Comparison of Effects of Valproate and Trans-2-en-Valproate on Different Forms of Epileptiform Activity in Rat Hippocampal and Temporal Cortex Slices

Summary: Purpose: Reducing the extracellular magnesium or calcium or increasing the extracellular potassium induces different patterns of epileptiform activity in the hippocampus and the entorhinal cortex. Although in the low Ca²⁺ and K⁺ models, seizure-like events (SLEs) develop in area CA1 of the hippocampus, only short recurrent discharges develop in the low Mg²⁺ model. In contrast, in low Mg²⁺, SLEs and late recurrent discharges (LRDs) are observed in the entorhinal cortex. Methods: We compared the effects of valproate (VPA) and its major metabolite, trans-2-en-VPA (TVPA), on all these different model activities using extracellular field potential measurements. We also investigated the equilibration time course of VPA in the slice by using VPA-sensitive microelectrodes. Results: Both drugs reversibly blocked most forms of epileptiform activity. The only exception was the LRDs in the entorhinal cortex. In paired experiments, TVPA appeared to be more effective than VPA bath applied with the same concentration to the same slice. With our measurements of the VPA concentrations in slices, we showed that the concentrations used were close to therapeutic drug levels. Conclusions: If TVPA stands the toxicological tests, it might be a useful alternative in the treatment of seizures.     

Effects of standard anticonvulsant drugs on different patterns of epileptiform discharges induced by 4-aminopyridine in combined entorhinal cortex-hippocampal slices

Application of 4-aminopyridine (4-AP) has previously been reported to produce different patterns of epileptiform discharges in entorhinal cortex (EC)-hippocampal slices: recurrent short discharges (RSDs) in hippocampal area CA1, seizure-like events (SLEs) and negative-going potentials (NGPs) in the medial entorhinal cortex (mEC). Using recordings of field potentials, we investigated the pharmacological effects of the clinically employed standard anticonvulsant drugs phenytoin (PHT), carbamazepine (CBZ), valproic acid (VPA) and phenobarbital (PHB) and those of pentobarbital (PB) on 4-AP-induced epileptiform activity. The anticonvulsant drugs showed different effects: SLEs were completely blocked by all tested drugs. Valproic acid, which suppressed all epileptiform activities, seemed to have the most fundamental effect of all drugs on 4-AP induced activity, because under phenytoin and carbamazepine, some epileptiform activity was still observable. The RSDs in hippocampal area CA1 of the hippocampus did not respond to the different anticonvulsants. In contrast, PB decreased the frequency of the RSDs in CA1 and enhanced the frequency of the NGPs in the EC. We propose that the activities induced by 4-AP in the combined entorhinal cortex-hippocampal slices may provide an in vitro model for the development of new drugs against difficult-to-treat focal epilepsy.     

Effects of standard anticonvulsant drugs on different patterns of epileptiform discharges induced by 4-aminopyridine in combined entorhinal cortex–hippocampal slices

Application of 4-aminopyridine (4-AP) has previously been reported to produce different patterns of epileptiform discharges in entorhinal cortex (EC)–hippocampal slices: recurrent short discharges (RSDs) in hippocampal area CA1, seizure-like events (SLEs) and negative-going potentials (NGPs) in the medial entorhinal cortex (mEC). Using recordings of field potentials, we investigated the pharmacological effects of the clinically employed standard anticonvulsant drugs phenytoin (PHT), carbamazepine (CBZ), valproic acid (VPA) and phenobarbital (PHB) and those of pentobarbital (PB) on 4-AP-induced epileptiform activity. The anticonvulsant drugs showed different effects: SLEs were completely blocked by all tested drugs. Valproic acid, which suppressed all epileptiform activities, seemed to have the most fundamental effect of all drugs on 4-AP induced activity, because under phenytoin and carbamazepine, some epileptiform activity was still observable. The RSDs in hippocampal area CA1 of the hippocampus did not respond to the different anticonvulsants. In contrast, PB decreased the frequency of the RSDs in CA1 and enhanced the frequency of the NGPs in the EC. We propose that the activities induced by 4-AP in the combined entorhinal cortex–hippocampal slices may provide an in vitro model for the development of new drugs against difficult-to-treat focal epilepsy.     

Model of frequent, recurrent, and spontaneous seizures in the intact mouse hippocampus

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.     

