March 2006
Lack of Interactions between Breast Cancer Resistance Protein (BCRP/ABCG2) and Selected Antiepileptic Agents
Lukas Cerveny, Petr Pavek, Jana Malakova, Frantisek Staud, and Zdenek Fendrich

A protein that normally plays a role in resistance to breast cancer (BCRP, ABCG2) has been found in blood vessels in the brain. Expression of this protein is especially strong in certain brain tumors.

Therefore, we hypothesized that the breast cancer protein could critically influence the bioavailability of drugs in these tumors and potentially contribute to failure of antiepileptic treatment. The aim of the present paper was to test whether some commonly used antiepileptic drugs (AEDs) are taken up by human BCRP. In particular, we focused on phenobarbital, phenytoin, ethosuximide, primidone, valproate, carbamazepine, clonazepam and lamotrigine. No obvious interactions of tested AEDs with BCRP transporter were observed. Therefore, these drugs in relevant therapeutic concentrations are neither substrates nor inhibitors of BCRP. Based on our in vitro data we can conclude that resistance to treatment with the tested antiepileptic agents is not probably caused by the overexpression of BCRP in epileptogenic brain tumors. Furthermore, tested AEDs in relevant therapeutic concentrations should not affect transport of co-administred drugs, which are BCRP substrates, across physiological barriers. Epilepsia 2006;47(3).

Levetiracetam: Antiepileptic Properties and Protective Effects on Mitochondrial Dysfunction in Experimental Status Epilepticus

Julie E. Gibbs, Matthew C. Walker, and Hannah R. Cock

Status epilepticus (SE) is a medical emergency, defined as seizure activity lasting at least 30 minutes. SE is associated with neuronal damage, particularly in the hippocampus. This damage is, in part, related to specific defects in energy metabolism that are known to occur after SE (mitochondrial dysfunction, since mitochondria are the “energy factories” of cells). Consequently, drugs that reduce these defects may reduce seizure-associated brain damage. This study assesses the anticonvulsant activity of the anti-epileptic drug, levetiracetam (LEV), in an animal model of SE and measures the consequence of LEV treatment on the mitochondrial dysfunction which occurs after SE. Rats were electrically stimulated, using electrodes implanted into the brain, to induce SE lasting 5 hours, with simultaneous brain wave (EEG) and behavioral monitoring. Stimulated rats were assigned to a treatment group, receiving injections of saline or LEV (200 mg/kg or 1000 mg/kg) during SE and at 3 time points over the next 44 hours. Animals were then sacrificed and the brain removed, and levels of key biochemical markers of mitochondrial dysfunction were measured in the hippocampus, and compared between treatment groups and sham-operated rats. LEV administration did not terminate seizures, though rats that received 1000 mg/kg LEV had less severe seizures. Typical defects in mitochondrial function were seen in saline treated stimulated rats compared to control untreated animals. However, rats that received 1000 mg/kg LEV had significantly improved biochemical parameters, often comparable to normal control levels. Despite continuing seizures, administration of LEV (1000 mg/kg) protects against mitochondrial dysfunction, indicating that LEV may have neuroprotective effects. Epilepsia 2006;47(3).

Status Epilepticus in 12-day-old Rats Leads to Temporal Lobe Neurodegeneration and Volume Reduction: A Histologic and MRI Study

Jaak Nairismägi, Asla Pitkänen, Mikko I. Kettunen, Risto A. Kauppinen, and Hana Kubova

