disease Models, Animal; Dose-Response Relationship, Drug; Epilepsy; Excitatory Amino Acid Antagonists; K Cl- Cotransporters; Male; Rats, Sprague-Dawley; Sodium Potassium Chloride Symporter Inhibitors; Symporters; Thiazoles; Thiazolidines; Thioglycolates; Tissue Culture Techniques

In hippocampal Cornu Ammonis 1 (CA1) neurons, a prolonged depolarization evokes a train of action potentials followed by a prominent afterhyperpolarizing potential (AHP), which critically dampens neuronal excitability. Because it is not known whether epileptiform activity alters the AHP and whether any alteration of the AHP is independent of inhibition, we acutely induced epileptiform activity by bath application of the GABA(A) receptor blocker gabazine (5 μM) in the rat hippocampal slice preparation and studied its impact on the AHP using intracellular recordings. Following 10 min of gabazine wash-in, slices started to develop spontaneous epileptiform discharges. This disinhibition was accompanied by a significant shift of the resting membrane potential of CA1 neurons to more depolarized values. Prolonged depolarizations (600 ms) elicited a train of action potentials, the number of which was not different between baseline and gabazine treatment. However, the AHP following the train of action potentials was significantly reduced after 20 min of gabazine treatment. When the induction of epileptiform activity was prevented by co-application of 6-cyano-7-nitroquinoxaline-2,3-dione disodium (CNQX, 10 μM) and D-(-)-2-amino-5-phosphonopentanoic acid (D-AP5, 50 μM) to block α-amino-3-hydroxy-5-methylisoxazolepropionate (AMPA) and N-methyl-d-aspartate (NMDA) receptors, respectively, the AHP was preserved despite of GABA(A) receptor inhibition suggesting that the epileptiform activity was required to suppress the AHP. Moreover, the AHP was also preserved when the slices were treated with the protein kinase blockers H-9 (100 μM) and H-89 (1 μM). These results demonstrate that the AHP following a train of action potentials is rapidly suppressed by acutely induced epileptiform activity due to a phosphorylation process-presumably involving protein kinase A.

Topics: 2-Amino-5-phosphonovalerate; 6-Cyano-7-nitroquinoxaline-2,3-dione; Action Potentials; Animals; CA1 Region, Hippocampal; Disease Models, Animal; Drug Interactions; Electric Stimulation; Enzyme Inhibitors; Epilepsy; Excitatory Amino Acid Antagonists; GABA Antagonists; In Vitro Techniques; Isoquinolines; Male; Neural Inhibition; Neurons; Pyridazines; Rats; Rats, Wistar; Statistics, Nonparametric; Sulfonamides; Time Factors

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

Epileptic seizures can induce pathological processes of plasticity in the brain that tend to promote the generation of further seizures. However, the immediate impact of epileptic seizures on cellular excitability remains poorly understood. In order to unravel such early mechanisms of epilepsy-induced plasticity, we studied synaptic transmission before and shortly after three ictal discharges induced by transient elevation of extracellular K(+) in mouse hippocampal slices. Discharges were initiated in the CA3 region and propagated via the Schaffer collaterals into CA1 where they were associated with sustained membrane depolarization and bursts of action potentials in CA1 pyramidal cells. Subsequently, discharges were followed by long-term potentiation (LTP) of Schaffer collateral-evoked field excitatory post-synaptic potentials (EPSPs) in the CA1. The ability to generate epileptiform activity in response to repetitive stimulation was enhanced during LTP. Changes in both inhibitory and excitatory synaptic transmission contributed to LTP in CA1 pyramidal cells. Discharges reduced gamma-aminobutyric acid-A receptor-mediated hyperpolarizing inhibitory post-synaptic potentials by shifting their reversal potentials in a positive direction. At the same time, the amplitudes of Schaffer collateral-evoked RS-alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor-mediated EPSPs and action potential-independent miniature EPSPs were enhanced. However, N-methyl-d-aspartate receptor-mediated EPSPs remained unchanged. Paired-pulse stimulation revealed a reduced probability of glutamate release. Together, these changes in synaptic transmission produce a sustained increase in hippocampal excitability. We conclude that a few seizure-like ictal episodes are sufficient to cause fast and lasting changes in the excitation/inhibition balance in hippocampal networks, and therefore may contribute to early phases of progressive epileptogenesis.

Topics: 6-Cyano-7-nitroquinoxaline-2,3-dione; Animals; Biophysics; Calcium; Electric Stimulation; Epilepsy; Excitatory Amino Acid Antagonists; Excitatory Postsynaptic Potentials; GABA Antagonists; GABA-B Receptor Antagonists; Hippocampus; In Vitro Techniques; Inhibitory Postsynaptic Potentials; Long-Term Potentiation; Magnesium; Male; Mice; Mice, Inbred C57BL; Neural Pathways; Neuronal Plasticity; Potassium; Pyridazines; Receptors, GABA-B; Receptors, N-Methyl-D-Aspartate; Sodium Channel Blockers; Synapses; Tetrodotoxin; Valine

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

There is evidence suggesting that protein kinase C (PKC) activation can prevent the enhanced network excitability associated with status epilepticus and group I metabotropic glutamate receptor (mGluR)-induced epileptogenesis. However, we observed no suppression of mGluR-induced burst prolongation in the guinea pig hippocampal slice when applied in the presence of the PKC activator phorbol-12,13-dibutyrate (PDBu). Furthermore, PDBu alone converted picrotoxin-induced interictal bursts into ictal-length discharges ranging from 2 to 6s in length. This effect could not be elicited by the inactive analog 4-alpha-PDBu and was suppressed with the PKC inhibitor chelerythrine, indicating PKC dependence. PKC activation can enhance neurotransmitter release, and both glutamate and acetylcholine are capable of eliciting similar prolonged synchronized discharges. However, neither mGluR1 nor NMDA receptor antagonist suppressed PDBu-driven burst prolongation, suggesting that increased glutamate release alone is unlikely to account for the PKC-induced expression of ictaform discharges. Similarly, atropine, a broad-spectrum muscarinic receptor antagonist, had no effect on PKC-induced burst prolongation. By contrast, AMPA/kainate receptor antagonist abolished PKC-induced burst prolongation, and mGluR5 antagonist significantly blunted the maximum burst length induced by PKC. These data suggest that PKC-induced prolongation of epileptiform bursts is dependent on changes specific to mGluR5 and AMPA/kainate receptors and not mediated simply by a generalized increase in transmitter release.

Topics: 2-Amino-5-phosphonovalerate; 6-Cyano-7-nitroquinoxaline-2,3-dione; Acetylcholine; Action Potentials; Animals; Atropine; Benzoates; Benzophenanthridines; Enzyme Activation; Epilepsy; Glutamic Acid; Glycine; Guinea Pigs; Hippocampus; In Vitro Techniques; Neurotransmitter Agents; Phorbol 12,13-Dibutyrate; Picrotoxin; Protein Kinase C; Pyridines; Receptor, Metabotropic Glutamate 5; Receptors, Kainic Acid; Receptors, Metabotropic Glutamate; Signal Transduction

Approximately 30% of epilepsy patients suffer from drug-resistant epilepsy. Direct electrical stimulation of the epileptogenic zone is a potential new treatment modality for this devastating disease. In this study, we investigated the effect of two electrical stimulation paradigms, sustained low-frequency stimulation and short trains of high-frequency stimulation, on epileptiform discharges in neocortical brain slices treated with either bicuculline or magnesium-free extracellular solution. Sustained low-frequency stimulation (5-30 min of 0.1- to 5-Hz stimulation) prevented both interictal-like discharges and seizure-like events in an intensity-, frequency-, and distance-dependent manner. Short trains of high-frequency stimulation (1-5 s of 25- to 200-Hz stimulation) prematurely terminated seizure-like events in a frequency-, intensity-, and duration-dependent manner. Roughly one half the seizures terminated within the 100-Hz stimulation train (P < 0.01 compared with control), whereas the remaining seizures were significantly shortened by 53 +/- 21% (P < 0.01). Regarding the cellular mechanisms underlying the antiepileptic effects of electrical stimulation, both low- and high-frequency stimulation markedly depressed excitatory postsynaptic potentials (EPSPs). The EPSP amplitude decreased by 75 +/- 3% after 10-min, 1-Hz stimulation and by 86 +/- 6% after 1-s, 100-Hz stimulation. Moreover, partial pharmacological blockade of ionotropic glutamate receptors was sufficient to suppress epileptiform discharges and enhance the antiepileptic effects of stimulation. In conclusion, this study showed that both low- and high-frequency electrical stimulation possessed antiepileptic effects in the neocortex in vitro, established the parameters determining the antiepileptic efficacy of both stimulation paradigms, and suggested that the antiepileptic effects of stimulation were mediated mostly by short-term synaptic depression of excitatory neurotransmission.

Topics: 2-Amino-5-phosphonovalerate; 6-Cyano-7-nitroquinoxaline-2,3-dione; Action Potentials; Analysis of Variance; Animals; Animals, Newborn; Bicuculline; Dose-Response Relationship, Radiation; Electric Stimulation; Epilepsy; Excitatory Amino Acid Antagonists; Excitatory Postsynaptic Potentials; GABA Antagonists; In Vitro Techniques; Neocortex; Rats; Rats, Wistar; Time Factors

We have previously reported that topical application of alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) to the rat neocortex prevents the effects of a subsequent application of N-methyl-d-aspartic acid (NMDA). Activation of NMDA receptors is involved in the pathogenesis of epileptic activity. Therefore, we examined if topically applied AMPA could affect changes in the somatosensory evoked potentials (SEPs) and electrocorticogram (ECoG) epileptic spikes caused by bicuculline. AMPA (50 microM) prevented the epileptiform activity to a level that was comparable to that caused by diazepam (3 mg/kg i.p.) or clomethiazole (100 mg/kg i.p.). Also, the epileptiform activity was suppressed by the AMPAR antagonist, CNQX, or the blocker of AMPAR desensitization, cyclothiazide. In the hippocampal slice, bicuculline-induced changes in the population spike potentials recorded from the CA1 cells were not affected by AMPA. We conclude that in the complex neuronal network of the rat neocortex, epileptiform activity can be suppressed in a variety of strategies that target the AMPA receptors: (1) blocking AMPA receptors, (2) promoting an apparent desensitization of AMPA receptors (possibly on the pyramidal neurons) or (3) reducing an apparent desensitization of AMPA receptors (possibly on the inhibitory GABA-ergic interneurons).

Topics: 6-Cyano-7-nitroquinoxaline-2,3-dione; alpha-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic Acid; Animals; Anticonvulsants; Benzothiadiazines; Bicuculline; Chlormethiazole; Diazepam; Electroencephalography; Epilepsy; Evoked Potentials, Somatosensory; Excitatory Amino Acid Agonists; Excitatory Amino Acid Antagonists; Hippocampus; In Vitro Techniques; Male; Neocortex; Neurons; Rats; Rats, Sprague-Dawley; Receptors, AMPA

We examined effects of the novel anticonvulsant lacosamide and its inactive isomer (SPM 6953) in an in vitro model of epileptiform activity. Focal field potential recordings (34+/-0.2 degrees C) were obtained from 17 to 22 day old rat brain slices. Physiological synaptic transmission (fEPSP amplitude and duration) in CA1 of rat hippocampus was not significantly altered (P > 0.05, n = 4) by lacosamide (1 microM-1 mM). Recording from visual cortex during application of 4-aminopyridine (4-AP; 100 microM) revealed both spontaneous and evoked 'ictal like' discharges. Spontaneous ictal like discharges in the visual cortex were blocked by 100 microM carbamazepine (CBZ), 100 microM pentobarbital and 200 microM phenobarbital (PHB) but were insensitive to the anti-absence drug ethosuximide (750 microM; n = 4, P > 0.05). Lacosamide reduced tonic duration and maximal firing frequency with EC(50)s of 41 and 71 microM, respectively. In contrast, the S stereoisomer (100-320 microM) produced no significant effect on spontaneous ictal activity (n = 3-4, P > 0.05). Seizures induced by high frequency (100 Hz, 1s) stimulation were selectively reduced in amplitude by PHB (200 microM) and frequency by CBZ (100 microM; n = 6) and lacosamide (100 microM; n = 4). GABAergic negative going potentials were attenuated by CBZ (irreversible with washing) and lacosamide (reversible) but not by PHB. We conclude that lacosamide blocks 4-AP induced epileptiform activity in the visual cortex. This novel anticonvulsant drug appears to inhibit epileptogenesis (seizure spread) by interacting with a stereoselective, but as yet unidentified, target site in rodent neocortex in the mid-micromolar range.

Topics: 2-Amino-5-phosphonovalerate; 4-Aminopyridine; 6-Cyano-7-nitroquinoxaline-2,3-dione; Acetamides; Animals; Anticonvulsants; Carbamazepine; Convulsants; Electrophysiology; Epilepsy; Excitatory Amino Acid Antagonists; Extracellular Space; Female; In Vitro Techniques; Lacosamide; Male; Membrane Potentials; Patch-Clamp Techniques; Phenobarbital; Picrotoxin; Potassium Channel Blockers; Prohibitins; Rats; Rats, Sprague-Dawley; Synaptic Transmission; Visual Cortex

Long-term application of Cs(+) (5 mM) induces an epileptiform field potential (Cs-FP) in area CA1 of the rat hippocampus, which is independent of N-methyl-D-aspartate (NMDA) and non-NMDA glutamate receptors and gamma-aminobutyric acid (GABA)(A) receptors. To gain insight into possible mechanisms for the induction of the Cs-FP, we investigated the postnatal development of the response. In brain slices prepared from rats at different ages, the Cs-FP was evoked by stimulation of the Schaffer-collateral-commisural pathway. We found that expression of this potential was clearly dependent on the postnatal age. Thus, the Cs-FP was completely absent at 1 week of age. By 2 weeks, a reduced form of the response was observed, whereas slices taken from 3-week-old rats, displayed full Cs-FP, which were indistinguishable in size and shape from the adult form. In the presence of 4-aminopyridine, potentials resembling the Cs-FP were evoked. These potentials showed a similar age-dependency as the Cs-FP. The Na(+)/K(+) pump inhibitor dihydroouabain (DHO), when present during wash-in of Cs(+), gave a partial block of the Cs-FP in adult slices. This effect was not seen when DHO was applied after development of the Cs-FP. The data indicate that the processes necessary for expression of the Cs-PF are absent at birth and develop during the second postnatal week. We propose that the Cs-FP depends on Cs(+) entry into presynaptic neurons, and that the Na(+)/K(+) pump contributes to this transport of Cs(+). The observed age-dependency could therefore, in part, reflect the delayed development of the Na(+)/K(+) pump.

Topics: 2-Amino-5-phosphonovalerate; 4-Aminopyridine; 6-Cyano-7-nitroquinoxaline-2,3-dione; Aging; Animals; Cesium; Electrophysiology; Epilepsy; Excitatory Amino Acid Antagonists; Excitatory Postsynaptic Potentials; Extracellular Space; GABA Antagonists; Hippocampus; In Vitro Techniques; Male; Membrane Potentials; Patch-Clamp Techniques; Potassium Channel Blockers; Rats; Rats, Wistar; Receptors, Glutamate; Sodium-Potassium-Exchanging ATPase

It is well known that excitatory synaptic transmission at the hippocampal CA3-CA1 synapse depends on the binding of released glutamate to ionotropic receptors. Here we report that during long-term application of Cs+ (5 mM), stimulation of the Schaffer collateral-commisural pathway evokes an epileptic field potential (Cs-FP) in area CA1 of the rat hippocampal slice, which is resistant to antagonists of ionotropic glutamate and GABA(A) receptors. The Cs-FP was blocked by N-type but not L-type Ca2+ channel antagonists and was attenuated by adenosine (0.5 mM), as expected for a synaptically mediated response. These properties make the Cs-FP fundamentally different from other types of Cs(+)-induced epileptiform activity. Replacement of Cs+ with antagonists of the hyperpolarization-activated nonselective cation current I(h) and inwardly rectifying potassium channels (K(IR)) or partial inhibition of the Na(+)/K+ pump did not cause Cs-FP-like potentials, which indicates that such actions of Cs+ were not responsible for the Cs-FP. The effect of Cs+ was partly mimicked by 4-aminopyridine (4-AP; 2 mM), suggesting that an increase in transmitter release is involved. The group I metabotropic glutamate receptor (mGluR) agonist (RS)-3,5-dihydroxyphenylglycine (DHPG) attenuated the Cs-FP. This effect was not, however, antagonized by group I mGluR antagonists. Selective and nonselective mGluR antagonists did not attenuate the Cs-FP. We conclude that long-term exposure to Cs+ induces a state where excitatory synaptic transmission can exist between area CA3 and CA1 in the hippocampus, independent of ionotropic and metabotropic glutamate receptors and GABA(A) receptors.