Reduced ictogenic potential of 4-aminopyridine in the perirhinal and entorhinal cortex of kainate-treated chronic epileptic rats

We investigated the potential of 4-AP (50-100 microM) to induce seizure-like events (SLEs) in combined entorhinal cortex-hippocampal slices from Sprague Dawley rats which developed spontaneous limbic seizures following kainic acid induced status epilepticus. Slices from control rats (n=8) displayed SLEs in the entorhinal and perirhinal cortex upon application of 50 or 100 microM 4-AP. By contrast, 4-AP failed to induce SLEs in slices from chronic epileptic rats (n=13) except for one slice from one rat. This animal displayed only minor cell loss in layer III of the entorhinal cortex, in contrast to the other epileptic rats for which layer III neuronal loss was extensive. In all slices from epileptic rats, 4-AP induced recurrent epileptiform discharges similar to the interictal activity observed in control rats. Combined application of 4-AP (100 microM) and bicuculline methiodide (30 microM) induced frequent and prolonged recurrent epileptiform discharges in both control and chronic epileptic rats. 4-AP at 50-100 microM likely affects potassium channels containing Kv1.4, Kv1.5, Kv3.1 or Kv3.2 subunits. Real-time PCR revealed no significant downregulation of Kv1.4, Kv1.5, Kv3.1 or Kv3.2 in the subiculum, entorhinal and perirhinal cortex from chronic epileptic rats compared to controls. However, the expression of Kv3.4, responding to 4-AP in mM range, was significantly reduced. Using sub-unit-specific antibodies, the real-time PCR findings were confirmed by immunocytochemistry. We suggest that after chronic epilepsy, reorganization in the entorhinal cortex is accompanied by adaptations in homeostatic plasticity with anticonvulsant consequences.     

Effects of retigabine (D-23129) on different patterns of epileptiform activity induced by low magnesium in rat entorhinal cortex hippocampal slices

PURPOSE: The objective of this study was to evaluate the effect of a new antiseizure drug, retigabine (D-23129; N-(2-amino-4-[fluorobenzylamino]-phenyl) carbamic acid ethyl ester) on low-Mg2+-induced epileptiform discharges in rat in vitro. METHODS: Three types of epileptiform discharges (recurrent short discharges in the hippocampus, seizure-like events, and late recurrent discharges in the entorhinal cortex) were elicited in rat combined entorhinal cortex-hippocampal slices by perfusion with low-Mg2+-artificial cerebrospinal fluid (ACSF). The antiepileptic properties of retigabine were evaluated as effect on the frequency and amplitude of the epileptiform activities as well as time of onset of the effect in the entorhinal cortex (EC) and in hippocampal area CA1 (CA1) by using extracellular recording techniques. RESULTS: Retigabine (20 microM) reversibly suppressed the recurrent short discharges otherwise sensitive only to high doses of valproate (VPA) but insensitive to standard antiepileptic drugs (AEDs) in CA1, whereas 10 microM reduced the frequency of discharges by 34+/-18.8%, with no significant effect on the amplitude. In EC, retigabine (50 microM) reversibly suppressed the seizure-like events, whereas 20 microM blocked seizure-like events in 71.5% of the slices. The seizure-like events were also sensitive to standard AEDs. Late recurrent discharges in EC that are not blocked by standard AEDs were reversibly suppressed by retigabine (100 microM), whereas 50 microM reduced the frequency of the discharges by 94.4+/-7.7%, and 20 microM, by 74.2+/-18.0%, with no significant effect on the amplitude. CONCLUSIONS: Retigabine is an effective AED with suppressive effects on recurrent short discharges and on late recurrent discharges normally insensitive to standard AEDs.     

Excitatory and inhibitory control of epileptiform discharges in combined hippocampal/entorhinal cortical slices

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 μM) were enough to induce epileptiform discharges in the three regions. These discharges were similar to those observed under high concentrations of bicuculline (>10 μM) 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.     