Status epilepticus (SE) is defined as epileptic activity lasting for more than 20 (or sometimes 30) minutes. It is a neurologic emergency, with higher incidence in infancy and childhood than in any other period of life. It remains controversial, however, whether SE causes injury to the developing brain. Causality between seizure activity and structural damage is difficult to investigate in clinical studies because of associated illnesses and treatments. The present study was designed to show whether experimentally induced SE in early infancy in originally healthy brain leads to temporal lobe neurodegeneration and volume reduction. SE was induced pharmacologically in 12-day-old rat pups, and damaged neurons were detected eight hours after the insult using specific histologic markers for neurodegeneration: Fluoro-Jade-B and silver staining. Chronic changes of volume of temporal structures were assessed with magnetic resonance (MRI) three months after SE and confirmed morphologically after the end of MRI study. There were three major findings in the present study. First, MRI revealed volume decline in several regions of temporal lobe (hippocampus, amygdala, and perirhinal cortex) in adult rats with SE early in postnatal development. Second, volume loss was apparent only in less than one third of animals. Third, volume loss detected in MRI appeared in the same regions as the degenerating neurons in Fluoro-Jade B and silver stained preparations eight hours after SE. The present data demonstrate that SE induces irreversible neuronal damage even in very immature rats. However, only a subpopulation of animals exhibit permanent volume loss of studied brain regions. Epilepsia 2006;47(3).

Beta and Gamma Range EEG Power Spectrum Correlation with Spiking Discharges in DBA/2J Mice Absence Model: Role of GABA B Receptors

Francesco Marrosu, Federico Santoni, Mauro Fà, Monica Puligheddu, Luigi Barberini, Fabrizio Genugu, Roberto Frau, Mario Manunta, and Giampaolo Mereu

The GABA-B receptor is a brain cell receptor for the inhibitory transmitter, GABA, although it is secondary in importance to the main GABA-A receptor. Several studies demonstrate the involvement of GABA-B mechanism in models of absence (formerly called petit mal) seizures. In particular, GABA-B activating drugs increase spontaneous EEG spiking activity; whereas, blockade of GABA-B receptors decreases spiking episodes. A genetically epileptic strain of mice called DBA2/J show spike-wave discharges (SWDs) similar to the pattern observed in other rodent models of absence seizures, for which GABA-B stimulation enhances SWDs. As the activation of thalamic GABA-B receptors may be critical in setting not only SWDs pattern but also the frequency of the entire thalamocortical network, we investigated the possible correlations between SWDs and the power spectrum frequency in relation to the modulation of GABA-B receptors. DBA2/J mice, tested with the GABA-B agonist, L-baclofen, followed by the GABA-B antagonist, SCH 50911, were recorded with digital EEG. The power spectra for delta (0.5-3 Hz), theta (3.5-7.5 Hz), alpha (8-12 Hz), beta (13-20 Hz), and gamma (21-50 Hz) frequencies were processed off line.

SWDs and power spectrum of beta activity were increased by up to 80% following administration of L-baclofen, while gamma power frequency decreased to the same extent. Moreover, following administration of SCH 5911, SWDs and beta power frequencies were markedly reduced, while gamma power increased.

These results confirm the potential usefulness of GABA-B antagonists in the treatment human absence seizures. Moreover, as gamma increase might correlate with optimised cortical binding during coherent percepts, it can be speculated that this mechanism could be involved in some aspects of “cognition-enhancing” effects already described in murine absence models following GABA-B antagonists. Epilepsia 2006;47(3).

February 2006
Influence of Lamotrigine and Topiramate on MDR1 Expression in Difficult to Treat Temporal Lobe Epilepsy
Ying Wang-Tilz, Christian Tilz, Bing Wang, Gernot P. Tilz, and Hermann Stefan

Some individuals with epilepsy appear resistant to most or all antiepileptic medications. One possible explanation for this resistance is presence of a gene called MDR1 that causes production in the brain of a substance called p-glycoprotein (P-gp). We studied a model of epilepsy in rats produced by repeated injections of Coriaria lactone, an epileptogenic plant toxin. The clinical antiepileptic drugs, topiramate and lamotrigine significantly reduced seizures. With these two drugs, P-gp was not increased in brain. In contrast, carbamazepine and valproate did increase brain P-gp, and these drugs were less beneficial against the seizures. These results suggest that attention could usefully be paid to whether medications activate multiple drug resistance genes in brain, since such an effect could counteract the effectiveness of a medication. Epilepsia 2006;47(2).