Topics: 6-Cyano-7-nitroquinoxaline-2,3-dione; Action Potentials; Animals; Epilepsy; Male; Rats; Rats, Wistar; Receptors, GABA; Receptors, Glutamate; Synapses

A paroxysmal depolarization shift (PDS) has been suggested to be a hallmark for epileptic activity in partial-onset seizures. By monitoring membrane potentials and currents in pairs of pyramidal neurons and astrocytes with dual patch-clamp recording and exocytosis of vesicles from astrocytes with two-photon laser scanning microscopy in hippocampal slices, we found that infusion of inositol 1,4,5-trisphosphate (IP(3)) into astrocytes by patch pipettes induced astrocytic glutamate release that triggered a transient depolarization (TD) and epileptiform discharges in CA1 pyramidal neurons. The TD is due to a tetrodotoxin (TTX)-insensitive slowly decaying transient inward current (STC). Astrocytic glutamate release simultaneously triggers both the STC in pyramidal neurons and a transport current (TC) in astrocytes. The neuronal STC is mediated by ionotropic glutamate receptors leading to the TD and epileptiform discharges; while the astrocytic TC is a glutamate reuptake current resulting from transporting released glutamate into the patched astrocyte. Fusion of a large vesicle in astrocytes was immediately followed by an astrocytic TC, suggesting that the fused vesicle contains glutamate. Both fusion of large vesicles and astrocytic TCs were blocked by tetanus toxin (TeNT), suggesting that astrocytic glutamate release is via SNARE-dependent exocytosis of glutamate-containing vesicles. In the presence of TTX, the epileptogenic reagent, 4-AP, also induced similar neuronal STCs and astrocytic TCs, suggesting that astrocytic glutamate release may play an epileptogenic role in initiation of epileptic seizures under pathological conditions. Our study provides a novel mechanism, astrocytic release of glutamate, for seizure initiation.

Topics: 2-Amino-5-phosphonovalerate; 4-Aminopyridine; 6-Cyano-7-nitroquinoxaline-2,3-dione; Animals; Animals, Newborn; Aspartic Acid; Astrocytes; Calcium; Diagnostic Imaging; Drug Interactions; Electric Stimulation; Epilepsy; Excitatory Amino Acid Antagonists; Glutamic Acid; Hippocampus; In Vitro Techniques; Inositol 1,4,5-Trisphosphate; Membrane Potentials; Microscopy, Confocal; Neurons; Neurotoxins; Patch-Clamp Techniques; Potassium Channel Blockers; Rats; Rats, Sprague-Dawley; Sodium Channel Blockers; Tetanus Toxin; Tetrodotoxin; Time Factors

This study presents a model of chronic, recurrent, spontaneous seizures in the intact isolated hippocampal preparation from mice aged P8-P25. Field activity from the CA1 pyramidal cell layer was recorded and recurrent, spontaneous seizure-like events (SLEs) were observed in the presence of low Mg2+ (0.25 mM) artificial cerebrospinal fluid (ACSF). Hippocampi also showed interictal epileptiform discharges (IEDs) of 0.9-4.2 Hz occurring between seizures. No age-specific differences were found in SLE occurrence (2 SLEs per 10 min, on average), duration, and corresponding frequencies. After long exposure to low Mg2+ ACSF (>3 h), SLEs were completely reversible within minutes with the application of normal (2 mM Mg2+) ACSF. The AMPA antagonist, CNQX, blocked all epileptiform activity, whereas the NMDA antagonist, APV, did not. The gamma-aminobutyric acid (GABA)A antagonist, bicuculline, attenuated and fragmented SLEs, implicating interneurons in SLE generation. The L-type Ca2+ blocker, nifedipine, enhanced epileptiform activity. Analysis of dual site recordings along the septotemporal hippocampus demonstrated that epileptiform activity began first in the temporal pole of the hippocampus, as illustrated by disconnection experiments. Once an SLE had been established, however, the septal hippocampus was sometimes seen to lead the epileptiform activity. The whole hippocampus with intact local circuitry, treated with low Mg2+, provides a realistic model of recurrent spontaneous seizures, which may be used, in normal and genetically modified mice, to study the dynamics of seizures and seizure evolution, as well as the mechanisms of action of anti-epileptic drugs and other therapeutic modalities.

Topics: 2-Amino-5-phosphonovalerate; 6-Cyano-7-nitroquinoxaline-2,3-dione; Action Potentials; Age Factors; Animals; Calcium Channel Blockers; Disease Models, Animal; Epilepsy; Excitatory Amino Acid Antagonists; GABA Antagonists; Hippocampus; Magnesium; Magnesium Deficiency; Mice; Mice, Inbred C57BL; Neural Pathways; Neurons; Organ Culture Techniques; Receptors, AMPA; Receptors, N-Methyl-D-Aspartate; Recurrence

Neuronal activity is thought to play an important role in refining patterns of synaptic connectivity during development and in the molecular maturation of synapses. In experiments reported here, a 2-week infusion of tetrodotoxin (TTX) into rat hippocampus beginning on postnatal day 12 produced abnormal synchronized network discharges in in vitro slices. Discharges recorded upon TTX washout were called 'minibursts', owing to their small amplitude. They were routinely recorded in area CA3 and abolished by CNQX, an AMPA receptor antagonist. Because recurrent excitatory axon collaterals remodel and glutamate receptor subunit composition changes after postnatal day 12, experiments examined possible TTX-induced alterations in recurrent excitation that could be responsible for network hyperexcitability. In biocytin-labelled pyramidal cells, recurrent axon arbors were neither longer nor more highly branched in the TTX infusion site compared with saline-infused controls. However, varicosity size and density were increased. Whereas most varicosities contained synaptophysin and synaptic vesicles, many were not adjacent to postsynaptic specializations, and thus failed to form anatomically identifiable synapses. An increased pattern of excitatory connectivity does not appear to explain network hyperexcitability. Quantitative immunoblots also indicated that presynaptic markers were unaltered in the TTX infusion site. However, the postsynaptic AMPA and NMDA receptor subunits, GluR1, NR1 and NR2B, were increased. In electrophysiological studies EPSPs recorded in slices from TTX-infused hippocampus had an enhanced sensitivity to the NR2B containing NMDA receptor antagonist, ifenprodil. Thus, increases in subunit protein result in alterations in the composition of synaptic NMDA receptors. Postsynaptic changes are likely to be the major contributors to the hippocampal network hyperexcitability and should enhance both excitatory synaptic efficacy and plasticity.

Topics: 6-Cyano-7-nitroquinoxaline-2,3-dione; Anesthetics, Local; Animals; Animals, Newborn; Axons; Disease Models, Animal; Epilepsy; Excitatory Amino Acid Antagonists; Excitatory Postsynaptic Potentials; Hippocampus; Immunoblotting; Immunohistochemistry; In Vitro Techniques; Lysine; Membrane Potentials; Microscopy, Confocal; Microscopy, Electron; Nerve Net; Patch-Clamp Techniques; Piperidines; Pyramidal Cells; Rats; Receptors, AMPA; Receptors, N-Methyl-D-Aspartate; Synapses; Synaptophysin; Tetrodotoxin; Time Factors

Slices of human cortical tissue from epilepsy surgery were investigated with intracellular recordings to elucidate the mechanisms contributing to augmented synaptic excitation and to repetitive activity. The analysis of single synaptic potentials revealed, amongst other differences to rodent cortex, a disturbance of GABAA inhibition, namely depolarizing responses. A tentative ionic mechanism, impaired KCl outward-transport (KCC2), was evaluated in a rat model (0-Mg hyperexcitability). The observed down-regulation of KCC2 mRNA after 0-Mg-ACSF exposure of slices may contribute to the depolarizations by GABA. The factors enabling repetitive activity were addressed with a paired-pulse paradigm. In slices from epilepsy surgery, synaptic responses were virtually constant with interstimulus intervals between 100 and 1000 ms. Tiagabine markedly prolonged the effects of released GABA at GABAA receptors, but paired-pulse behaviour was only slightly affected. We demonstrate that bicuculline-induced paroxysmal activity of rat cortex is frequency-limited (to about <1 Hz) by presynaptic GABAB receptors. The lack of frequency limitation of synaptic events suggests an impaired GABAB receptor function in the human epileptogenic cortex. The data are discussed regarding the pivotal role of KCl transport in epileptic disorders of various origins and the role of GABAB receptors in the frequency limitation of paroxysmal activity.

Topics: 6-Cyano-7-nitroquinoxaline-2,3-dione; Animals; Anticonvulsants; Bicuculline; Drug Resistance; Electric Stimulation; Epilepsy; Excitatory Amino Acid Antagonists; Guinea Pigs; Humans; Neocortex; Nerve Tissue Proteins; Neurons; Rats; Receptors, AMPA; Receptors, GABA-A; Receptors, Kainic Acid; Receptors, N-Methyl-D-Aspartate; Species Specificity; Synaptic Transmission

We examined whether epileptiform activity can be induced and prevented by mild reduction of GABA(A) receptor-mediated inhibition and non-NMDA receptor-mediated excitation, respectively, in different regions of combined hippocampal/entorhinal cortical slices from juvenile rats (P15-21). We used the receptor antagonists bicuculline (GABA(A)) and CNQX (non-NMDA) as tools to investigate the sensitivities of the CA1, the subiculum (SUB) and the medial entorhinal cortex (MEC) for generating epileptiform discharges upon extracellular stimulation. We found that low concentrations of bicuculline (<3.5 microM) were enough to induce epileptiform discharges in the three regions. These discharges were similar to those observed under high concentrations of bicuculline (>10 microM) and consisted of stereotyped population bursts, recorded both extra- and intracellularly. Interestingly, the CA1 and SUB were more susceptible to generate discharges compared to the MEC in the same slices. We also found that non-NMDA excitation was critical in controlling discharges, as they were blocked by CNQX in a concentration-dependent manner. The sensitivity of the CA1 region to CNQX was lower than that of the SUB and MEC. Based on these regional differences, we show that epileptiform activity can be pharmacologically isolated within the CA1 region in the hippocampal-entorhinal circuitry in vitro.

Topics: 6-Cyano-7-nitroquinoxaline-2,3-dione; Animals; Bicuculline; Dose-Response Relationship, Drug; Electric Stimulation; Entorhinal Cortex; Epilepsy; Excitatory Amino Acid Antagonists; GABA Antagonists; GABA-A Receptor Antagonists; Hippocampus; In Vitro Techniques; Membrane Potentials; Neural Inhibition; Patch-Clamp Techniques; Rats; Rats, Wistar

Spontaneous activity was monitored during pharmacological blockade of GABA(A) receptor function in the CA1 minislice (CA3 was cut off). Synaptic inhibition was blocked by competitive GABA(A) antagonists bicuculline-methiodide (Bic) or GABAZINE (GBZ) and the chloride channel blocker picrotoxin (PTX). Extra- and intracellular recordings using sharp electrodes were carried out in stratum radiatum and pyramidale. At low antagonist concentrations (Bic, GBZ: 1-10 microM; PTX: < 100 microM), synchronized bursts (< 500 ms in duration, interictal activity) were seen as described previously. However, in the presence of high concentrations (Bic, GBZ: 50-100 microM; PTX: 100-200 microM), seizure-like, ictal events (duration 4-17 s) were observed in 67 of 88 slices. No other experimental measures to increase excitability were applied: cation concentrations ([Ca2+]o = 2 mM, [Mg2+]o = 1.7 mM, [K+]o = 3 mM) and recording temperature (30-32 degrees C) were standard and GABA(B)-mediated inhibition was intact. In whole-slice recordings prominent interictal activity, but fewer ictal events were observed. A reduced ictal activity was also observed when interictal-like responses were evoked by afferent stimulation. Ictal activity was reversibly blocked by antagonists of excitatory transmission, CNQX (40 microM) or D-AP5 (50 microM). Disinhibition-induced ictal development did not rely on group I mGluR activation as it was not prevented in the presence of group I mGluR antagonists (AIDA or 4CPG). (RS)-3,5-DHPG prevented the induction and reversed the tertiary component of the ictal event through a group I mGluR-independent mechanism.

Topics: 6-Cyano-7-nitroquinoxaline-2,3-dione; Action Potentials; Age Factors; Animals; Bicuculline; Epilepsy; Excitatory Amino Acid Antagonists; Excitatory Postsynaptic Potentials; GABA Antagonists; Glycine; Guinea Pigs; Hippocampus; Neural Inhibition; Neurons, Afferent; Organ Culture Techniques; Picrotoxin; Pyridazines; Receptors, AMPA; Receptors, GABA-A; Receptors, Metabotropic Glutamate; Receptors, N-Methyl-D-Aspartate; Resorcinols

Knockout Otx1 mice present a microcephalic phenotype mainly due to reduced deep neocortical layers and spontaneous recurrent seizures. We investigated the excitable properties of layer V pyramidal neurons in neocortical slices prepared from Otx1-/- mice and age-matched controls. The qualitative firing properties of the neurons of Otx1-/- mice were identical to those found in wild-type controls, but the proportion of intrinsically bursting (IB) neurons was significantly smaller. This is in line with the lack of the Otx1 gene contribution to the generation and differentiation of neurons destined for the deep neocortical layers, in which IB neurons are located selectively in wild-type rodents. The pyramidal neurons recorded in Otx1-/- mice responded to near-threshold electrical stimulation of the underlying white matter, with aberrant polysynaptic excitatory potentials often leading to late action potential generation. When the strength of the stimulus was increased, the great majority of the Otx1-/- neurons (78%) responded with a prominent biphasic inhibitory postsynaptic potential that was significantly larger than that observed in the wild-type mice, and was often followed by complex postinhibitory depolarizing events. Both late excitatory postsynaptic potentials and postinhibitory excitation were selectively suppressed by NMDA receptor antagonists, but not by AMPA antagonists. We conclude that the cortical abnormalities of Otx1-/- neocortex due to a selective loss of large projecting neurons lead to a complex rearrangement of local circuitry, which is characterized by an excess of N-methyl-d-aspartate-mediated polysynaptic excitation that is counteracted by GABA-mediated inhibition in only a limited range of stimulus intensity. Prominent postsynaptic inhibitory potentials may also act as a further pro-epileptogenic event by synchronizing abnormal excitatory potentials.