Effects of taurine and glycine on epileptiform activity induced by removal of Mg2+ in combined rat entorhinal cortex-hippocampal slices

PURPOSE: The imbalance between neuronal inhibition and excitation contributes to epileptogenesis. Inhibition in the central nervous system (CNS) is mediated by gamma-aminobutyric acid (GABA) and glycine. Recent studies indicate the expression of glycine receptor (GlyR) in hippocampus and neocortex. However, the function of GlyR in these regions is not clarified completely. The aim of this study was to investigate whether the GlyR agonists glycine and taurine promote an anticonvulsive effect. METHODS: We induced epileptiform discharges by reducing extracellular Mg2+ concentration in combined rat entorhinal cortex-hippocampal slices (400 micro m). Epileptiform discharges were detected by using extracellular recording techniques. RESULTS: Seizure-like events were suppressed by taurine, exhibiting a half-maximal inhibitory effect (IC50) of 0.9 mM. Suppression of late recurrent discharges in the medial entorhinal cortex and recurrent short discharges in the hippocampus was obtained at an IC50 value of 1.6 and 2.1 mM, respectively. Strychnine at concentrations <1 micro M abolished these effects. Likewise glycine, after an initial proconvulsant effect, suppressed epileptiform discharges. CONCLUSIONS: These findings show that GlyR agonists, in particular taurine, could serve as potential anticonvulsants and suggest an important role of GlyR in cortical function and dysfunction.     

Electrical and chemical long-term depression do not attenuate low-Mg2+-induced epileptiform activity in the entorhinal cortex

PURPOSE: Low-frequency electrical and magnetic stimulation of cortical brain regions has been shown to reduce cortical excitability and to decrease the susceptibility to seizures in humans and in vivo models of epilepsy. The induction of long-term depression (LTD) or depotentiation of a seizure-related long-term potentiation has been proposed to be part of the underlying mechanism. With the low-Mg(2+)-model of epilepsy, this study investigated the effect of electrical LTD, chemical LTD, and depotentiation on the susceptibility of the entorhinal cortex to epileptiform activity. METHODS: The experiments were performed on isolated entorhinal cortex slices obtained from adult Wistar rats and mice. With extracellular recording techniques, we studied whether LTD induced by (a) three episodes of low-frequency paired-pulse stimulation (3 x 900 paired pulses at 1 Hz), and by (b) bath-applied N-methyl-D-aspartate (NMDA, 20 microM) changes time-to-onset, duration, and frequency of seizure-like events (SLEs) induced by omitting MgSO(4) from the artificial cerebrospinal fluid. Next we investigated the consequences of depotentiation on SLEs themselves by applying low-frequency stimulation after onset of low-Mg(2+)-induced epileptiform activity. RESULTS: LTD, induced either by low-frequency stimulation or by bath-applied NMDA, had no effect on time-to-onset, duration, and frequency of SLEs compared with unconditioned slices. Low-frequency stimulation after onset of SLEs did not suppress but induced SLEs that lasted for the time of stimulation and were associated with a simultaneous increase of the extracellular K(+) concentration. CONCLUSIONS: Our study demonstrates that neither conditioning LTD nor brief low-frequency stimulation decreases the susceptibility of the entorhinal cortex to low-Mg(2+)-induced epileptiform activity. The present study does not support the hypothesis that low-frequency brain stimulation exerts its anticonvulsant effect via the induction of LTD or depotentiation.     

Epileptiform propagation patterns mediated by NMDA and non-NMDA receptors in rat neocortex

PURPOSE: 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. METHODS: 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. RESULTS: 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. CONCLUSIONS: 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.     

The epileptiform activity induced by 4-aminopyridine in rat amygdala slices: antagonism by non-N-methyl-D-aspartate receptor antagonists

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.     

Epileptiform activity induced by 4-aminopyridine in entorhinal cortex hippocampal slices of rats with a genetically determined absence epilepsy (GAERS)

Patients with absence epilepsy frequently develop convulsions later in life. We were therefore interested whether tissue from rats with a genetic absence epilepsy is more prone to seizure generation than normal animals. We compared the epileptiform activities induced by 4-aminopyridine (4-AP) induced in hippocampal–entorhinal cortex slices from genetic absence epilepsy rats of Strasbourg (GAERS, age 6 months) in which absence seizures have been present for about 4 months and from control non epileptic rats (NE). 4-AP induced short recurrent discharges in area CA1 of rat hippocampus, seizure-like events and interictal discharges in the entorhinal cortex. The various epileptiform discharges did not differ between the two strains in amplitude, duration and frequency. However, the latency for induction of different epileptiform activities by 50 μM 4-AP was significantly shorter in GAERS (about 16 min) than in NE rats (about 25 min). We also analysed differences in evoked field potentials (fp) in hippocampal area CA1 before, during and after application of 4-AP. Before application of 4-AP, responses to stimulation of Schaffer collateral were smaller in GAERS than in NE rats. Paired pulse potentiation was significantly larger in GAERS than in NE rats. 4-AP in the bath augmented the size of the evoked field potentials and this increase was larger in GAERS than in NE rats. Our findings show a greater excitability of hippocampal area CA1 in GAERS rats and a greater ability to develop 4-AP-induced epileptiform activity in combined hippocampal–enthorhinal cortex slices in GAERS than in NE rats.     