Serotonin Depletion Attenuates AY-9944 Mediated Atypical Absence Seizures

Eduard Bercovici, Miguel A. Cortez, Xiaomei Wang, and O. Carter Snead III

Atypical absence seizures (AAS) are one of the most debilitating types of childhood epilepsies. They usually coexist with other seizure types, are refractory to medications, and associated with cognitive deficits. AAS are reproducible in rats and mice by administration of the cholesterol inhibitor AY-9944 (AY) to developing pups, leading to chronic seizures with slow-spike-wave discharge (SSWD) morphology. Seizures in AY treated rats are worsened during slow wave sleep, where SSWD become continuous and myoclonic jerks appear more frequently. This effect of sleep suggests that serotonin (5-HT) may be involved in modulating epileptic seizures in the AY model. To investigate the role of serotonin we studied AY-treated rats with implanted cortical electrodes, and depleted serotonin via administration of para-chlorophenylalanine (PCPA). SSWD were measured before and after serotonin depletion, following which brains were removed for biochemical analysis via high-performance liquid chromatography (HPLC). We found that PCPA decreased the total duration of SSWD in AY rats as compared to controls. In addition, we confirmed that PCPA depleted serotonin and its metabolite (5-HIAA) in all brain regions studied. Furthermore, we showed that naïve AY rats had elevated 5-HT and 5-HIAA as compared to controls. Our data show that serotonin depletion protects against atypical absence seizures and suggest that high serotonin levels may be involved in the pathogenesis of these seizures. These results could suggest strategies to develop antiepileptic medications that reduce serotonin effects, and also could introduce a caution in using drugs that increase serotonin in patients with atypical absence epilepsy. Epilepsia 2006;47(2).

A Brief Period of Epileptiform Activity Strengthens Excitatory Synapses in the Rat Hippocampus in Vitro

Dominique Debanne, Scott M. Thompson, and Beat H. Gähwiler

A sizeable fraction of all human epilepsies are acquired by otherwise healthy individuals as the result of injury or illness. In many of these acquired epilepsies, the initial insult is associated with a brief period of seizures which is then followed by a long latent period before the patient becomes truly epileptic. We examined here whether a very short period of epileptiform (seizure) activity could produce lasting modifications of synaptic strength and network properties in the rat hippocampus slice, kept alive in a dish. Hippocampus is useful to study because it is the most seizure-prone part of brain, and its circuitry is well studied. Synaptic transmission at two types of synapse was monitored in hippocampal slice cultures before and after a very brief episode of epileptiform activity (20-180 sec) induced with bicuculline methochloride, a blocker of inhibitory synaptic transmission. We show here that a very brief period of epileptiform activity induces long-lasting (>20 minutes) potentiation of excitatory synaptic transmission. This potentiation was also observed at synapses formed by pairs of connected neurons. It was dependent upon activation of the N-methyl-D-aspartate (NMDA) subtype of the glutamate receptor, a receptor that is involved in learning and memory. Interestingly, the enhancement of synaptic transmission could be reversed by a specific regime of stimulation. Recruitment of synaptic networks within the hippocampus was facilitated after epileptiform activity, indicating that the induced potentiation enhanced overall hippocampal network excitability. These changes in synaptic transmission may contribute to the genesis of epilepsy and to seizure-associated memory deficits. Epilepsia 2006;47(2).

Reeler Homozygous Mice Exhibit Enhanced Susceptibility to Epileptiform Activity

Peter R. Patrylo, Ronald A. Browning, and Scott Cranick

Humans with diffuse cortical malformations (CM) are observed frequently to have impaired cognitive development, epilepsy, and other neurological disorders. Several animal models are available currently that exhibit diffuse CNS disruptions. However, in contrast to the human condition, enhanced seizure susceptibility has been reported in only a handful of these models. Whether this disparity reflects a difference in the phenotypic properties of these models that can be used to delineate the mechanisms involved with increasing seizure susceptibility, or reflects a paucity of knowledge is unclear. Consequently, in this study, a combination of in vivo and in vitro techniques were used to determine whether seizure susceptibility is altered in reeler mutant mice (rl/rl); a model of CM which exhibits anatomical alterations and a mode of inheritance similar to that found in a human condition (lissencephaly with cerebellar hypoplasia; LCH). In vivo experiments revealed that rl/rl mice, compared to controls, exhibit a lower threshold for seizures and a higher incidence of seizures when subjected to comparable stimuli. Studies of brain slices maintained alive in a dish revealed that compared to controls, malformed cortical regions from rl/rl mice were more likely to generate spontaneous epileptiform activity when inhibition was compromised, and that the duration of these events was longer. Considered together, these data indicate that rl/rl mice have enhanced seizure susceptibility that is in part intrinsic to the malformed cortical regions. Moreover, these findings indicate that the reeler mutant mouse may provide a valuable model of CM due to their similarities with the human condition of LCH. Epilepsia 2006;47(2).