Topics: 2-Amino-5-phosphonovalerate; 6-Cyano-7-nitroquinoxaline-2,3-dione; Action Potentials; Animals; Cell Size; Cerebral Cortex; Electric Stimulation; Epilepsy; Excitatory Amino Acid Antagonists; gamma-Aminobutyric Acid; Gene Expression Regulation, Developmental; Homeodomain Proteins; Mice; Mice, Knockout; Nerve Tissue Proteins; Nervous System Malformations; Neural Inhibition; Otx Transcription Factors; Pyramidal Cells; Rats; Receptors, AMPA; Receptors, N-Methyl-D-Aspartate; Synaptic Transmission; Transcription Factors

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

The action of somatostatin on GABA-mediated transmission was investigated in cat and rat thalamocortical neurons of the dorsal lateral geniculate nucleus and ventrobasal thalamus in vitro. In the cat thalamus, somatostatin (10 microM) had no effect on the passive membrane properties of thalamocortical neurons and on the postsynaptic response elicited in these cells by bath or iontophoretic application of (+/-)baclofen (5-10 microM) or GABA, respectively. However, somatostatin (1-10 microM) decreased by a similar amount (45-55%) the amplitude of electrically evoked GABA(A) and GABA(B) inhibitory postsynaptic potentials in 71 and 50% of neurons in the lateral geniculate and ventrobasal nucleus, respectively. In addition, the neuropeptide abolished spontaneous bursts of GABA(A) inhibitory postsynaptic potentials in 85% of kitten lateral geniculate neurons, and decreased (40%) the amplitude of single spontaneous GABA(A) inhibitory postsynaptic potentials in 87% of neurons in the cat lateral geniculate nucleus. Similar results were obtained in the rat thalamus. Somatostatin (10 microM) had no effect on the passive membrane properties of thalamocortical neurons in this species, or on the outward current elicited by puff-application of (+/-)baclofen (5-10 microM). However, in 57 and 22% of neurons in the rat lateral geniculate and ventrobasal nuclei, respectively, somatostatin (1 microM) reduced the frequency, but not the amplitude, of miniature GABA(A) inhibitory postsynaptic currents by 31 and 37%, respectively. In addition, the neuropeptide (1 microM) decreased the amplitude of evoked GABA(A) inhibitory postsynaptic currents in 20 and 55% of rat ventrobasal neurons recorded in normal conditions and during enhanced excitability, respectively: this effect was stronger on bursts of inhibitory postsynaptic currents(100% decrease) than on single inhibitory postsynaptic currents (41% decrease). These results demonstrate that in the sensory thalamus somatostatin inhibits GABA(A)- and GABA(B)-mediated transmission via a presynaptic mechanism, and its action is more prominent on bursts of GABAergic synaptic currents/potentials.

Topics: 2-Amino-5-phosphonovalerate; 6-Cyano-7-nitroquinoxaline-2,3-dione; Animals; Baclofen; Bicuculline; Cats; Cell Membrane; Epilepsy; Evoked Potentials; Excitatory Amino Acid Antagonists; GABA Agonists; GABA Antagonists; gamma-Aminobutyric Acid; Geniculate Bodies; Hormones; Male; Membrane Potentials; Neural Inhibition; Patch-Clamp Techniques; Presynaptic Terminals; Rats; Rats, Wistar; Receptors, GABA-A; Receptors, GABA-B; Sleep; Somatostatin; Synaptic Transmission; Tetrodotoxin; Ventral Thalamic Nuclei

We examined the generation, propagation and pharmacology of 4-aminopyridine (4-AP)-induced epileptiform activity (EA) in the intact interconnected limbic structure of the newborn (P0-7) rat in vitro. Whole-cell recordings of CA3 pyramidal cells and multisite field potential recordings in CA3, CA1, dentate gyrus, and lateral and medial entorhinal cortex revealed 4-AP-induced EA as early as P0-1. At this age, EA was initiated in the CA3 region and propagated to CA1, but not to the entorhinal cortex. Starting from P3-4, EA propagated from CA3 to the entorhinal cortex. Along the CA3 septo-temporal axis, EA arose predominantly from the septal pole and spread towards the temporal site. Whereas the onset of 4-AP-induced EA decreased with age from 21.2 +/- 1.6 min at P0-1 to 4.7 +/- 0.63 min at P6-7, the seizure duration increased in the same age groups from 98 +/- 14 s to 269.4 +/- 85.9 s, respectively. The EA was blocked by 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) but not by DL-2-amino-5-phosphonovaleric acid (APV), (+)-MK-801 hydrogen maleate (MK-801) or (+/-)-alpha-methyl-4-carboxyphenylglycine (MCPG), suggesting that they were mediated by alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA)/kainate receptor activation. We conclude that: (i) the septal pole of the hippocampal CA3 region plays a central role in the generation of EA in the neonatal limbic system; and (ii) AMPA/kainate receptor-mediated EA can be generated in CA3 already at birth. Therefore, the recurrent collateral synapses and circuits required for the generation of EA are developed earlier than previously suggested on the basis of studies on hippocampal slices.

Topics: 2-Amino-5-phosphonovalerate; 4-Aminopyridine; 6-Cyano-7-nitroquinoxaline-2,3-dione; Action Potentials; Age Factors; Animals; Animals, Newborn; Benzoates; Dizocilpine Maleate; Entorhinal Cortex; Epilepsy; Excitatory Amino Acid Antagonists; Glycine; Hippocampus; In Vitro Techniques; Neurons; Patch-Clamp Techniques; Rats; Rats, Wistar; Receptors, Glutamate

Paired intracellular recordings were used to investigate recurrent excitatory transmission in layers II, III and V of the rat entorhinal cortex in vitro. There was a relatively high probability of finding a recurrent connection between pairs of pyramidal neurons in both layer V (around 12%) and layer III (around 9%). In complete contrast, we have failed to find any recurrent synaptic connections between principal neurons in layer II, and this may be an important factor in the relative resistance of this layer in generating synchronized epileptiform activity. In general, recurrent excitatory postsynaptic potentials in layers III and V of the entorhinal cortex had similar properties to those recorded in other cortical areas, although the probabilities of connection are among the highest reported. Recurrent excitatory postsynaptic potentials recorded in layer V were smaller with faster rise times than those recorded in layer III. In both layers, the recurrent potentials were mediated by glutamate primarily acting at alpha-amino-3-hydroxy-5-methyl-4-isoxazole receptors, although there appeared to be a slow component mediated by N-methyl-D-aspartate receptors. In layer III, recurrent transmission failed on about 30% of presynaptic action potentials evoked at 0.2Hz. This failure rate increased markedly with increasing (2, 3Hz) frequency of activation. In layer V the failure rate at low frequency was less (19%), and although it increased at higher frequencies this effect was less pronounced than in layer III. Finally, in layer III, there was evidence for a relatively high probability of electrical coupling between pyramidal neurons. We have previously suggested that layers IV/V of the entorhinal cortex readily generate synchronized epileptiform discharges, whereas layer II is relatively resistant to seizure generation. The present demonstration that recurrent excitatory connections are widespread in layer V but not layer II could support this proposal. The relatively high degree of recurrent connections and electrical coupling between layer III cells may be a factor in it's susceptibility to neurodegeneration during chronic epileptic conditions.

Topics: 2-Amino-5-phosphonovalerate; 6-Cyano-7-nitroquinoxaline-2,3-dione; Action Potentials; Animals; Entorhinal Cortex; Epilepsy; Excitatory Amino Acid Antagonists; Excitatory Postsynaptic Potentials; In Vitro Techniques; Lysine; Male; Neural Pathways; Pyramidal Cells; Quinoxalines; Rats; Rats, Wistar; Receptors, AMPA; Receptors, Kainic Acid; Receptors, N-Methyl-D-Aspartate; Synaptic Transmission

Although the peptide somatostatin (SST) has been speculated to function in temporal lobe epilepsy, its exact role is unclear, as in vivo studies have suggested both pro- and anticonvulsant properties. We have shown previously that SST has multiple inhibitory cellular actions in the CA1 region of the hippocampus, suggesting that in this region SST should have antiepileptic actions. To directly assess the effect of SST on epileptiform activity, we studied two in vitro models of epilepsy in the rat hippocampal slice preparation using extracellular and intracellular recording techniques. In one, GABA-mediated neurotransmission was inhibited by superfusion of the GABAA receptor antagonist bicuculline. In the second, we superfused Mg2+-free artificial cerebrospinal fluid to remove the Mg2+ block of the N-methyl-D-aspartate (NMDA) subtype of glutamate receptor. We show here that SST markedly reduces the intensity of evoked epileptiform afterdischarges and the frequency of spontaneous bursts in both CA1 and CA3. SST appears to act additively in the two regions to suppress the transmission of epileptiform events through the hippocampus. We further examined SST's actions in CA3 and found that SST dramatically reduced the frequency of paroxysmal depolarizing shifts (PDSs) recorded intracellularly in current clamp, as well as increasing the threshold for evoking "giant" excitatory postsynaptic currents (EPSCs), large polysynaptically mediated EPSCs that are the voltage-clamp correlate of PDSs. We also examined the actions of SST on pharmacologically isolated EPSCs generated at both mossy fiber (MF) and associational/commissural (A/C) synapses. SST appears to act specifically to reduce recurrent excitation between CA3 neurons because it depresses A/C- but not MF-evoked EPSCs. SST also increased paired-pulse facilitation of A/C EPSCs, suggesting a presynaptic site of action. Reciprocal activation of CA3 neurons through A/C fibers is critical for generation of epileptiform activity in hippocampus. Thus SST reduces feedforward excitation in rat hippocampus, acting to "brake" hyperexcitation. This is a function unique from that described for other hippocampal neuropeptides, which affect more standard neurotransmission. Our results suggest that SST receptors could be a unique, selective clinical target for treatment of limbic seizures.

Topics: 6-Cyano-7-nitroquinoxaline-2,3-dione; Action Potentials; Animals; Bicuculline; Dendrites; Epilepsy; Excitatory Amino Acid Antagonists; Excitatory Postsynaptic Potentials; GABA Antagonists; Hippocampus; Hormone Antagonists; Male; Mossy Fibers, Hippocampal; Organ Culture Techniques; Phosphinic Acids; Propanolamines; Rats; Rats, Sprague-Dawley; Somatostatin

The GABA(A) receptor antagonist bicuculline methiodide (BMI, 10 microM) transformed the evoked synaptic responses, recorded intracellularly from the CA3 area of neonatal (postnatal days 3-7, P3-P7), juvenile (P8-P20) and adult hippocampal slices, into long-lasting paroxysmal depolarizations (PDs), with repetitive action potentials (APs). In the same preparation, GABA(A)-mediated fast-IPSPs were depolarizing at resting membrane potential (RMP), with a reversal potential shifting to a hyperpolarizing direction with age (n=15, P6-P17). BMI provoked also spontaneous PDs in juvenile (20/30) and adult (7/10) but not in neonatal (0/12) neurons. PDs were depressed by either the NMDA receptor antagonist CPP (10 microM) or the non-NMDA antagonist CNQX (10 microM), but were blocked only by the combination of the two (n=6), indicating that activation of either NMDA or non-NMDA receptors can independently sustain PDs in immature hippocampus. In conclusion, these findings show that endogenous GABA tonically inhibits CA3 synaptic responses in neonatal life despite the depolarizing nature of GABA(A)-mediated potentials. Moreover, they suggest that during the 1st postnatal week, disinhibition alone is not sufficient to provoke spontaneous epileptiform discharges in CA3 hippocampal area.

Topics: 6-Cyano-7-nitroquinoxaline-2,3-dione; Action Potentials; Aging; Animals; Animals, Newborn; Bicuculline; Epilepsy; Excitatory Amino Acid Antagonists; GABA Antagonists; GABA-A Receptor Antagonists; gamma-Aminobutyric Acid; In Vitro Techniques; N-Methylaspartate; Piperazines; Pyramidal Cells; Rats; Rats, Sprague-Dawley; Receptors, GABA-A; Receptors, N-Methyl-D-Aspartate

In vivo studies suggest that ontogenesis of limbic seizures is determined by the development of the limbic circuit. We have now used the newly-developed in vitro intact interconnected neonatal rat limbic structures preparation to determine the developmental profile of kainate-induced epileptiform activity in the hippocampus and its propagation to other limbic structures. We report gradual alterations in the effects of kainate during the first postnatal week on an almost daily basis; from no epileptiform activity at birth, through interictal seizures around postnatal day (P) 2 and ictal seizures by the end of the first week. The developmental profile of kainate-induced hippocampal seizures is paralleled by the expression of postsynaptic kainate receptor-mediated currents in CA3 pyramidal cells. Intralimbic propagation of the hippocampal seizures is also age-dependent: whereas seizures readily propagate to the septum and to the contralateral hippocampus via the commissures on P2, propagation to the entorhinal cortex only takes place from P4 onwards. Finally, repeated brief applications of kainate to the hippocampus induce recurrent spontaneous glutamatergic ictal and interictal discharges which persist for several hours after the kainate is washed away and which replace the physiological pattern of network activity. Paroxysmal activities are thus generated by kainate in the hippocampus at an early developmental stage and are initially restricted to this structure. Before the end of the first week of postnatal life, kainate generates the epileptiform activities that may perturb activity-dependent mechanisms that modulate neuronal development. Although at this stage neurons are relatively resistant to the pathological effects of kainate, the epileptiform activities that it generates will perturb activity-dependent mechanisms that modulate neuronal development.

Topics: 6-Cyano-7-nitroquinoxaline-2,3-dione; Animals; Animals, Newborn; Benzodiazepines; Calcium; Electrophysiology; Entorhinal Cortex; Epilepsy; Excitatory Amino Acid Agonists; Excitatory Amino Acid Antagonists; Hippocampus; Kainic Acid; Limbic System; Male; Organ Culture Techniques; Potassium; Rats; Rats, Wistar; Septal Nuclei; Synapses; Tetrodotoxin

The neocortex can generate various forms of epileptiform activity, including one that depends on N-methyl-D-aspartate (NMDA)-type glutamate receptors (NMDARs), and another dependent on non-NMDA-type (AMPA) glutamate receptors (AMPARs). Previous work in vitro suggests that both forms of activity are initiated by neurons of layer 5, but the spatial patterns of horizontal propagation have been studied only for the AMPAR form. We have tested the hypothesis that both types of epileptiform activity spread via common pathways in one cortical layer, suggesting that lamina-specific intervention might selectively interrupt both.. Slices of rat somatosensory cortex were maintained in vitro and treated with the gamma-aminobutyric acid type A (GABA(A))-receptor antagonist picrotoxin. Single all-or-none epileptiform discharges were evoked with an electrical stimulus, and extracellular microelectrodes were used to track the vertical and lateral spread of the discharges.. In both high and low concentrations of picrotoxin, the non-NMDAR antagonist 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) completely blocked propagation, whereas the NMDAR antagonist D-2-amino-5-phosphonovaleric acid (DAPV) only shortened the duration of discharges. When extracellular [Mg2+] was reduced in the presence of picrotoxin and CNQX, NMDAR-dependent epileptiform discharges could be initiated. NMDAR-dependent discharges spread at about one fifth the conduction velocity of AMPAR-dependent events. Analysis of spatiotemporal field-potential patterns suggested that both NMDAR- and AMPAR-mediated propagation involved early activity in layers 5 and 6, followed by larger-amplitude activity in upper cortical layers along the path of propagation.. Our results imply that a common pathway mediates the propagation of these two forms of epileptiform activity, and suggests that lamina-specific surgical intervention might maximize anticonvulsant effect while minimally disrupting cortical function.