The epileptiform activity induced by 4-aminopyridine in rat amygdala slices: antagonism by non-N-methyl-d-aspartate receptor anatagonists

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 μM. In addition, CNQX which is a specificnon-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, thed,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 of 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.     

Early developmental alterations of low-Mg2+ -induced epileptiform activity in the intact corticohippocampal formation of the newborn mouse in vitro

The generation, propagation and pharmacological properties of low-Mg2+ -induced epileptiform activity were examined in the intact corticohippocampal formation (CHF) of the newborn (P0-4) mouse in vitro. Multi-site field potential recordings in dentate gyrus (DG), CA3, CA1, entorhinal cortex (EC) and temporal cortex (TC) revealed in 0.2 mM Mg2+ -containing ACSF a stable pattern of spontaneous epileptiform activity consisting of recurrent ictal-like events (ILEs) and interictal events (IEs). Although this activity could be consistently observed as early as P0, ILEs were smaller in amplitude, less frequent and showed a slower onset in P0-2 as compared to P3-4 animals. In all age groups, epileptiform events were largest in CA3 and smallest in EC and TC. A specific pacemaker region could not be identified since ILEs appeared simultaneously at all recording sites. Reducing the extracellular Mg2+ concentration to 0.1 mM or nominally zero caused an increase in ILE frequency. Pharmacological studies in the P3-4 age group with 0.2 mM Mg2+ revealed a complete blockade of the ILEs by an NMDA receptor antagonist and a pronounced suppression of epileptiform activity by an AMPA/kainate antagonist. Application of a GABA-A receptor antagonist induced repetitive bursts of interictal discharges, which persisted for at least 1.5 h after washout of the antagonist. Our data demonstrate that the intact CHF in vitro preparation of the newborn mouse offers a most valuable model to study epileptiform activity in the immature limbic system.     

Propagation dynamics of epileptiform activity acutely induced by bicuculline in the hippocampal-parahippocampal region of the isolated Guinea pig brain

PURPOSE: Aim of the study is to investigate the involvement of parahippocampal subregions in the generation and in the propagation of focal epileptiform discharges in an acute model of seizure generation in the temporal lobe induced by arterial application of bicuculline in the in vitro isolated guinea pig brain preparation. METHODS: Electrophysiological recordings were simultaneously performed with single electrodes and multichannel silicon probes in the entorhinal, perirhinal, and piriform cortices and in the area CA1 of the hippocampus of the in vitro isolated guinea pig brain. Interictal and ictal epileptiform discharges restricted to the temporal region were induced by a brief (3-5 min) arterial perfusion of the GABA(A) receptor antagonist, bicuculline methiodide (50 microM). Current source density analysis of laminar field profiles performed with the silicon probes was carried out at different sites to establish network interactions responsible for the generation of epileptiform potentials. Nonlinear regression analysis was conducted on extracellular recordings during ictal onset in order to quantify the degree of interaction between fast activities generated at different sites, as well as time delays. RESULTS: Experiments were performed in 31 isolated guinea pig brains. Bicuculline-induced interictal and ictal epileptiform activities that showed variability of spatial propagation and time course in the olfactory-temporal region. The most commonly observed pattern (n = 23) was characterized by the initial appearance of interictal spikes (ISs) in the piriform cortex (PC), which propagated to the lateral entorhinal region. Independent and asynchronous preictal spikes originated in the entorhinal cortex (EC)/hippocampus and progressed into ictal fast discharges (around 25 Hz) restricted to the entorhinal/hippocampal region. The local generation of fast activity was verified and confirmed both by CSD and phase shift analysis performed on laminar profiles. Fast activity was followed by synchronous afterdischarges that propagated to the perirhinal cortex (PRC) (but not to the PC). Within 1-9 min, the ictal discharge ceased and a postictal period of depression occurred, after which periodic ISs in the PC resumed. Unlike preictal ISs, postictal ISs propagated to the PRC. CONCLUSIONS: Several studies proposed that reciprocal connections between the entorhinal and the PRC are under a very efficient inhibitory control (1). We report that ISs determined by acute bicuculline treatment in the isolated guinea pig brain progress from the PC to the hippocampus/EC just before ictal onset. Ictal discharges are characterized by a peculiar pattern of fast activity that originates from the entorhinal/hippocampal region and only secondarily propagates to the PRC. Postictal propagation of ISs to the PRC occurred exclusively when an ictal discharge was generated in the hippocampal/entorhinal region. The results suggest that reiteration of ictal events may promote changes in propagation pattern of epileptiform discharges that could act as trigger elements in the development of temporal lobe epilepsy.     