Diacylglycerol Kinase Epsilon Modulates Rapid Kindling Epileptogenesis

Alberto Musto and Nicolas G. Bazan

The development of mesial temporal lobe epilepsy progresses from partial complex seizures and is followed by secondary generalized seizures. In turn, this can lead to seizure recurrence that causes mental and learning disabilities, psychosocial problems, and unresponsiveness to anti-epileptic drugs.

Neuronal signal transduction mediated by the neurotransmitter glutamate is believed to be a major pathway by which seizures develop and become more severe. A family of enzymes known as the diacylglycerol kinases participates in glutamate-mediated signaling through the regulation of a fatty (lipid) signaling system based on the compound, inositol. One form of the diacylglycerol kinase enzyme, called the epsilon isoform, specifically recognizes inositol lipids that contain arachidonic acid. It has long been known that arachidonic acid is liberated from brain cell membranes during seizures, stroke, and other forms of neural injury, and this free fatty acid is rapidly converted to a cascade of signaling molecules that propagate and magnify the initial cellular injury. Mice deficient in the diacylglycerol kinase gene subjected to an experimental model of mesial temporal lobe epilepsy displayed attenuated development and progression of epileptic seizures. Furthermore, the microscopic alterations in brain synapses that are characteristic of epileptogenesis are decreased or absent when this gene is not active. The significance of this discovery lies in its identification of a molecular pathway that potentially can be used for the targeting of anti-seizure medications. Epilepsia 2006;47(2).

Decrease of Hippocampal GABA B Receptor Mediated Inhibition after Hyperthermia Induced Seizures in Immature Rats

Min-Lan Tsai and L. Stan Leung

We studied the consequences of heat-induced seizures in immature rat pups, 13 to 15 days after birth, as a model of febrile seizures in children. We hypothesize that neuronal inhibition in the hippocampus, one of the most seizure-prone areas of the brain, is decreased by heat-induced seizures. A single seizure was induced by heating with an infrared lamp (fast heat), or repeated seizures were induced in rat pups (9 seizures over 3 days) by a hair dryer (slow heat). Brain wave and behavioral recordings indicate that the hair-dryer induced seizures were relatively localized to the amygdala and hippocampus, while an infrared lamp seizure was more severe and involved extensive areas of the brain perhaps including the brainstem. However, irrespective of seizure types, long-term physiological recordings of the rats after the heat-induced seizures indicated that a late neuronal inhibition was lost in certain key regions of the hippocampus. The late neuronal inhibition, shown to be mediated by GABAB receptors, a brain receptor that produces long-lasting inhibitory potentials, was lost for 14 to 30 days after a single or repeated seizures. Since GABAB receptors play a protective role in neurons mainly by controlling excitability, we infer that neuronal excitability in the hippocampus is compromised for at least 14 days after heat-induced seizures. This is the first time that heat-induced seizures in immature rats have been shown to decrease late neuronal inhibition in the brain. Epilepsia 2006;47(2).