Topics: 2-Amino-5-phosphonovalerate; 6-Cyano-7-nitroquinoxaline-2,3-dione; Animals; Dose-Response Relationship, Drug; Epilepsy; Evoked Potentials; Magnesium; Neocortex; Neural Pathways; Picrotoxin; Rats; Rats, Sprague-Dawley; Receptors, AMPA; Receptors, N-Methyl-D-Aspartate; Somatosensory Cortex

Cesium has been widely used to study the roles of the hyperpolarization-activated (I(h)) and inwardly rectifying potassium (K(IR)) channels in many neuronal and nonneuronal cell types. Recently, extracellular application of cesium has been shown to produce epileptiform activity in brain slices, but the mechanisms for this are not known. It has been proposed that cesium blocks the K(IR) in glia, resulting in an abnormal accumulation of potassium in the extracellular space and inducing epileptiform activity. This hypothesis has been tested in hippocampal slices and cultured hippocampal neurons using potassium-sensitive microelectrodes. In the present study, application of cesium produced spontaneous epileptiform discharges at physiological extracellular potassium concentration ([K(+)](o)) in the CA1 and CA3 regions of hippocampal slices. This epileptiform activity was not mimicked by increasing the [K(+)](o). The epileptiform discharges induced by cesium were not blocked by the N-methyl-D- aspartate (NMDA) receptor antagonist AP-5, but were blocked by the non-NMDA receptor antagonist CNQX. In the dentate gyrus, cesium induced the appearance of spontaneous nonsynaptic field bursts in 0 added calcium and 3 mM potassium. Moreover, cesium increased the frequency of field bursts already present. In contrast, ZD-7288, a specific I(h) blocker, did not cause spontaneous epileptiform activity in CA1 and CA3, nor did it affect the field bursts in the dentate gyrus, suggesting that cesium induced epileptiform activity is not directly related to blockade of the I(h). When potassium-sensitive microelectrodes were used to measure [K(+)](o), there was no significant increase in [K(+)](o) in CA1 and CA3 after cesium application. In the dentate gyrus, cesium did not change the baseline level of [K(+)](o) or the rate of [K(+)](o) clearance after the field bursts. In cultured hippocampal neurons, which have a large and relatively unrestricted extracellular space, cesium also produced cellular burst activity without significantly changing the resting membrane potential, which might indicate an increase in [K(+)](o). Our results suggest that cesium causes epileptiform activity by a mechanism unrelated to an alteration in [K(+)](o) regulation.

Topics: 2-Amino-5-phosphonovalerate; 6-Cyano-7-nitroquinoxaline-2,3-dione; Animals; Cells, Cultured; Cesium; Dentate Gyrus; Electric Stimulation; Epilepsy; Excitatory Amino Acid Antagonists; Extracellular Space; Female; Hippocampus; In Vitro Techniques; Male; Membrane Potentials; Potassium; Potassium Channels; Rats; Rats, Sprague-Dawley; Receptors, N-Methyl-D-Aspartate

1. Interictal and ictal epileptiform discharges induced by 4-aminopyridine (4AP, 50 microM) were studied in the rat lateral entorhinal cortex with field potential and intracellular recordings in an in vitro slice preparation. Both types of discharge disappeared in layer II, but continued to occur in layers IV-VI after a knife cut separation was made at approximately 600 micro(m) from the pia (n = 4 slices). 2. Interictal depolarizations recorded in layer IV-VI cells (amplitude, 29.4 +/- 8.6 mV; duration, 386 +/- 177.4 ms, means +/- s.d.; n = 17) were capped by action potential bursts, while smaller interictal depolarizations in layer II cells (amplitude, 11.7 +/- 5.8 mV; duration, 192.6 +/- 47.9 ms; n = 10) were associated with single action potentials and were terminated by a hyperpolarization. Ictal discharges were initiated by an interictal discharge; they were characterized by a depolarization of 31.5 +/- 6.2 mV (n = 12) in layer IV-VI and 11.6 +/- 3.5 mV (n = 7) in layer II neurones. 3. Slow, presumptive Ca2+-mediated spikes occurred in layer II (n = 4) and IV-VI (n = 6) cells loaded with the Na+ channel blocker QX-314 (50 mM). These events were synchronized with population spikes during interictal and ictal discharges, and were abolished by Ni2+ (1 mM, n = 4 cells) along with the 4AP-induced synchronous activity. 4. The N-methyl-D-aspartate (NMDA) receptor antagonist 3, 3-(2-carboxypiperazine-4-yl)-propyl-1-phosphonate (CPP, 10 microM) abolished ictal discharges and reduced interictal depolarizations in layer IV-VI neurones (n = 4). The non-NMDA receptor antagonist 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX, 10 microM) abolished both interictal and ictal activity (n = 4 cells). 5. These findings provide evidence for a role played by NMDA-mediated mechanisms in the generation of epileptiform discharges in the entorhinal cortex. Lack of an NMDA-mediated component along with presence of inhibition in layer II neurones results in attenuation of epileptiform activity at this site. Moreover Ca2+-mediated spikes may contribute to the appearance of epileptiform discharges in this model.

Topics: 4-Aminopyridine; 6-Cyano-7-nitroquinoxaline-2,3-dione; Action Potentials; Animals; Calcium; Electrophysiology; Entorhinal Cortex; Epilepsy; Excitatory Amino Acid Antagonists; Male; Neurons; Organ Culture Techniques; Piperazines; Rats; Rats, Sprague-Dawley; Receptors, N-Methyl-D-Aspartate

To elucidate the mechanism underlying epileptiform discharges in kindled rats, synaptic responses in kindled basolateral amygdala neurons in vitro were compared with those from control rats by using intracellular and whole cell patch-clamp recordings. In kindled neurons, electrical stimulation of the stria terminalis induced epileptiform discharges. The resting potential, apparent input resistance, current-voltage relationship of the membrane, and the threshold, amplitude, and duration of action potentials in kindled neurons were not different from those in control neurons. The electrical stimulation of stria terminalis elicited excitatory postsynaptic potentials (EPSPs) and DL-2-amino-5-phosphonopentanoic acid (AP5)-sensitive and 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX)-sensitive excitatory postsynaptic currents (EPSCs). The amplitude of evoked EPSPs and of evoked AP5-sensitive and CNQX-sensitive EPSCs were enhanced markedly, whereas fast and slow inhibitory postsynaptic potentials (IPSPs) induced by electrical stimulation of lateral amygdaloid nucleus were not significantly different. The rise time and the decay time constant of the evoked CNQX-sensitive EPSCs were shortened, whereas the rise time of the evoked AP5-sensitive EPSCs was shortened, but the decay time constants were not significantly different. In both tetrodotoxin (TTX)-containing medium and low Ca2+ and TTX-containing medium, the frequency and amplitude of spontaneous EPSCs were increased in kindled neurons. These increases are presumably due to nearly synchronous multiquantal events resulted from the increased probability of Glu release at the nerve terminals. The rise time of evoked CNQX- and AP5-sensitive EPSCs and the decay time constant of evoked CNQX-sensitive EPSCs were shortened, suggesting that excitatory synapses at the proximal dendrite and/or the soma in kindled neurons may contribute more effectively to generate evoked EPSCs than those at distal dendrites. In conclusion, the increases in the amplitudes of spontaneous and evoked EPSCs and in the frequency of spontaneous EPSCs may contribute to the epileptiform discharges in kindled neurons.

Topics: 2-Amino-5-phosphonovalerate; 6-Cyano-7-nitroquinoxaline-2,3-dione; Amygdala; Animals; Baclofen; Bicuculline; Epilepsy; Excitatory Amino Acid Antagonists; Excitatory Postsynaptic Potentials; GABA Antagonists; gamma-Aminobutyric Acid; Kindling, Neurologic; Male; Neural Inhibition; Neurons; Patch-Clamp Techniques; Rats; Rats, Wistar; Receptors, N-Methyl-D-Aspartate; Synaptic Transmission; Tetrodotoxin

A freezing probe was placed on the skull of postnatal day (PN) 1 rats to induce formation of a cerebrocortical microsulcus. Experimental studies were performed on PN days 21-24. At that time point, Nissl-stained sections revealed the presence of a microsulcus similar to that described in human dysplastic cortex. Immunocytochemical staining for parvalbumin, calretinin and calbindin indicated a significant decrease in the number of immunoreactive neurons within the microsulcus and non-significant decreases in regions adjacent to the microsulcus. Staining for the glial markers GFAP and vimentin was increased near the microsulcus. Using in vitro brain slices, recordings were made in cortex adjacent to the microsulcus. Epileptiform activity was observed in response to electrical stimulation near the microsulcus. Analysis of the voltage dependence of evoked epileptiform discharges suggested the presence of an inhibitory component. As previously observed in non-lesioned animals, bath application of 4-aminopyridine induced bicuculline-sensitive spontaneous burst discharges in the presence of excitatory amino acid antagonists. These results suggest that cortical freeze lesions associated with abnormal neuronal migration produce a chronic hyperexcitable state. The findings are consistent with a mechanism involving an alteration, not loss, of inhibition in this model.

Topics: 2-Amino-5-phosphonovalerate; 6-Cyano-7-nitroquinoxaline-2,3-dione; Animals; Animals, Newborn; Brain; Cerebral Cortex; Electric Stimulation; Epilepsy; Freezing; Functional Laterality; Humans; In Vitro Techniques; Membrane Potentials; N-Methylaspartate; Neocortex; Nerve Tissue Proteins; Rats; Rats, Sprague-Dawley

The concept of dormant interneurons is proving to be hard to define precisely. We argue here that the term is best used as an operational description of interneurons which are not lost from the epileptic brain, but which fail to perform adequately. We present evidence for the existence of functionally dormant interneurons in the tetanus toxin model of chronic epilepsy, and we explore the roles of a partial dormancy (and also of charge-screening) in the acute low magnesium model of epilepsy.

Topics: 2-Amino-5-phosphonovalerate; 6-Cyano-7-nitroquinoxaline-2,3-dione; Animals; Baclofen; Bicuculline; Computer Simulation; Epilepsy; GABA Antagonists; Hippocampus; In Vitro Techniques; Interneurons; Male; Models, Neurological; Neurons; Pyramidal Cells; Rats; Tetanus Toxin

An in vitro model of epileptiform activity was developed to study the role of excitatory and inhibitory neurotransmitters in the epileptogenesis. Intracellular recordings were obtained from rat neocortical slices exposed to 4-aminopyridine in a magnesium-free solution. Spontaneous epileptiform activity consisting of paroxysmal depolarization shifts with associated spontaneous depolarizing postsynaptic potentials were observed. The paroxysmal depolarization shifts were blocked either by D,L-2-amino-5-phosphonovalerate (50 microM), an N-methyl-D-aspartate receptor antagonist, or by 6-cyano-7-nitroquinoxaline-2.3-dione (10 microM), a non-N-methyl-D-aspartate receptor antagonist. These glutamate receptor antagonists also reduced the occurrence of spontaneous depolarizing postsynaptic potentials. Bicuculline methiodide, an antagonist of GABAA receptors, suppressed spontaneous depolarizing postsynaptic potentials, while it reduced the frequency of paroxysmal depolarization shifts and increased their duration. Hyperpolarization of the membrane potential by continuous current injection increased the frequency of paroxysmal depolarization shifts and reduced their duration, but it reduced the occurrence of spontaneous postsynaptic potentials. Paroxysmal depolarization shifts were blocked by tetrodotoxin (1 microM). The duration and the frequency of paroxysmal depolarization shift were reduced by dopamine (30-300 microM) in a dose-dependent manner. Our model suggests a different involvement of excitatory and inhibitory processes in the generation of epileptiform activity.

Topics: 2-Amino-5-phosphonovalerate; 4-Aminopyridine; 6-Cyano-7-nitroquinoxaline-2,3-dione; Animals; Cells, Cultured; Culture Media; Dendrites; Dopamine; Epilepsy; Excitatory Amino Acid Antagonists; GABA-A Receptor Antagonists; gamma-Aminobutyric Acid; Magnesium; Male; Membrane Potentials; Neocortex; Neurons; Presynaptic Terminals; Rats; Rats, Wistar; Receptors, GABA-A; Receptors, N-Methyl-D-Aspartate

Using microcultured neurons and hippocampal slices, we found that under conditions that completely block AMPA receptors, kainate induces a reduction in the effectiveness of GABAergic synaptic inhibition. Evoked inhibitory postsynaptic currents (IPSCs) were decreased by kainate by up to 90%, showing a bell-shaped dose-response curve similar to that of native kainate-selective receptors. The down-regulation of GABAergic inhibition was not affected by antagonism of metabotropic receptors, while it was attenuated by CNQX. Kainate increased synaptic failures and reduced the frequency of miniature IPSCs, indicating a presynaptic locus of action. In vivo experiments using brain dialysis demonstrated that kainate reversibly abolished recurrent inhibition and induced an epileptic-like electroencephalogram (EEG) activity. These results indicate that kainate receptor activation down-regulates GABAergic inhibition by modulating the reliability of GABA synapses.

Topics: 2-Amino-5-phosphonovalerate; 6-Cyano-7-nitroquinoxaline-2,3-dione; Animals; Bicuculline; Cells, Cultured; Electric Stimulation; Electroencephalography; Embryo, Mammalian; Epilepsy; Evoked Potentials; Female; Functional Laterality; GABA Antagonists; gamma-Aminobutyric Acid; Hippocampus; In Vitro Techniques; Kainic Acid; Neurons; Pyramidal Cells; Rats; Rats, Sprague-Dawley; Rats, Wistar; Receptors, Kainic Acid; Receptors, Presynaptic; Synaptic Transmission; Tetrodotoxin

Epileptogenesis following neocortical trauma from two sources of disinhibition. J. Neurophysiol. 78: 2804-2810, 1997. Intracellular and field potential recordings were obtained from superficial and deep neurons from both intact coronal rat somatosensory slices, and from slices which had been acutely divided into a superficial strip of cortex ( approximately 450 micron from the pia) and a deep segment. Membrane properties for cells in the traumatized slices were similar to those of their counterparts in intact slices. However, synaptic hyperexcitability developed in the deep segments in which a majority of cells likely underwent dendrotomy. This hyperexcitability was manifested by epileptiform activity in 54% of traumatized slices. Measurements of fastGABAergic inhibitory strength showed these slices were disinhibited. Superficial delivery of tetrodotoxin to the upper 450 micron of intact slices led to disinhibition of fast GABAergic transmission as well as an attendant increase in excitatory postsynaptic potential strength but not epileptogenesis. Pharmacological maneuvers aimed at preventing glutamate-triggered increases in intracellular calcium [glutamate ionotropic antagonists, dantrolene, and bis-(o-aminophenoxy)-N,N,N', N'-tetraacetic acid (BAPTA)-AM] showed that a 1 h treatment in these agents conferred protection against epileptogenesis. These results demonstrate that the seizure-like activity developing in deep dendrotomized cortical segments resulted from two sources of GABAergic disinhibition: the physical removal of important superficial inhibitory circuits and glutamate-triggered increases in intracellular calcium.

Topics: 6-Cyano-7-nitroquinoxaline-2,3-dione; Animals; Calcium; Cell Membrane; Chelating Agents; Dantrolene; Egtazic Acid; Epilepsy; Excitatory Amino Acid Antagonists; Excitatory Postsynaptic Potentials; gamma-Aminobutyric Acid; In Vitro Techniques; Models, Neurological; Neocortex; Neurons; Rats; Rats, Sprague-Dawley; Somatosensory Cortex; Synaptic Transmission; Virulence Factors, Bordetella

Application of 4-aminopyridine (4AP, 50 microM) to combined slices of adult rat hippocampus-entorhinal cortex-induced ictal and interictal epileptiform discharges, as well as slow field potentials that were abolished by the mu-opioid agonist [D-Ala2,N-Me-Phe4,Gly-ol5] enkephalin (DAGO, 10 microM) or the GABAA receptor antagonist bicuculline methiodide (BMI, 10 microM); hence, they represented synchronous GABA-mediated potentials. Ictal discharges originated in the entorhinal cortex and propagated to the hippocampus, whereas interictal activity of CA3 origin was usually recorded in the hippocampus. The GABA-mediated potentials had no fixed site of origin or modality of propagation; they closely preceded (0.2-5 sec) and thus appeared to initiate ictal discharges. Only ictal discharges were blocked by the antagonist of the NMDA receptor 3,3-(2-carboxypiperazine-4-yl)propyl-1-phosphonate (CPP, 10 microM), whereas the non-NMDA receptor antagonist 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX, 10 microM) abolished all epileptiform activities. The GABA-mediated potentials continued to occur synchronously in all regions even after concomitant application of CNQX and CPP. [K+]o elevations were recorded in the entorhinal cortex during the ictal discharge (peak values = 13.9 +/- 0.9 mM) and the synchronous GABA-mediated potentials (peak values = 4.2 +/- 0.1 mM); the latter increases were presumably attributable to postsynaptic GABAa-receptor activation because they were abolished by DAGO or BMI. Their role in initiating ictal activity was demonstrated by using DAGO, which abolished both GABA-mediated synchronous potentials and ictal discharges. These data indicate that NMDA-mediated ictal discharges induced by 4AP originate in the entorhinal cortex; such a conclusion is in line with clinical evidence obtained in temporal lobe epilepsy patients. 4AP also induces GABA-mediated potentials that spread within the limbic system when excitatory transmission is blocked and may play a role in initiating ictal discharge by increasing [K+]o.