Phenytoin, phenobarbital, and midazolam fail to stop status epilepticus-like activity induced by low magnesium in rat entorhinal slices, but can prevent its development

Objectives – It was shown previously that low-Mg²⁺-induced epileptiform activity in rat entorhinal cortex slices changes with time from a pattern of serial seizure-like events (SLEs) to a state of continuously recurring epileptiform activity. Valproic acid blocked the early SLEs but not the late activity. It was proposed that the late activity is a model for pharmacoresistant status epilepticus since it was also refractory to phenytoin, carbamazepine, phenobarbital, and midazolam. In the present study, it is demonstrated that phenytoin (50 μM, n =6), phenobarbital (150 μM, n =7), and midazolam (50 μM, n =5) were able to block the early SLEs but not the late activity at the same concentrations. Carbamazepine (50 μM) reduced the duration of the SLEs from 21±5 s to 4±3 s ( P <0.01), the interictal interval from 123±27 s to 27±19 s ( P <0.01), the SLE-associated rise of [K⁺]₀ from 7.7±0.5 mM to 5.7±0.8 mM ( n =4, P <0.05), and the spread of the SLE between entorhinal cortex and neocortex from 4.0±0.6 s to 0.8±0.1 s ( n =4, P <0.05). Lower concentrations of phenytoin (5 and 10 μM, n =5), carbamazepine (10 μM, n =6), and phenobarbital (50 μM, n =4) had no effect. In conclusion, the hypothesis is supported that low-Mg²⁺-induced epileptiform activity in rat entorhinal cortex is an in vitro model for the transition from pharmacosensitive to pharmacoresistant status epilepticus.     

Interictal-ictal interactions and limbic seizure generation.

Interictal discharges are used in clinical practice to localize the epileptogenic focus in patients with partial epilepsy. However, the interaction between interictal and ictal discharges remains debatable. For instance, interictal events may lead to seizure onset in some models of epileptiform discharge. By contrast, in other models, disappearance of interictal activity (for example by activation of GABAB receptors) induces or potentiates ictal events. We have recently obtained new evidence for a control exerted by interictal discharges on ictal activity in rodent combined slices of hippocampus-entorhinal cortex. In this preparation continuous application of 4-aminopyridine induces: (i) interictal activity which initiates in CA3 and propagates via CA1 and subiculum to the entorhinal cortex, and return to the hippocampus through the dentate gyrus; and (ii) ictal discharges, which originate in the entorhinal cortex and propagate via the dentate gyrus to the hippocampus. Ictal discharges disappear over time, while synchronous interictal discharges continue to occur. Lesioning the Schaffer collaterals abolishes interictal discharges in CA1, entorhinal cortex and dentate gyrus and discloses entorhinal ictal discharges that propagate, via the dentate gyrus, to the CA3 subfield. Interictal activity of CA3 origin also prevents the occurrence of ictal events recorded in the entorhinal cortex in Mg(2+)-free medium. Moreover, in both models, ictal discharge generation in the entorhinal cortex after Schaffer collateral cut is prevented by mimicking CA3 activity through rhythmic electrical stimulation of CA1 hippocampal outputs. Hence, our data demonstrate that hippocampus interictal discharges control the expression of electrographic seizures in entorhinal cortex. Sectioning the Schaffer collaterals may model the epileptic condition in which cell damage in the CA3 subfield results in loss of CA3 control over the entorhinal cortex. Hence, the functional integrity of hippocampal CA3 neurons may represent a critical control point in temporal lobe epilepsy.     