Neocortical Microenvironment in Patients with Intractable Epilepsy: Potassium and Chloride Concentrations

Ali Gorji, Nina Stemmer, Bernhard Rambeck, Uwe Jürgens, Theodor May, Heinz Wolfgang Pannek, Friedrich Behne, Alois Ebner, Hiedrun Straub, and Erwin-Josef Speckmann

The regulation of extracellular ion concentrations plays an important role in neuronal function and epileptogenesis. Despite the many studies into the mechanisms of epileptogenesis in human experimental models, there are no data available regarding the fluctuations of extracellular potassium ([K+]o) and chloride ([Cl-]o), which could underlie seizure susceptibility, in human chronically epileptic tissues in vivo. Using cerebral microdialysis during surgical resection of epileptic foci, the basic [K+]o and [Cl-]o as well as their changes after epicortical electric stimulation were studied in samples of dialysates obtained from 11 patients by ion selective microelectrodes. The mean basal values of [K+]o and [Cl-]o in all patients were 3.83 ± 0.08 mM and 122.9 ± 2.6 mM, respectively. However, significant differences were observed in both the basal levels of [K+]o and [Cl-]o between different patients. Statistically, there was no correlation between basal [K+]o or [Cl-]o and ECoG spike activity but in one patient dramatically lowered baseline [Cl-]o was accompanied by enhanced ECoG spike activity. Application of epicortical electrical stimulation increased [K+]o but not [Cl-]o in all cases. According to the velocity as well as spatial distribution of [K+]o reduction to the pre-stimulation levels, three different types of responses were observed: slow decline, fast decline, and slow and fast declines at adjacent sites. Present data may represent abnormalities in ion homeostasis of the epileptic brain. Epilepsia 2006;47(2).

January 2006
Pharmacodynamic and Pharmacokinetic Characterization of Interactions between Levetiracetam and Numerous Antiepileptic Drugs in the Mouse Maximal Electroshock Seizure Model: An Isobolographic Analysis
Jarogniew J. Luszczki, Marta M. Andres, Piotr Czuczwar, Anna Cioczek-Czuczwar, Neville Ratnaraj, Philip N. Patsalos, and Stanislaw J. Czuczwar

Approximately 30% of patients with epilepsy do not experience satisfactory seizure control with current front-line antiepileptic drug (AED) monotherapy and often require polytherapy. The potential usefulness of AED combinations, in terms of efficacy and adverse effects, is therefore of major importance. The present study sought to identify potentially useful AED combinations with that of levetiracetam (LEV), recently introduced as an effective antiepileptic medication for refractory partial seizures. The mouse maximal electroshock (MES)-induced seizure model was investigated with regards to the anticonvulsant effects of carbamazepine (CBZ), phenytoin, phenobarbital (PB), valproate, lamotrigine, topiramate (TPM) and oxcarbazepine (OXC), administered singly and in combination with LEV. Acute adverse effects were ascertained by use of the chimney test, evaluating motor performance, and the passive avoidance task, assessing long-term memory. Brain AED concentrations were determined so as to ascertain any pharmacokinetic contribution to the observed anti-seizure effect. LEV in combination with TPM exerted supra-additive (synergistic) interactions in the MES-test. Likewise, the combinations of LEV with CBZ and OXC were supra-additive in this test. In contrast, all other LEV/AED combinations displayed additivity. Furthermore, none of the investigated combinations altered motor performance and long-term memory. LEV brain concentrations were unaffected by concomitant AED administration and also LEV had no significant effect on brain concentrations of concomitant AEDs. These preclinical data would suggest that LEV in combination with TPM is associated with beneficial anticonvulsant pharmacodynamic interactions. Similar, but less profound effects were seen with OXC and CBZ. Epilepsia 2006;47(1).

Evidence Supporting a Role of Serotonin in Modulation of Sudden Death Induced by Seizures in DBA/2 Mice