Topics: 4-Aminopyridine; 6-Cyano-7-nitroquinoxaline-2,3-dione; Analgesics; Animals; Disease Models, Animal; Electrophysiology; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Enkephalins; Entorhinal Cortex; Epilepsy; Excitatory Amino Acid Antagonists; gamma-Aminobutyric Acid; Hippocampus; Limbic System; Male; Membrane Potentials; N-Methylaspartate; Nerve Fibers; Piperazines; Potassium; Rats; Rats, Sprague-Dawley; Receptors, N-Methyl-D-Aspartate; Serotonin Receptor Agonists

In order to determine whether changes in synaptic inhibition are involved in chronic models of epilepsy, it is necessary to understand the factors which determine the kinetics of fast gamma-aminobutyric acid (GABA)ergic inhibition. For this purpose, we analyzed the decaying phase of isolated inhibitory postsynaptic currents (IPSC) in rats CA1 pyramidal cells. Reduction of GABA release (by reducing [Ca2+]o or paired-pulse stimulation) or blockade of GABA uptake (with tiagabine) led to the conclusion that small changes in the amount of GABA available for postsynaptic binding have little effect on the peak amplitude, but have marked effect on the duration of the IPSC. We then studied isolated GABAA receptor-mediated inhibition in area CA1 of the El mouse strain, which is genetically predisposed to epilepsy. Results were compared with the non-epileptogenic mother strain, ddY. Inhibitory postsynaptic potentials (IPSPs) in El mice (IPSPEl) were not significantly different in amplitude of those from ddY mice (IPSPddY). However, the rise-time and duration of IPSPEl were respectively about 25% and 50% shorter than those of IPSPddY. With appropriate pharmacological manipulation of GABA release or uptake, IPSPEl could be made to resemble the IPSPddY and vice versa. It is concluded that the synaptic release of GABA in area CA1 of the El mouse is decreased compared to that of the ddY mouse.

Topics: 6-Cyano-7-nitroquinoxaline-2,3-dione; Animals; Anticonvulsants; Baclofen; Disease Models, Animal; Electric Stimulation; Epilepsy; gamma-Aminobutyric Acid; Hippocampus; Interneurons; Mice; Mice, Mutant Strains; Neural Inhibition; Nipecotic Acids; Receptors, GABA; Species Specificity; Synaptic Transmission; Tiagabine

Blocking inhibition provides one of the most common experimental means of triggering epileptic activity in hippocampus and neocortex. However, it has proved much more difficult to show that chronic models of epilepsies are due to disinhibition. One problem is knowing how much inhibition needs to be blocked to provide a sufficient mechanism for epileptic activity. We have found that inhibitory (GABAA) transmission, estimated from evoked monosynaptic IPSCs, must be reduced to 17% of their control amplitude (by 4-7 microM bicuculline) before hippocampal slices generate all-or-none epileptic discharges. Similar estimates of inhibition in chronic epileptic foci induced by intrahippocampal injection of tetanus toxin showed that monosynaptic IPSCs dropped to 10% of control in the injected hippocampus during the first 2 weeks after injection. At all other stages of the active epileptic foci in the two hippocampi the reduction in IPSCs was not alone sufficient for epileptic activity; at 4-6 weeks IPSCs were normal despite continued epileptic activity. One likely mechanism for the late epileptic activity is a reduction of either the intrinsic excitability, or the synaptic excitation, of inhibitory interneurons so they fail to be recruited normally. Alternative mechanisms include the formation of new excitatory connections, as found at modest levels in the dentate gyrus. Several mechanisms may play a part in chronic foci such as those induced by tetanus toxin, either acting together, or sequentially during the progression of the epileptic focus.

Topics: 6-Cyano-7-nitroquinoxaline-2,3-dione; Animals; Bicuculline; Cerebral Cortex; Disease Models, Animal; Epilepsy; Functional Laterality; gamma-Aminobutyric Acid; Hippocampus; Interneurons; Male; Neural Inhibition; Rats; Rats, Sprague-Dawley; Synaptic Transmission; Tetanus Toxin

1. Intracellular and extracellular recordings were obtained from neocortical brain slices of immature rats (postnatal days 9-16) maintained in vitro. Spontaneous and evoked epileptiform discharges (termed paroxysmal depolarizing shifts or PDSs) were recorded from upper cortical laminae (layers II-III) after exposure to the gamma-aminobuturic acid-A receptor antagonist, bicuculline methiodide. The effects of mGluR activation on PDS duration, spontaneous frequency, and threshold for evoking a PDS were determined. Putative mGluR agonists and antagonists also were tested. 2. Bath application of the mGluR agonist (1S,3R)-1-aminocyclopentane-1,3-dicarboxylate (ACPD, 50-200 mM) elicited biphasic, time-dependent effects on evoked and spontaneous epileptiform discharges. At times early in drug wash-in, ACPD increased PDS duration and spontaneous PDS frequency. In > 60% of the slices, the spontaneous PDSs became regular. Subsequently, ACPD reduced PDS duration and increased the stimulus threshold for evoking a PDS, suggesting that the actions of ACPD were dose dependent. 3. Investigation of the concentration-dependence revealed that sustained low ACPD concentrations (5 microM) elicited only facilitatory actions, whereas higher concentrations were suppressive. These observations suggest the activation of different mGluR subtypes, which may be localized differentially at pre- and postsynaptic sites. 4. Bath application of the mGluR agonists, L-2-amino-4-phosphonobutyrate or (2S,3S,4S)-alpha-(carboxycyclopropyl) glycine, produced only suppressive effects on epileptiform activity in the immature neocortex. L-2-amino-3-phosphonopropionate was an ineffective antagonist of ACPD-mediated modulation of epileptiform activity. Application of the putative antagonist, alpha-methyl-4-carboxyphenylglycine (MCPG), failed to antagonize the biphasic actions of ACPD. MCPG had suppressive effects of epileptiform activity, suggesting activation of mGluRs by endogenous agonists. 5. Simultaneous recordings from deeper and upper cortical layers indicated that the initial negativity of both evoked and spontaneous PDSs began in deeper cortical layers under control conditions and in the presence of ACPD. Intracellular records from neurons in deeper layers displayed two distinct patterns of activity during mGluR activation. Most deep layer neurons received a barrage of excitatory postsynaptic potentials before a spontaneous PDS during ACPD application. A small population of neurons depolarize

Topics: 6-Cyano-7-nitroquinoxaline-2,3-dione; Animals; Cerebral Cortex; Epilepsy; Evoked Potentials; Humans; Infant, Newborn; N-Methylaspartate; Neurons; Rats; Receptors, Metabotropic Glutamate

An in vitro model of persistent epileptiform activity was developed to study the mechanisms involved in epileptogenesis. Extracellular recordings were obtained from rat neocortical slices exposed to magnesium-free solution for 2 h. During exposure to magnesium-free solution spontaneous epileptiform activity consisting of interictal bursting and ictal-like discharges were observed. Interestingly, this activity persisted for hours after the slices were returned to magnesium-containing control solution. The N-methyl-D-aspartate (NMDA) receptor antagonist CPP prevented the development of the epileptiform activity, while the non-NMDA receptor antagonist CNQX abolished the epileptiform discharge that persisted after slices were returned to control solution. These findings suggest there are two distinct phases in the development of epileptic activity in this model, namely, induction (mediated by NMDA receptor activity) and maintenance (supported largely by non-NMDA receptor activity). The similarities and possible parallels between the mechanisms underlying this epileptogenesis and other forms of use-dependent modification of synaptic excitation, such as long-term potentiation, are discussed. This in vitro model of neocortical epileptogenesis may provide insights into the events underlying the development of clinical partial epilepsy.

Topics: 6-Cyano-7-nitroquinoxaline-2,3-dione; Animals; Cerebral Cortex; Disease Models, Animal; Epilepsy; In Vitro Techniques; Magnesium; Rats; Rats, Sprague-Dawley; Time Factors

Lowering extracellular magnesium induces different patterns of epileptiform activity in rat hippocampus and entorhinal cortex. Short recurrent epileptiform discharges in the hippocampus are stable over time, whereas seizure-like events (SLEs) in the entorhinal cortex, the subiculum, and the neighboring neocortex develop into late recurrent discharges which are not blocked by clinically employed antiepileptic drugs. We tested the sensitivity of the different epileptiform discharge patterns to N-methyl-D-aspartate (NMDA)- and non-NMDA-receptor antagonists. As NMDA-receptor antagonist we used dextrorphan, ketamine, and 2-aminophosphonovalerate (2APV); as alpha-amino-3-hydroxy-5-methyl-4-isoxazole-propionic acid (AMPA)-receptor antagonist we employed the quinoxaline derivative glutamate 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX). The findings show that the different patterns of epileptiform activity, including the late recurrent discharges, are sensitive to all NMDA-receptor antagonists. However, when dextrorphan was employed to suppress seizure-like events, later recurrent discharges did not develop during the remaining time course of the experiment. CNQX reversibly suppressed recurrent discharges in the hippocampus and SLEs in the entorhinal cortex. However, late recurrent discharges become insensitive to CNQX, even at a high concentration of 60 microns. This finding suggests a prominent role for NMDA receptors in the generation of late recurrent discharges.

Topics: 2-Amino-5-phosphonovalerate; 6-Cyano-7-nitroquinoxaline-2,3-dione; Animals; Dextrorphan; Epilepsy; Evoked Potentials; Extracellular Space; Female; Hippocampus; Ion Channels; Magnesium; Quinoxalines; Rats; Rats, Wistar; Receptors, AMPA; Receptors, N-Methyl-D-Aspartate; Temporal Lobe

Selective excitatory amino acid- and GABAB-receptor antagonists were used to examine the role these receptors play in epileptiform burst discharge elicited by blocking GABAA receptor-mediated inhibition in hippocampal slice cultures of the rat. Application of bicuculline caused a single ictal burst followed by interictal bursting. The N-methyl-D-aspartate receptor antagonist, D-2-amino-5-phosphonovalerate, reduced the depolarizing envelope underlying interictal discharge, and accentuated the appearance of concomitant slow oscillatory potentials, which occurred synchronously in all CA3 cells. The non-N-methyl-D-aspartate receptor antagonists, 6-nitro-7sulphamoyl-benzo(F) quinoxaline and 6-cyano-7-nitro-quinoxaline-2,3-dione, blocked interictal bursting at high concentrations, and low concentrations of 6-cyano-7-nitro-quinoxaline-2,3-dione selectively eliminated the slow oscillations in an all-or-none manner, leaving the depolarizing envelope. No effects of either metabotropic glutamate receptor antagonists or of dihydropyridine Ca2+ channel agonists or antagonists on evoked interictal discharge were observed. 6-Cyano-7-nitro-quinoxaline-2,3-dione-resistant interictal-like discharge could be obtained in the presence of bicuculline when the external Mg2+ concentration was reduced from 1.5-0.5 mM. The GABAB receptor antagonist CGP 35348 prolonged individual evoked interictal bursts, and caused the appearance of spontaneous ictal-like discharges. The implications of these results are discussed with regard to the mechanisms of epileptogenesis and to potential therapeutic intervention.

Topics: 6-Cyano-7-nitroquinoxaline-2,3-dione; Animals; Bicuculline; Calcium Channel Agonists; Calcium Channel Blockers; Calcium Channels; Electrophysiology; Epilepsy; Excitatory Amino Acid Antagonists; GABA-B Receptor Antagonists; Hippocampus; In Situ Hybridization; Organ Culture Techniques; Pyramidal Cells; Rats; Receptors, GABA-B; Receptors, Glutamate; Synapses

1. The effects of unilateral gamma-ray irradiation at birth on the properties of adult CA3 pyramidal neurons have been studied in hippocampal slices. 2. Neonatal gamma-ray irradiation reduced by 80% the number of granule cells and prevented the formation of mossy fiber synapses without reducing the number of CA3 pyramidal cells. The destruction of the mossy fibers was also confirmed with extracellular recordings. 3. Excitatory and inhibitory postsynaptic potentials (EPSPs and IPSPs) evoked by stimulation of the stratum radiatum had similar properties in nonirradiated and irradiated hippocampi: the EPSP reversed polarity near 0 mV, was reduced in amplitude by 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX, 10 microM) and D(-)-2-amino-5-phosphonovalerate (APV, 50 microM); the fast and slow IPSPs reversed at -75 and -100 mV, were blocked by bicuculline (10 microM), and reduced by phaclofen (0.5 mM), respectively. 4. Bath application of kainate (300-500 nM) evoked epileptiform activity in 81.5% of nonirradiated hippocampal CA3 regions and only in 29% of the irradiated CA3 regions. In contrast, bath application of high potassium (7 mM) and bicuculline (10 microM) generated spontaneous and evoked epileptiform activity in both nonirradiated and irradiated CA3 regions. 5. In nonirradiated and irradiated CA3 regions, kainate (200-300 nM) reduced the amplitude of the fast and slow IPSPs, reduced spike accommodation, and increased the duration of the action potential generated by a depolarizing pulse. 6. The postsynaptic responses of CA3 neurons to bath application of glutamatergic agonists were similar in nonirradiated and irradiated hippocampi in terms of amplitude, reversal potential, and pharmacology. 7. It is concluded that the most conspicuous effect of neonatal gamma-ray irradiation is to prevent the epileptic action of kainate. We propose that kainate generates epileptiform activity in the intact CA3 region by activating high-affinity binding sites located on the mossy fiber terminals.