Differential effects of NMDA and AMPA/kainate receptor antagonists on superoxide production and MnSOD activity in rat brain following intrahippocampal injection

The involvement of NMDA and AMPA/kainate receptors in the induction of superoxide radical production in the rat brain was examined after injection of kainate, non-NMDA receptor agonist, kainate plus 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX), selective AMPA/kainate receptor antagonist, or kainate plus 2-amino-5-phosphonopentanoic acid (APV), selective NMDA receptor antagonist. Competitive glutamate receptor antagonists were injected with kainate unilaterally into the CA3 region of the rat hippocampus. We investigated superoxide production and mitochondrial MnSOD activity after injection. The measurements took place at different times (5, 15 min, 2, 48 h and 7 days) in the ipsi- and contralateral hippocampus, forebrain cortex, striatum, and cerebellum homogenates. Used glutamate antagonists APV and CNQX both expressed sufficient neuroprotection in sense of decreasing superoxide production and increasing MnSOD levels, but with differential effect in mechanisms and time dynamics. Our findings suggest that NMDA and AMPA/kainate receptors are differentially involved in superoxide production. Following intrahippocampal antagonists injection they, also, interpose different neuroprotection effect on the induction of MnSOD activity in distinct brain regions affected by the injury, which are functionally connected via afferents and efferents. It suggests that MnSOD protects the cells in these regions from superoxide-induced damage and therefore may limit the retrograde and anterograde spread of neurotoxicity.     

Entorhinal cortex entrains epileptiform activity in CA1 in pilocarpine-treated rats

Layer III neurons of the medial entorhinal cortex (mEC) project to CA1 via the temporoammonic pathway and exert a powerful feed-forward inhibition of CA1 pyramidal neurons. The present study evaluates the hypothesis that disrupted inhibition of CA1 pyramidal neurons causes an eased propagation of entorhinal seizures to the hippocampus via the temporoammonic pathway. Using a method to induce a confined epileptic focus in brain slices, we investigated the spread of epileptiform activity from the disinhibited mEC to CA1 in control and pilocarpine-treated rats that had displayed status epilepticus and spontaneous recurrent seizures. In pilocarpine-treated rats, the mEC showed a moderate layer III cell loss and an enhanced susceptibility to epileptiform discharges compared to control animals. Entorhinal discharges propagated to CA1 in pilocarpine-treated rats but not in controls. Disconnecting CA3 from CA1 did not affect the spread of epileptiform activity to CA1 excluding its propagation via the trisynaptic hippocampal loop. Mimicking the invasion of epileptiform discharges by repetitive stimulation of the temporoammonic pathway caused a facilitation of field potentials in CA1 that were contaminated by population spikes and afterdischarges in pilocarpine-treated but not control rats. Single cell recordings of CA1 pyramidal neurons revealed a dramatic loss of feed-forward inhibition and the occurrence of strong postsynaptic excitatory potentials in pilocarpine-treated rats. Excitatory responses in CA1 were characterized by multiple NMDA receptor-mediated afterdischarges and a strong paired-pulse facilitation in response to activation of the temporoammonic pathway. Our results suggest that, irrespective of the enhanced seizure-susceptibility of the mEC in epileptic rats, the loss of feed-forward inhibition and the enhanced NMDA receptor-mediated excitability CA1 pyramidal cells ease the spread of epileptiform activity from the mEC to CA1 via the temporoammonic pathway bypassing the classical trisynaptic hippocampal loop.     

Search for the Structures Initiating Seizures Triggered by Intraventricular Injection of the μ opioid Agonist Dermorphin in Rats

Summary: Free-moving rats received intraventricular (i.c.v.) or intravenous (i.v.) injections of the μ opioid agonist dermorphin (DRM). The EEG activity of the cortex and of several structures near the injected lateral ventricle was recorded. The intravenous injections of DRM did not induce epileptiform activity. The intracerebroventricular injections of DRM triggered several types of electrical seizures and interictal spikes. With the aim of determining which structure gave rise to the epileptiform discharges, we compared the time relationships of epileptiform phenomena occurring in different structures. Epileptiform discharges, at once generalized, appeared first in the CA3 area of the ventral hippocampus, with involvement of the CA1 area of ventral hippocampus, the entorhinal cortex and the amygdala following immediately. We conclude that, after intracerebroventricular in-jection of a p opiate agonist, epileptiform activity originates in the CA3 area of the ventral hippocampus.