Srinivasan Tupal and Carl L. Faingold

Sudden unexpected death in epilepsy (SUDEP) is a serious concern for epileptic patients and their families. DBA/2 mice may be a useful animal model of SUDEP. This strain of mouse inherits susceptibility to sound-induced seizures, and most DBA/2 mice stop breathing immediately following seizure. Breathing difficulties are also implicated in some cases of human SUDEP. Normal breathing is controlled partly by a brain neurochemical called serotonin. We evaluated whether drugs that either increase or decrease the effect of serotonin can change whether these mice stop breathing after seizures. Although most DBA/2 mice (75%) stopped breathing after seizure, nearly 99% survive if promptly resuscitated. DBA/2 mice that stopped breathing and were resuscitated were then given a drug that increases serotonin called fluoxetine. Thirty minutes after fluoxetine injection (15-25 mg/kg) most mice no longer stopped breathing after seizure, and the highest dose also reduced seizure severity. These effects were gone 72 hours after drug. Ten percent of untreated DBA/2 mice do not stop breathing after seizure, and these mice were given a drug that blocks serotonin called cyproheptadine. Thirty minutes after injection of cyproheptadine (1-2 mg/kg) most of these mice stopped breathing after seizure and needed resuscitation. This effect was gone 72 hours after drug. These findings show that fluoxetine prevented breathing stoppage after seizures in most DBA/2 mice, suggesting that this drug, which is widely used to treat depression, should be tested for SUDEP prevention in patients likely to show SUDEP. The findings also suggest that cyproheptadine should be avoided in this patient population. Epilepsia 2006;47(1).

Chronic Bilateral Stimulation of the Anterior Thalamus of Kainate Treated Rats Increases Seizure Frequency

Fred A. Lado

Electrical stimulation of the anterior nucleus of the thalamus (ANT) is receiving increased attention and interest as a potentially novel means of controlling intractable epilepsy, and has entered human clinical trials. All animal data supporting the anticonvulsant benefit of ANT stimulation, however, has been obtained from acute chemoconvulsant models of epilepsy rather than models of chronic epilepsy with spontaneous seizures. It is unknown whether ANT stimulation is effective in models of chronic epilepsy. To address this question, the effects of bilateral ANT stimulation were evaluated in rats with chronic epilepsy following acute status epilepticus (SE) produced by systemic kainic acid (KA) administration. In the course of this study, it was necessary to monitor the evolution of epilepsy following KA SE using continuous video-EEG recordings. Following KA SE, most rats have 2-8 seizures per day, and the average seizure rate increases over time, doubling over the course of 14 weeks. Behavioral seizure severity, after the initial development of epilepsy, remains stable. ANT stimulation consistently exacerbated seizure frequency. The effects of stimulation were specific to stimulation applied to the ANT, since stimulation applied to electrodes placed outside the ANT did not significantly worsen seizure frequency. Epilepsia 2006;47(1).

Amygdala Kindling in the WAG/Rij Rat Model of Absence Epilepsy

Rezzan Gülhan Aker, Hasan Raci Yananl?, Ayten Azizova Gurbanova, Aydan Ergün Özkaynakç?, Nurbay Ates, Gilles van Luijtelaar, and Filiz Ylmaz Onat

Experimental models are suitable for studying mechanisms involved in the propagation and generalization of seizure activity in the convulsive and non-convulsive components of epilepsy. In the present study, we compared the widely used model of temporal lobe epilepsy called amygdala kindling in normal rats and in WAG/Rij rats, a model of absence epilepsy. Electrodes were implanted into the amygdala of rats for stimulation and recording and into the cortex for recording. After a recovery period, the animals were stimulated electrically via the electrodes. EEG was recorded to analyze spike-and wave-discharges, which characterize absence epilepsy and after-discharge durations, which characterize the kindling process. The seizure severity was evaluated using Racine’s 5-stage scale. All non-epileptic control animals and four out of seven WAG/Rij animals reached a stage 5, generalized convulsive seizure state (the most intense seizure stage); whereas, three animals failed and stayed at stage 2 after application of 30 stimulations. Interestingly, kindling resistant WAG/Rij rats demonstrated a significantly longer duration of spike-and-wave discharges on the first day of the experiment before kindling stimulation than did the kindled WAG/Rij animals. Additionally, the cumulative total duration and the number of spike-and-wave discharges after the kindling stimulation were statistically increased compared to spike-and-wave discharges before kindling stimulation. The results of our study demonstrate that the progress of amygdala kindling is changed in rats with genetic absence epilepsy, perhaps as a consequence of the hundreds of daily spike-and-wave discharges. Epilepsia 2006;47(1).