Topics: 2-Amino-5-phosphonovalerate; 6-Cyano-7-nitroquinoxaline-2,3-dione; Animals; Animals, Newborn; Bicuculline; Electric Stimulation; Electrophysiology; Epilepsy; Gamma Rays; gamma-Aminobutyric Acid; Hippocampus; In Vitro Techniques; Kainic Acid; Male; Membrane Potentials; Nerve Fibers; Neurons, Afferent; Potassium; Pyramidal Cells; Quinoxalines; Rats; Rats, Wistar

When hippocampal slices are exposed to GABAA antagonists, area CA3 pyramidal cells and dentate hilar "mossy" cells discharge in synchronized epileptiform bursts (Müller and Misgeld, 1991; Scharfman, 1994b). Dentate interneurons are excited simultaneously, but the degree of discharge varies (Scharfman, 1994b). This study primarily examined the activity of dentate granule cells simultaneous to the epileptiform bursts of pyramidal cells and mossy cells. EPSPs followed by large GABAB receptor-mediated IPSPs were generated in granule cells during all epileptiform bursts of pyramidal cells and mossy cells, regardless of whether they were evoked or spontaneous. By simultaneous recording it was determined that granule cell EPSPs began several milliseconds after the start of pyramidal cell bursts (n = 48 simultaneous recordings) and immediately after the first action potential of a mossy cell burst (n = 77). Interneurons were similar to granule cells in the timing of their depolarizations relative to the onset of pyramidal cell (n = 24; Scharfman, 1994b) and mossy cell (n = 9) bursts. All excitatory activity was blocked by bath application of the glutamatergic AMPA/kainate receptor antagonist CNQX (5 microM, n = 5), but not the NMDA receptor antagonist D-APV (25-50 microM, n = 9). Granule cell EPSPs were decreased after focal application of CNQX to the molecular layer at a site close to the impaled granule cell (n = 5), whereas D-APV had no effect (n = 3). EPSPs also decreased after focal application of CNQX to the hilus, in two of four slices tested. The extracellularly recorded EPSP of granule cells was maximal in the inner molecular layer (n = 33), the site of the mossy cell axonal plexus. Severing the junction of the dentate gyrus and area CA3 blocked all spontaneous and evoked activity of dentate neurons without affecting burst discharges in area CA3a and CA3b (n = 6). None of the excitatory activity of any cell type was affected by cholinergic antagonists (atropine and mecamylamine, 25 microM each, n = 5; pirenzipine and dihydro-beta-erythroidine, 25 microM each, n = 5). The results suggest that there is a glutamatergic, AMPA/kainate receptor-mediated, excitatory pathway from area CA3 to the dentate gyrus in disinhibited slices. The pharmacological results, analyses of latency, as well as the known axonal projections of the sampled cells, suggest that the excitatory pathway begins within area CA3 and leads to granule cells via mossy cells. The data also sugg

Topics: 2-Amino-5-phosphonovalerate; 6-Cyano-7-nitroquinoxaline-2,3-dione; Animals; Atropine; Epilepsy; Evoked Potentials; Hippocampus; In Vitro Techniques; Male; Mecamylamine; Membrane Potentials; Pyramidal Cells; Rats; Rats, Sprague-Dawley; Synaptic Transmission

1. Injecting twelve mouse minimum lethal doses of tetanus toxin into one hippocampus of a rat leads to the development of chronic epileptic foci in both hippocampi. These generate intermittent epileptic discharges for 6-8 weeks. Here we compare GABAergic inhibition, 10-18 days after injection, in slices prepared from the injected and contralateral hippocampi (respectively the primary and the secondary or 'mirror' foci), using both neurochemical and electrophysiological methods. 2. Epileptic activity was recorded from slices of both hippocampi from all tetanus toxin-injected rats. Evoked epileptic discharges were similar on the two sides, but spontaneous epileptic discharges were more common contralaterally. 3. Ca(2+)-dependent, K(+)-stimulated (synaptic) release of radiolabelled GABA was depressed in slices from the injected hippocampus, compared with vehicle-injected controls. In contrast, slices from the contralateral hippocampus had normal levels of Ca(2+)-dependent, K(+)-stimulated GABA release, even though adjacent slices were epileptogenic. 4. Intracellular recordings revealed that both fast and slow stimulus-evoked inhibitory postsynaptic potentials (IPSPs) were abolished in CA3 pyramidal cells in the primary focus. In the secondary focus, however, fast IPSPs were seen in seven of twenty-five cells, and slow IPSPs were seen in all cells if the stimulus was strong enough. 5. Monosynaptic IPSPs were isolated pharmacologically by blocking glutamatergic excitatory postsynaptic potentials (EPSPs) with 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) and D(-)-2-amino-5-phosphopentanoic acid (AP-5). No monosynaptic IPSPs were uncovered in cells from the primary focus at any stimulus strength. Monosynaptic IPSPs were evoked in all cells from both the secondary focus and control slices. The estimated conductances of monosynaptic fast IPSPs were similar in cells from the secondary focus and from the controls, although the former required twice the stimulus strength. 6. Slow IPSPs were found in the secondary focus and in controls, but not in the primary focus. They were sensitive to 3-amino-2-(4-chlorophenyl)-2-hydroxy-propylsulphonic acid (2-OH saclofen). The estimated conductances of slow IPSPs evoked by weak stimuli in the secondary focus were much smaller than in the controls. However, stimuli that could trigger epileptic discharges in the secondary focus, evoked 2-OH saclofen-sensitive slow IPSPs with estimated conductances approaching the controls. This mark

Topics: 2-Amino-5-phosphonovalerate; 6-Cyano-7-nitroquinoxaline-2,3-dione; Animals; Calcium; Electrophysiology; Epilepsy; Evoked Potentials; gamma-Aminobutyric Acid; Hippocampus; In Vitro Techniques; Injections; Male; Potassium; Pyramidal Tracts; Quinoxalines; Rats; Rats, Sprague-Dawley; Stereotaxic Techniques; Synapses; Tetanus Toxin

Many studies suggest that the dentate gyrus (DG) is a control point for hippocampal epileptogenesis. However, the importance of GABAergic inhibition in the DG is not quite clear. Intracellular recordings were obtained from granule cells (GC) of the rat DG. In addition to GABAA-mediated spontaneous postsynaptic potentials (PSPs), some GC exhibited spontaneous slow hyperpolarizations (SH). The SH were more commonly observed in a high concentration of external potassium. 2-Hydroxysaclofen, a GABAB antagonist, reduced the SH. Focal stimulation of the perforant path (PP) in the subiculum with a single pulse evoked a depolarization followed by a SH, which were both abolished by the excitatory amino acid (EAA) blockers, 6-cyano-7-nitroquinoxaline-2,3 dione (CNQX) and 2-amino-5-phosphonovaleric acid (APV). When evoked with a train of pulses, the SH was unaffected by the EAA blockers in 40% of the cells, suggesting either the existence of a GABAergic PP, or an unidentified polysynaptic mechanism. In control, the synaptic response to PP stimulation was superficially similar whether the stimulus was applied in the subiculum or stratum moleculare. However, in presence of bicuculline, the subicular PSP was followed by a train of PSPs occurring at a constant frequency of 25 Hz. This 'reverberating' effect of bicuculline was decreased in presence of APV and was abolished in slices in which the excitatory transmission had been interrupted downstream from CA3 neurons, suggesting that reverberation required the integrity of the hippocampo-entorhinal loop. By contrast, bicuculline decreased the amplitude of the stratum moleculare PSP. It is concluded that GC receive tonic inhibition from GABA acting at GABAA and GABAB receptors. The role of GABAB receptors is unclear; by contrast, GABAA-mediated inhibition prevents GC from reverberated excitation. The probability of occurrence of reverberation is higher during activation of the whole temporo-ammonic pathway and is partly dependent on the activation of N-methyl-D-aspartate (NMDA) receptors. Thus, the in vitro brain slice can be used as a model to study reverberation which has been recently demonstrated to underlie epileptiform discharges in the whole brain preparation.

Topics: 2-Amino-5-phosphonovalerate; 6-Cyano-7-nitroquinoxaline-2,3-dione; Animals; Baclofen; Bicuculline; Electric Stimulation; Epilepsy; Evoked Potentials; gamma-Aminobutyric Acid; Hippocampus; In Vitro Techniques; Male; Membrane Potentials; Pyramidal Tracts; Quinoxalines; Rats; Rats, Sprague-Dawley; Receptors, GABA-A; Receptors, GABA-B; Synapses

We studied some of the physiological and pharmacological properties of an in vitro model of epileptic seizures induced by elevation of [K+]0 (to 8 mM and 10 mM) in combination with lowering of [Mg2+]0 (to 1.4 mM and 1.6 mM) and [Ca2+]0 (to 0.7 mM and 1 mM) in rat hippocampal slices. These concentrations correspond to the ionic constitution of the extracellular microenvironment during seizures in vivo. The resulting activity was rather variable in appearance. In area CA3 recurrent discharges were observed which resulted in seizure-like events with either clonic-like or tonic-clonic-like ictaform events in area CA1. With ion-sensitive electrodes, we measured the field potential and the changes in extracellular ion concentrations which accompany this activity. The recurrent discharges in area CA3 were accompanied by small fluctuations in [K+]0 and [Ca2+]0. The grouped clonic-like discharges in area CA1 were associated with moderate increases in [K+]0 and small decreases in [Ca2+]0 in the order of 2 mM and 0.2 mM, respectively. Large, negative field-potential shifts and increases in [K+]0 to 13 mM, as well as decreases in [Ca2+]0 by up to 0.4 mM, accompanied the tonic phase of ictaform events. The ictaform events were not blocked by D-2-aminophosphonovalerate (2-APV) but were sensitive to 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) alone and in combination with 2-APV and ketamine. In order to determine the pharmacological characteristics of the ictaform events we bath-applied most clinically employed anticonvulsants (carbamazepine, phenytoin, valproate, phenobarbital, ethosuximide, trimethadione) and some experimental anticonvulsants (losigamone, vinpocetine, and apovincaminic acid). Carbamazepine, phenytoin, valproate, and phenobarbital were effective at clinically relevant doses. The data suggest that the high-K+ model of epileptiform activity is a good model of focal convulsant activity.

Topics: 2-Amino-5-phosphonovalerate; 6-Cyano-7-nitroquinoxaline-2,3-dione; Animals; Calcium; Carbachol; Electrophysiology; Epilepsy; Evoked Potentials; Hippocampus; In Vitro Techniques; Magnesium; Membrane Potentials; Microelectrodes; N-Methylaspartate; Neurons; Potassium; Pyramidal Tracts; Quaternary Ammonium Compounds; Quinoxalines; Quisqualic Acid; Rats; Rats, Wistar; Temperature

We used an in vitro model similar to kindling to examine the processes underlying epileptogenesis. A 60 Hz train was applied every 5-10 min to the Schaffer collateral pathways in guinea pig hippocampal slices until epileptiform bursting was elicited in the CA3 region. The resultant alterations in both spontaneous and evoked activities were studied using intracellular recordings from CA3 pyramidal cells. An attempt was made to elucidate the synaptic modifications responsible for the conversion to this state of enhanced excitability. Analyses revealed that the emergence of epileptiform discharge was accompanied by a long-term depression of evoked inhibitory conductances. This tetanus-induced reduction of inhibition involved both the early and late phases of the evoked hyperpolarization, suggesting modification of both the GABAA and GABAB receptor-mediated events. Previous studies have suggested that NMDA receptor activation plays an important role in the induction of epileptiform activity in this model. Our data, showing that depression of inhibition can be induced in the presence of CNQX, is consistent with this hypothesis. The parallel development of long-term depression of inhibition and epileptiform bursting following tetanic stimulation suggests that plasticity of the inhibitory transmission process is a potential source of vulnerability contributing to epileptogenesis.

Topics: 6-Cyano-7-nitroquinoxaline-2,3-dione; Action Potentials; Animals; Electric Stimulation; Epilepsy; gamma-Aminobutyric Acid; Guinea Pigs; Hippocampus; In Vitro Techniques; Pyramidal Cells; Quinoxalines; Synapses; Synaptic Transmission; Tetany

The morphological and functional consequences of epileptic activity were investigated by applying the convulsants bicuculline and/or picrotoxin to mature rat hippocampal slice cultures. After 3 days, some cells in all hippocampal subfields showed signs of degeneration, including swollen somata, vacuolation, and dendritic deformities, whereas others displayed only a massive reduction in the number of their dendritic spines. Intracellular recordings from CA3 pyramidal cells revealed a decrease in the amplitude of evoked excitatory synaptic potentials. gamma-Aminobutyric acid-releasing interneurons and inhibitory synaptic potentials were unaffected. Seven days after withdrawal of convulsants, remaining cells possessed a normal number of dendritic spines, thus demonstrating a considerable capacity for recovery. The pathological changes induced by convulsants are similar to those found in the hippocampi of human epileptics, suggesting that they are a consequence, rather than a cause, of epilepsy.

Topics: 2-Amino-5-phosphonovalerate; 6-Cyano-7-nitroquinoxaline-2,3-dione; Animals; Animals, Newborn; Bicuculline; Dendrites; Electric Stimulation; Epilepsy; Evoked Potentials; Hippocampus; Membrane Potentials; Organ Culture Techniques; Organophosphorus Compounds; Pyramidal Tracts; Quinoxalines; Rats; Synapses

Extracellular field potential recordings were used to study the epileptiform activity evoked by tetraethylammonium (TEA) in the CA3 subfield of hippocampal slices obtained from young (12-18 day-old) and adult (> 60-day-old) rats. During TEA application (5-10 mM), young slices generated both ictal-like (duration: up to 28 s, rate of occurrence 1-3 x 10(-2) s-1) and interictal-like (duration: 1.5-2 s; rate of occurrence: 1-3 x 10(-1) s-1) activity. In adult slices only interictal-like activity was induced by TEA (3-10 mM). Depending on the concentrations of TEA, these events lasted 80-600 ms and occurred at 5-60 x 10(-2) s-1. Both the N-methyl-D-aspartate (NMDA) receptor antagonist 3-3(2-carboxypiperazine-4-yl)propyl-1-phosphonate (5-10 microM; CPP) and the non-NMDA receptor antagonist 6-cyano-7-nitroquinoxaline-2,3-dione (5-10 microM; CNQX) were necessary to suppress ictal-likeand interictal-like discharges in young slices. By contrast, interictal-like activity in adult slices was reduced and eventually blocked by CNQX (0.5-3 microM) alone. Furthermore the pattern of epileptiform discharges seen in young slices was modified by CPP (i.e. decrease in the rate of occurrence of ictal events and reduction in the duration of interictal discharges), while the activity recorded in adult slices was resistant to this NMDA antagonist. Bicuculline methiodide (5 microM; BMI) enhanced the duration of epileptiform activities in both young and adult slices. Our data demonstrate that the epileptiform discharges induced by TEA in the CA3 subfield of the rat hippocampus display age-dependent patterns of activity.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: 6-Cyano-7-nitroquinoxaline-2,3-dione; Aging; Animals; Bicuculline; Dose-Response Relationship, Drug; Epilepsy; Evoked Potentials; GABA-A Receptor Antagonists; Hippocampus; In Vitro Techniques; Male; Piperazines; Pyramidal Tracts; Quinoxalines; Rats; Rats, Sprague-Dawley; Receptors, N-Methyl-D-Aspartate; Tetraethylammonium; Tetraethylammonium Compounds

Injecting 0.5-1.0 microgram of cholera toxin into rat hippocampus induces a chronic epileptic focus which generates interictal discharges and brief epileptic seizures intermittently over the following seven to 10 days. Here we examined the electrophysiological properties of hippocampal slices prepared from these rats three to four days after injection, at the height of the epileptic syndrome. These slices generated epileptic discharges in response to electrical stimulation of afferent pathways. In many cases epileptic discharges occurred spontaneously in the CA3 subregion; these usually lasted < 200 ms, but they could last < 0.6 s. Intracellular recordings from pyramidal layer cells revealed depolarization shifts synchronous with the epileptic field potentials. These depolarization shifts had slow onsets compared with those induced by blocking inhibition with bicuculline (depolarizations started a mean of 57 ms before, and reached 5.2 mV by, the onset of the cholera toxin epileptic field potential, compared with 12 ms and 3.6 mV respectively for 70 microM bicuculline methiodide). Extracellular unit recordings showed that the slow predepolarization seen in the cholera toxin focus was associated with an acceleration of the firing of other pyramidal layer neurons. The epileptic activity in this model cannot be attributed to the loss of synaptic inhibition, because inhibitory postsynaptic potentials could be evoked when the synchronous bursts were blocked by increasing [Ca2+]o from 2 to 8 mM. Observations of monosynaptic inhibitory postsynaptic currents isolated by application of 20 microM 6-cyano-7-nitroquinoxaline-2,3-dione, 50 microM DL-2-amino-5-phosphonovaleric acid and 100-200 microM 3-amino-2-(4-chlorophenyl)-2-hydroxy-propylsulphonic acid showed a small effect of the toxin only on the time course of the inhibitory postsynaptic current. On the other hand, there were significant changes in the intrinsic properties of individual neurons. The membrane potentials of cells in the cholera toxin focus did not differ from those in slices from rats injected with vehicle solution, but their input resistances were significantly increased. Unlike the other cellular changes in this model, the increase in input resistance was not seen in slices exposed acutely to 1 micrograms/ml cholera toxin for 30 min, suggesting there may be morphological changes in the chronic focus. Action potential accommodation and the slow afterhyperpolarization were depressed in both acute

Topics: 2-Amino-5-phosphonovalerate; 6-Cyano-7-nitroquinoxaline-2,3-dione; Action Potentials; Animals; Baclofen; Bicuculline; Cholera Toxin; Chronic Disease; Epilepsy; Hippocampus; Injections; Male; Neurons; Quinoxalines; Rats; Rats, Wistar; Receptors, GABA-A; Synaptic Transmission

Extracellular field recordings were performed in guinea-pig and rat neocortical slice preparations maintained in vitro. Bath application of the convulsant drug 4-aminopyridine (4-AP, 100 microM) induced spontaneous epileptiform potentials in 80% of the guinea-pig neocortical slices and only in 6% of the neocortical slices from rat. In both species spontaneous epileptiform activity consisted of a 4-16 s long ictal-like discharge that recurred with a frequency range of 0.01-0.02 Hz. In rat neocortical slices stimulus-induced responses resembled the spontaneous occurring epileptiform events. Ictal-like discharges in guinea-pig neocortical slices were blocked by the N-methyl-D-aspartate (NMDA) receptor antagonist 3-((+/-)-2-carboxypiperazine-4-yl)propyl-1-phosphonic acid (5 microM), while those in the rat disappeared during perfusion with the non-NMDA excitatory amino acid receptor antagonist 6-cyano-7-nitroquinoxaline-2,3-dione (1-3 microM). These results indicate that the neocortex of guinea-pig has a higher propensity to generate 4-AP-induced spontaneous epileptiform activity than that of rat. Furthermore the epileptiform activity in these two species requires a different involvement of excitatory amino acid receptors.

Topics: 4-Aminopyridine; 6-Cyano-7-nitroquinoxaline-2,3-dione; Animals; Anticonvulsants; Cerebral Cortex; Electric Stimulation; Electrophysiology; Epilepsy; Guinea Pigs; In Vitro Techniques; Piperazines; Quinoxalines; Rats; Receptors, N-Methyl-D-Aspartate

Spontaneous episodes of spreading depression (SD) were observed in the CA3 subfield of immature or young (2-30 days postnatally) hippocampal slices perfused with medium containing 4-aminopyridine (4-AP, 50 microM). SD appeared in 34% of the hippocampal slices examined and was more frequently observed in slices obtained from 11 to 20-day-old animals. SD studied with extracellular field potential recordings consisted of large amplitude (18.7 +/- 1.1 mV, mean +/- S.E.M.) negative DC shifts that lasted 30-250 s. Unlike the epileptiform activity that was concomitantly seen during 4-AP application, SD was blocked by the NMDA receptor antagonist 3-((RS)-2-carboxypiperazine-4-yl)-propyl-1-phosphonic acid (CPP, 2-10 microM). In contrast, 6-cyano-7-nitro-quinoxaline-2,3-dione (CNQX, 5 microM), a non-NMDA-type receptor antagonist, blocked the epileptiform activity but only increased the interval between SD episodes. These results demonstrate that immature hippocampal tissue is susceptible to SD episodes, when perfused with 4-AP-containing medium, and that the occurrence of these episodes presumably depends on the activation of the NMDA receptor. In addition these findings indicate that SD shows a sensitivity to excitatory amino acid receptor antagonists that differs from that of the epileptiform activity recorded simultaneously.

Topics: 4-Aminopyridine; 6-Cyano-7-nitroquinoxaline-2,3-dione; Aging; Animals; Animals, Newborn; Anticonvulsants; Dendrites; Epilepsy; Evoked Potentials; Hippocampus; In Vitro Techniques; Membrane Potentials; Models, Biological; Piperazines; Pyramidal Tracts; Quinoxalines; Rats; Rats, Inbred Strains; Receptors, N-Methyl-D-Aspartate

1. Synaptic transmission mediated by the N-methyl-D-aspartate (NMDA)-receptor type was studied in neocortex from children undergoing surgical treatment for intractable epilepsy. Intracellular recordings from pyramidal cells were obtained in slices of neocortical tissue by use of microelectrodes. Synaptic responses were induced by electrical stimulation and studied with current-clamp and single-electrode voltage-clamp techniques. The NMDA-receptor-mediated component of the synaptic responses was isolated by addition of 10 microM bicuculline and 30 microM 6-cyano-2,3-dihydroxy-7-nitroquinoxaline (CNQX) in the perfusion solution. 2. In the presence of bicuculline and CNQX, electrical stimulation evoked an excitatory postsynaptic potential (EPSP) in every recorded cell. The amplitude of this EPSP increased when membrane potential was depolarized with injected current. 3. All cells studied in voltage clamp were recorded with microelectrodes containing Cs+ and QX 314. To avoid contamination of the responses from voltage-dependent Ca2+ conductances, membrane potential was held at depolarized potentials until Ca2+ spiking inactivated completely. The evoked excitatory postsynaptic currents (EPSCs) measured at resting membrane potential ranged from 100 to 400 pA. The NMDA receptor-selective antagonist DL-2-amino-5-phosphonopentanoic acid (AP-5) reversibly decreased the current amplitude by 60% for 10 microM and 80% for 30 microM. 4. The current-voltage (I-V) relation showed a region of negative slope conductance between -100 and -20 mV. The largest currents (-250 to -900 pA) were recorded in the range of -45 to -20 mV and reversed between -10 and +10 mV. Removing Mg2+ from the perfusion solution decreased the negativity of the slope, which is consistent with a reduction in the voltage-dependent Mg2+ block of the NMDA-receptor channel. 5. The I-V plots obtained from cells recorded in the most abnormal tissue were averaged and compared with those from the least abnormal tissue. No significant difference was found between these two groups. The averaged plots from the youngest patients (8 and 10 mo old) and those from the oldest (5-15 yr old) patients were also compared, and the results from these two groups were not significantly different.(ABSTRACT TRUNCATED AT 400 WORDS)

Topics: 2-Amino-5-phosphonovalerate; 6-Cyano-7-nitroquinoxaline-2,3-dione; Adolescent; Aging; Bicuculline; Cerebral Cortex; Child; Child, Preschool; Electric Stimulation; Electrophysiology; Epilepsy; Evoked Potentials; Female; Humans; Infant; Male; Membrane Potentials; Microelectrodes; N-Methylaspartate; Neurons; Quinoxalines; Receptors, GABA-A; Receptors, N-Methyl-D-Aspartate; Synapses; Synaptic Transmission

The effect of ethanol on the epileptiform activity induced by Mg(++)-free solution was studied in rat amygdalar slices using intracellular recording techniques. The spontaneous and evoked epileptiform discharges consisting of an initial burst followed by afterdischarges were observed 20-30 min after switching to Mg(++)-free medium. Superfusion with ethanol (20-100 mM) reversibly reduced the duration of spontaneous and evoked bursting discharges in a concentration-dependent manner. Synaptic response mediated by N-methyl-D-aspartate (NMDA) receptor activation was isolated by application of a solution containing the non-NMDA receptor antagonist 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) and either in Mg(++)-free solution or in the presence of 50 microM bicuculline. Application of ethanol reversibly suppressed the duration of NMDA receptor-mediated synaptic response. These results suggest that intoxicating concentrations of ethanol possess anticonvulsant activity through blocking the NMDA receptor-mediated synaptic excitation. In addition, the observed effect of ethanol on NMDA receptor-mediated synaptic response could be relevant to the cognitive and behavioral manifestations seen in some alcoholics.

Topics: 6-Cyano-7-nitroquinoxaline-2,3-dione; Amygdala; Animals; Anticonvulsants; Bicuculline; Depression, Chemical; Epilepsy; Ethanol; In Vitro Techniques; Magnesium; Male; Membrane Potentials; Quinoxalines; Rats; Rats, Inbred Strains; Receptors, N-Methyl-D-Aspartate; Synapses; Synaptic Transmission

The AMPA/KA receptor antagonist 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) and the NMDA receptor antagonist 2-amino-5-phosphonovalerate (D-APV) were used to investigate the contribution of excitatory amino acid (EAA) receptors to graded bursting activity recorded in the CA1 region of the rat hippocampal slice following bath application of the convulsant drug bicuculline methiodide (BIC, 2-3 microM). CNQX (5-9 microM) significantly antagonised the burst in a reversible, concentration-dependent manner (n = 5). The effect involved a reduction in the amplitude but not the number of population spikes of the burst and also a depression of the underlying burst excitatory post-synaptic potential (EPSP). D-APV (5-25 microM), in contrast, reduced the amplitude and number of spikes in the burst but had no effect on the burst EPSP (n = 5). Following a single concentration of CNQX (5 microM), applied in the presence of bicuculline, it was observed that the components of epileptiform response which remained could be completely abolished with D-APV (10 microM; n = 10). It was also shown that, following elimination of synaptic transmission with CNQX (5 microM), application of bicuculline (2-3 microM) induced a small burst that could be reversibly antagonised with D-APV (10 microM). These results show that evoked epileptiform activity witnessed in the presence of bicuculline involves the activation of both AMPA and NMDA receptors, the AMPA receptor activation making the major contribution. The burst mediated by NMDA receptors is not dependent on prior activation of AMPA receptors.

Topics: 2-Amino-5-phosphonovalerate; 6-Cyano-7-nitroquinoxaline-2,3-dione; Animals; Bicuculline; Epilepsy; Hippocampus; In Vitro Techniques; Male; Quinoxalines; Rats; Rats, Wistar; Receptors, AMPA; Receptors, N-Methyl-D-Aspartate; Receptors, Neurotransmitter; Synapses; Synaptic Transmission

Conventional intracellular recordings were made from regular-spiking cells located in layers II-IV to examine the involvement of excitatory amino acid receptors in synaptic transmission in epileptogenic human neocortical slices maintained in vitro. Extracellular stimuli that were below the threshold for generating action potentials evoked an excitatory postsynaptic potential (EPSP) with short latency to onset (0.8-4 ms). When suprathreshold stimuli were delivered, 95% of the neurons fired a single action potential. In 5% of the population, however, an all-or-none bursting discharge was observed. The EPSP and the bursting discharge were tested with the N-methyl-D-aspartate (NMDA) antagonist 3-((+/-)-2-carboxypiperazin-4-yl)propyl-1-phosphonate (CPP, 5 microM) or the non-NMDA antagonist 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX, 4 microM). In the presence of CNQX the peak amplitude of the EPSP was reduced by 85% and the bursting discharge was abolished completely. By contrast, CPP reduced the peak amplitude of the EPSP by 52%, attenuated the late phase of the bursting discharge and increased its threshold. These results indicate that excitatory amino acids function as excitatory transmitters in the human brain. While the involvement of non-NMDA receptors in the EPSP is in line with data from normal neocortical slices of other mammals, the participation of NMDA-mediated conductances to the EPSP appears peculiar to the epileptogenic human neocortex. This evidence, together with the contribution of NMDA and non-NMDA receptors to the all-or-none bursting discharge suggests that excitatory amino acid-mediated transmission might be modified in the epileptogenic human neocortex.

Topics: 6-Cyano-7-nitroquinoxaline-2,3-dione; Action Potentials; Adult; Cerebral Cortex; Epilepsy; Evoked Potentials; Humans; Membrane Potentials; Organ Culture Techniques; Piperazines; Psychosurgery; Quinoxalines; Receptors, N-Methyl-D-Aspartate; Synaptic Transmission

1. Intracellular recordings were obtained from neurons in layer II-III of rat frontal cortex maintained in vitro. The role of excitatory amino acid receptors in generation of picrotoxin (PTX)-induced epileptiform activity was investigated with the use of D-2-amino-5-phosphonovaleric acid (D-APV) and 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) as selective antagonists of N-methyl-D-aspartate (NMDA) and non-NMDA receptors, respectively. 2. Bath application of PTX resulted in a decrease in evoked inhibitory postsynaptic potentials (IPSPs) in neocortical neurons and a concomitant increase in a polysynaptic late excitatory postsynaptic potential (IEPSP). Epileptiform burst responses, termed paroxysmal depolarizing shifts (PDSs), subsequently developed. Based on response duration, two types of PDSs were identified. Long PDSs were greater than 100 ms in duration, whereas short PDSs lasted less than 50 ms. An early depolarizing potential preceded both types of epileptiform burst response. 3. The NMDA receptor antagonist D-APV reduced the peak amplitude and duration of the PDS. D-APV-insensitive portions of the PDS were greatly attenuated or abolished by CNQX. The non-NMDA antagonist also increased the latency to PDS onset and reduced its duration without affecting peak amplitude. CNQX-insensitive components of the PDS, when present, were abolished by D-APV. 4. Short-duration PDSs could be blocked by CNQX. In these neurons, increasing the stimulation strength produced epileptiform responses of reduced amplitude. 5. Under control conditions, PDS amplitude was a linear function of membrane potential, increasing with hyperpolarization and diminishing on depolarization.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: 2-Amino-5-phosphonovalerate; 6-Cyano-7-nitroquinoxaline-2,3-dione; Action Potentials; Animals; Cerebral Cortex; Electricity; Epilepsy; Female; Male; N-Methylaspartate; Picrotoxin; Quinoxalines; Rats; Rats, Inbred Strains; Reaction Time; Receptors, Amino Acid; Receptors, Cell Surface; Receptors, N-Methyl-D-Aspartate

1. Intracellular recordings were made from layer V/VI neurons of the guinea pig anterior cingulate cortex to investigate postsynaptic potentials (PSPs) evoked by electrical stimulation of the subcortical white matter (forceps minor). 2. Four distinct types of PSPs were recorded (at the resting potential) under normal physiological conditions; 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX)-sensitive excitatory postsynaptic potentials (EPSPs) were followed by bicuculline- or picrotoxin-sensitive depolarizing or hyperpolarizing inhibitory postsynaptic potentials (IPSPs), which were further followed by phaclofen-sensitive, long-lasting hyperpolarizing postsynaptic potentials (LPSPs). The average times-to-peak for the EPSP, depolarizing and hyperpolarizing IPSPs, and LPSP were 10, 22, 28, and 146 ms, respectively. 3. In the presence of CNQX and bicuculline, high-intensity electrical stimulation elicited a longer lasting EPSP with a time-to-peak of 21 ms. The amplitude and duration of the EPSP decreased with membrane hyperpolarization and increased with membrane depolarization. The EPSP was reversibly abolished by D,L-2-amino-5-phosphonovaleric acid (D,L-APV). 4. The bicuculline- or picrotoxin-sensitive depolarizing and hyperpolarizing IPSPs and the phaclofen-sensitive LPSP were markedly suppressed by CNQX, suggesting that glutamate (Glu) and/or aspartate nerve terminals project to GABAergic interneurons, and that the GABAergic interneurons are activated mainly by non-N-methyl-D-aspartate (non-NMDA) receptors. 5. In the presence of picrotoxin, the average reversal potential for the compound EPSP was 0 mV, which was similar to that (-6 mV) for the Glu-induced depolarization. In a solution containing D,L-APV at low concentrations, the average reversal potentials for the depolarizing and hyperpolarizing IPSPs and for the early and late components of the gamma-aminobutyric acid (GABA)-induced responses were -62, -72, -70, and -61 mV, respectively. Thus the value for the depolarizing IPSP was similar to that for the late response to GABA, whereas the value for the hyperpolarizing IPSP was almost the same as that for the early response to GABA. The average reversal potential of -90 mV for the LPSP was similar to -93 mV for the baclofen-induced hyperpolarization and to -94 mV for the spike afterhyperpolarization. 6. Application of phaclofen decreased the interspike interval of the spontaneous firing and reversed the increase in the interspike interval after subcortical s

Topics: 2-Amino-5-phosphonovalerate; 6-Cyano-7-nitroquinoxaline-2,3-dione; Animals; Baclofen; Bicuculline; Electrophysiology; Epilepsy; gamma-Aminobutyric Acid; Glutamates; Glutamic Acid; Guinea Pigs; Gyrus Cinguli; Magnesium; Neurons; Quinoxalines; Synapses

The selective excitatory amino acid receptor antagonists, 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) and 2-amino-5-phosphonovalerate (D-APV), have been used to investigate the identity of the receptors involved in evoked epileptiform bursting activity in the chronic kainic acid lesioned hippocampus. Comparisons have been made with the acute bursting activity induced by bicuculline. Presented data suggest there are two possible mechanisms contributing to epileptiform bursting activity in the kainic acid lesioned hippocampus. One of these is probably a product of disinhibition, and generates a predominantly non-NMDA receptor mediated burst which is blocked by CNQX (2 microM). The second synaptic mechanism involves a major (or total) contribution by NMDA receptors to the epileptiform burst, and is blocked by D-APV (10 microM).

Topics: 2-Amino-5-phosphonovalerate; 6-Cyano-7-nitroquinoxaline-2,3-dione; Animals; Bicuculline; Chronic Disease; Electric Stimulation; Electrophysiology; Epilepsy; Hippocampus; In Vitro Techniques; Kainic Acid; Male; Quinoxalines; Rats; Rats, Inbred Strains; Receptors, N-Methyl-D-Aspartate

Conventional intracellular recording techniques were used to investigate the N-methyl-D-aspartate (NMDA) and non-NMDA mediated synaptic mechanisms underlying the stimulus-induced paroxysmal depolarization shift (PDS) generated by cells in rat neocortical slices treated with bicuculline methiodide (BMI). The NMDA receptor antagonists CPP or MK-801 were ineffective in abolishing the PDS. However, both drugs were able to attenuate the late phase of the PDS and delay its time of onset. In contrast, the non-NMDA receptor blocker CNQX demonstrated potent anticonvulsant property by reducing the PDS into a depolarizing potential that was graded in nature. This CNQX-resistant depolarizing potential was readily blocked by CPP. Voltage-response analysis of the PDS indicated that the entire response (including its NMDA-mediated phase) displayed conventional voltage characteristics reminiscent of an excitatory postsynaptic potential that is mediated by non-NMDA receptors. We conclude that the activation of non-NMDA receptors is necessary and sufficient to induce epileptiform activity in the neocortex when the GABAergic inhibitory mechanism is compromised. The NMDA receptors contribute to the process of PDS amplification by prolonging the duration and reducing the latency of each epileptiform discharge. However, the participation of NMDA receptors is not essential for BMI-induced epileptogenesis, and their partial involvement in the PDS is dependent upon the integrity of the non-NMDA mediated input. The lack of NMDA-like voltage dependency observed in the PDS's late phase might reflect an uneven distribution of NMDA receptors along the cell and/or an association of this excitatory amino acid receptor subtype in the polysynaptic pathways within the neocortex.

Topics: 6-Cyano-7-nitroquinoxaline-2,3-dione; Amino Acids; Animals; Bicuculline; Cerebral Cortex; Dizocilpine Maleate; Electric Stimulation; Epilepsy; In Vitro Techniques; Male; Membrane Potentials; N-Methylaspartate; Neurons; Piperazines; Quinoxalines; Rats; Rats, Inbred Strains; Receptors, Amino Acid; Receptors, Cell Surface; Receptors, N-Methyl-D-Aspartate

Extracellular field potentials and [K+]o were recorded in slices of human epileptogenic neocortex maintained in vitro during perfusion with Mg(2+)-free artificial cerebrospinal fluid (ACSF). The human neocortex was obtained during neurosurgical procedures for the relief of seizures that were resistant to medical treatment. Spontaneous epileptiform activity and episodes of spreading depression appeared within 1.5 to 2 hours of perfusion with Mg(2+)-free ACSF. The epileptiform discharges consisted of negative field potential shifts (amplitude, 0.8-10 mV) that lasted 2.5 to 80 seconds and recurred at intervals ranging between 4 and 160 seconds. Both duration and frequency of occurrence of epileptiform events were not significantly different when measured in slices obtained from spiking tissue compared with those gathered from nonspiking neocortical areas. Transient increases in [K+]o of up to 10.5 mM were associated with each epileptiform discharge; these changes were maximal and fastest in the middle neocortical layers. Spreading depression episodes were characterized by 20 to 30-mV negative shifts that lasted up to 200 seconds and were accompanied by increases in [K+]o of approximately 100 mM. Epileptiform discharges and spreading depressions did not occur during perfusion with Mg(2+)-free ACSF that contained either competitive or noncompetitive antagonists of the N-methyl-D-aspartate (NMDA) receptor subtype. In contrast, pharmacological blockade of non-NMDA receptors did not influence the epileptiform activity observed in Mg(2+)-free ACSF. These findings demonstrate that decreasing [Mg2+]o leads to the appearance of both spontaneous epileptiform discharges and spreading depression in the human epileptogenic neocortex.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: 2-Amino-5-phosphonovalerate; 6-Cyano-7-nitroquinoxaline-2,3-dione; Action Potentials; Cerebral Cortex; Electric Stimulation; Epilepsy; Humans; In Vitro Techniques; Magnesium; Microelectrodes; Quinoxalines; Receptors, N-Methyl-D-Aspartate

1. Neocortical brain slices were prepared from animals 8-15 days of age and maintained in vitro. Intracellular recordings were obtained from neurons in cortical layers 2-3. The role of synaptic activity and excitatory amino acid receptors in generation of picrotoxin-induced ictal-like epileptiform activity in the immature neocortex was investigated. D-2-amino-5-phosphonovaleric acid (D-APV) and 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) were used as selective antagonists of N-methyl-D-aspartate (NMDA) and non-NMDA receptors, respectively. 2. Ictal-like epileptiform discharges were induced by bath application of the GABAA-receptor antagonist picrotoxin. Paroxysmal discharges, 7-25 s in duration, occurred spontaneously or could be evoked by electrical stimulation. These events consisted of an initial paroxysmal depolarizing shift (PDS) followed by a long-duration depolarization (LLD) with superimposed late PDSs. 3. The amplitudes of the initial PDS, LLD, and late PDSs were linearly dependent on membrane potential, increasing with hyperpolarization and diminishing on depolarization. All responses reversed polarity near 0 mV. Under voltage-clamp conditions, both transient and sustained currents were observed, coincident with PDSs and the LLD, respectively. The duration of the ictal-like events was similar under current- and voltage-clamp conditions, suggesting activation of intrinsic membrane currents did not significantly prolong epileptiform discharges. 4. Bath application of D-APV (20 microM) decreased the amplitude and duration of both the initial PDS and LLD without affecting the time-to-onset of epileptiform activity.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: 2-Amino-5-phosphonovalerate; 6-Cyano-7-nitroquinoxaline-2,3-dione; Aging; Animals; Cerebral Cortex; Epilepsy; Evoked Potentials; In Vitro Techniques; Membrane Potentials; Neurons; Picrotoxin; Quinoxalines; Rats; Receptors, N-Methyl-D-Aspartate; Synapses; Time Factors

The relationship between epileptiform events in the medial entorhinal cortex (MEC) and the dentate gyrus was investigated using a slice preparation from rat brain. Simultaneous intracellular recordings were made from neurones in layer II of the MEC and neurones in the granule cell layer of the dentate gyrus (DGC). Epileptiform activity was induced by perfusion with Mg+(+)-free medium or GABAA-receptor blockers. Epileptiform discharges in MEC cells were reflected on a one-to-one basis and at a latency of 1-3 ms by depolarizing events in DGC. The latter rarely gave rise to action potentials. Bath perfusion of the N-methyl-D-aspartate (NMDA) receptor blocker, 2-aminophosphonovalerate (2-AP5) abolished the Mg+(+)-free induced events in MEC cells and the corresponding depolarizations in the DGC but local application of 2-AP5 to the dentate gyrus only reduced the depolarizations. The non-NMDA-receptor antagonist 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX), whether bath applied or applied locally to the DG, had little effect on the cortical events but strongly reduced the depolarizations of the DGC. The discharges induced in MEC cells by GABA-blockers were reduced by bath applied 2-AP5 but abolished by CNQX. These effects were mirrored in the dentate gyrus by a reduction in the depolarizing events by 2-AP5 and their abolition by CNQX. Local application of either antagonist to the dentate gyrus reduced but did not abolish the depolarizations. Thus, Mg+(+)-free induced events in MEC depend mainly on enhanced NMDA-receptor activity, while events induced by bicuculline are primarily dependant on non-NMDA receptors. The depolarizing events in the DGC which reflect the activity in the EC are mediated by both types of receptor, although non-NMDA receptors play a much greater role.

Topics: 2-Amino-5-phosphonovalerate; 6-Cyano-7-nitroquinoxaline-2,3-dione; Action Potentials; Animals; Bicuculline; Cerebral Cortex; Epilepsy; Hippocampus; In Vitro Techniques; Magnesium; Male; Quinoxalines; Rats; Rats, Inbred Strains; Receptors, Amino Acid; Receptors, Cell Surface; Receptors, N-Methyl-D-Aspartate; Receptors, Neurotransmitter

The effects of excitatory amino acid receptor antagonists kynuretic acid and 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) on epileptiform activity induced by 4-aminopyridine (4-AP) were studied in rat amygdala slices using intracellular recording techniques. Five to 10 min after switching to 4-AP-containing solution, spontaneous epileptiform bursts were observed in 35 out of 45 slices studied. The spontaneous epileptiform events consisted of an initial burst followed by a number of afterdischarges. Superfusion with kynuretic acid, a broad-spectrum excitatory amino acid receptor antagonist, reversibly reduced the duration of the spontaneous bursting discharges in a dose-dependent manner. The frequency of spontaneous bursting was also decreased. The IC50, estimated from the graph of the concentration-response relationship, was approximately 130 microM. In addition, CNQX which is a specific non-N-methyl-D-aspartate (NMDA) receptor antagonist blocked the spontaneous and evoked epileptiform bursting. In 11 out of 15 neurons tested, there was a residual depolarizing component remained. This depolarizing component was reversibly blocked by specific NMDA receptor antagonist, D,L-2-amino-5-phosphonovaleate (D,L-APV). Relative to the CNQX-sensitive component, the D,L-APV-sensitive component is much smaller in amplitude and shorter in duration indicating that NMDA receptor plays only a minor role in this process. These data suggest that the generation or propagation of spontaneous epileptiform events induced by 4-AP in the neurons of basolateral amygdala is mediated by excitatory amino acids and that activation of non-NMDA receptors is of primary importance.

Topics: 4-Aminopyridine; 6-Cyano-7-nitroquinoxaline-2,3-dione; Amygdala; Animals; Electrophysiology; Epilepsy; In Vitro Techniques; Male; Membrane Potentials; Neurons; Quinoxalines; Rats; Rats, Inbred Strains; Receptors, N-Methyl-D-Aspartate

The involvement of the N-methyl-D-aspartate (NMDA) receptor in the epileptiform activity induced by 4-aminopyridine (4-AP) was studied in rat amygdala slices using intracellular recording techniques. Stimulation of the ventral endopyriform nucleus evoked an excitatory postsynaptic potential (EPSP). After exposure to 4-AP (200 microM) the amygdala slices usually exhibited spontaneous and evoked epileptiform activity. The epileptiform events had an average duration of 522 +/- 78 ms with a frequency of 0.5-8.5 bursts/min. Superfusion of 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX), a selective non-NMDA receptor antagonist, practically abolished the epileptiform bursting. However, there remained a residual depolarizing component in 13 out of 18 neurons. This CNQX-resistant component was markedly enhanced both in amplitude and duration when extracellular Mg2+ was removed and could be reversibly blocked by the specific NMDA receptor antagonist, DL-2-amino-5-phosphonovaleate (DL-APV). Compared with the CNQX-sensitive component, the APV-sensitive component had a much smaller amplitude shorter duration. These data suggest that the NMDA receptor is likely to play only a minor role, and activation of the NMDA receptor may contribute to but is not required, for the generation of these bursts.

Topics: 2-Amino-5-phosphonovalerate; 4-Aminopyridine; 6-Cyano-7-nitroquinoxaline-2,3-dione; Amygdala; Animals; Calcium; Electric Stimulation; Epilepsy; Evoked Potentials; In Vitro Techniques; Male; Membrane Potentials; Microelectrodes; Neurons; Quinoxalines; Rats; Rats, Inbred Strains; Receptors, N-Methyl-D-Aspartate

The effect of 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX), a specific non-N-methyl-D-aspartate (non-NMDA) receptor antagonist, on NMDA-independent epileptiform activity induced by Mg2(+)-free medium was studied in rat basolateral amygdala (BLA) neurons using intracellular recording techniques. Twenty to 30 min after switching to Mg2(+)-free medium, spontaneous and evoked epileptiform activity were observed in 16 out of 18 amygdala slices. Superfusion of D-2-amino-5-phosphonovalerate (D-APV), a selective NMDA receptor antagonist, reduced the duration of epileptiform activity by an average of 83%. However, there was a residual depolarizing component which remained in the presence of D-APV. This D-APV-resistant component could be completely blocked by CNQX suggesting that it is mediated by non-NMDA receptors.

Topics: 2-Amino-5-phosphonovalerate; 6-Cyano-7-nitroquinoxaline-2,3-dione; Amygdala; Animals; Culture Media; Electric Stimulation; Epilepsy; In Vitro Techniques; Magnesium Deficiency; Male; Neurons; Osmolar Concentration; Quinoxalines; Rats; Rats, Inbred Strains; Receptors, N-Methyl-D-Aspartate

The ability of the non-NMDA receptor antagonist 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) to suppress picrotoxin-induced epileptiform burst activity was examined. Intracellular recordings were obtained from hippocampal CA1 and CA3 pyramidal neurons maintained in vitro. Bath application of CNQX (5 microM) significantly reduced or abolished evoked paroxysmal depolarizing shifts (PDSs) in all CA1 and CA3 neurons tested. In cells where a CNQX-insensitive component in the PDS was manifest, this remaining activity was abolished by the N-methyl-D-aspartate (NMDA) receptor antagonist D-2-amino-5-phosphonovaleric acid (20 microM), suggesting the existence of a NMDA-mediated synaptic potential. Our results indicate that non-NMDA receptor antagonists are capable of markedly reducing picrotoxin-induced epileptiform activity and that these receptors play an important role in generation of PDSs.

Topics: 6-Cyano-7-nitroquinoxaline-2,3-dione; Animals; Anticonvulsants; Epilepsy; Hippocampus; In Vitro Techniques; Picrotoxin; Quinoxalines; Rats; Rats, Inbred Strains; Receptors, Amino Acid; Receptors, Cell Surface

1. Two quinozalinediones, FG9041 and FG9065, which had previously been shown to displace binding to the quisqualate receptor, were tested on rat neocortex and frog spinal cord in vitro against depolarizations induced by quisqualate, kainate and N-methyl-D-aspartate (NMDA). In both preparations effects of quisqualate were reduced the most and those of NMDA the least. 2. The near unitary slopes of the Schild plots were consistent with a competitive type of interaction. pA2 values for FG9041 were estimated to be 6.6, 6.1 and 5.1 in frog cord and 5.9, 5.3 and and about 4 in the rat neocortex for quisqualate, kainate and NMDA antagonism, respectively. FG9065 gave equivalent pA2 values of 6.2, 5.6 and 4.5. 3. At concentrations, which were without effect on depolarizations induced by NMDA, FG9041 and FG9065 reduced or blocked synaptically-evoked field potentials in hippocampal and neocortical slices superfused with normal magnesium-containing medium. Since these synaptic components are also insensitive to NMDA antagonists, these results are consistent with their mediation by postsynaptic receptors of the quisqualate (or kainate) type. 4. By contrast, quinoxalinediones had only limited effects on spontaneous epileptiform activity seen in both neocortical and hippocampal preparations when superfused with magnesium-free medium. These burst discharges were, however, abolished by NMDA antagonists. 5. In the frog spinal cord the early component of the dorsal root to ventral root reflexes was selectively reduced by FG9041 whereas NMDA antagonists reduced the longer latency components. 6. Our results suggest that the quinoxalinediones are likely to be useful pharmacological probes for elucidating the role of non-NMDA receptors in the vertebrate central nervous system.

Topics: 6-Cyano-7-nitroquinoxaline-2,3-dione; Animals; Anura; Cerebral Cortex; Electric Stimulation; Epilepsy; Evoked Potentials; Hippocampus; In Vitro Techniques; Kainic Acid; Neuromuscular Depolarizing Agents; Oxadiazoles; Quinoxalines; Quisqualic Acid; Rats; Rats, Inbred Strains; Receptors, Kainic Acid; Receptors, Neurotransmitter; Spinal Cord; Synapses