Hippocampal T2 Signal Change on Serial MR Imaging During Amygdala Kindling Epileptogenesis

Bianca Jupp, John P. Williams, Yasvir A.Tesiram, Milosh Vosmansky, and Terence J. O’Brien

Electrical amygdala kindling in the rat, where repeated small electrical stimulations are applied to the brain resulting in a progressive and permanent change in the epileptic threshold, is one of the most widely studied animal models of temporal lobe epilepsy (TLE). However the processes underlying the development of epilepsy in this model remain incompletely understood. Magnetic resonance imaging (MRI) is a powerful technique that enables sequential visualization of structural and functional changes occurring in live animals. This makes it possible to uncover the contribution of such changes to the development of epilepsy. Here we report, for the first time, serial MR image acquisitions during the development of epilepsy in this model of TLE. We demonstrated MR signal changes (reflecting increased water content) in restricted parts of the hippocampus, an area of the brain particularly vulnerable to epileptic insults. This signal change occurred without a change in volume of the structure, suggesting that it may occur independent of cell loss in this region. This is important given cell loss had previously been proposed to be a critically important for the development of TLE. This study validates a method for acquiring serial MRI during kindling in the rat, and with the detection of changes in restricted regions of the hippocampus. These regions may be important sites for the neurobiological changes that contribute to the development of epilepsy in this model, and in human TLE. Epilepsia 2006;47(1).

Epileptiform Activity Induced by Pharmacological Reduction of M-current in the Developing Hippocampus in Vitro

Fernando Peña and Noé Alavez-Pérez

Potassium ion (K+) channels play major roles in neuronal activity. This is well exemplified by a genetic form of epilepsy called benign familial neonatal convulsions (BFNC), which is caused by a K+ channel malfunction produced by mutations in the KCNQ2 and KCNQ3 genes. Mutations are carried throughout the whole life, but BFNC is expressed only in neonates. Here, we attempted to develop an in vitro model to study this disease. Knowing that channels formed by mutated KCNQ2 and KCNQ3 subunits have a reduced function, we tested whether pharmacological reduction of the native channels, with their specific blocker linopirdine, induces seizure-like activity in brain slices. We also tested if there were differences in the effects of linopirdine throughout postnatal age, as occurs for clinical manifestations of BFNC. Using brain slices obtained from animals at different ages we observed that linopirdine induced seizure-like activity only in slices from neonatal rats. In contrast, on slices from adult rats linopirdine induced erratic abnormal activity. Our results show that pharmacological blockade of the KCNQ2 and KCNQ3 channels induces seizure-like activity with an age pattern that closely resembles the developmental characteristics of BFNC. We believe this could be an in vitro model that can be used to study the cellular mechanisms of developing epilepsy and the developmental pattern of BFNC, as well as to develop some therapeutic strategies. Epilepsia 2006;47(1).

Open Field Behaviors and Water Maze Learning in the F Substrain of Ihara Epileptic Rats

Yoko Okaichi, Shigeru Amano, Nobuo Ihara, Yoneko Hayase, Tooru Tazumi, and Hiroshige Okaichi

enetically epileptic model rats of the Ihara epileptic rat (IER/F) sub-strain, have neuropathological abnormalities and develop generalized convulsive seizures when they reach approximately five months of age. Structural abnormalities are centered in the hippocampus, which is the most seizure-prone part of brain, and also a part involved in memory in rats of positions in space. Therefore, we expected to observe spatial cognitive deficits in the model rats. The present study aimed to evaluate learning ability of the F sub-strain of IER. To determine whether deficits are caused by inborn structural brain abnormalities or by repeated generalized convulsions, we tested nine 6-12-week-old IER/F rats that had not yet experienced seizures (Experiment 1) and nine 7-9-month-old IER/F rats that had repeatedly experienced seizures (Experiment 2) with identical tasks. These tasks comprised an open field test to assess spontaneous behaviors and the Morris water maze place and cue tasks to assess spatial cognition and learning abilities. Both groups of IER/Fs showed behaviors that were different from those of normal rats in the open field test. Extensive learning impairments were seen in both the place task, which requires spatial cognition, and the cue task, which does not require spatial cognition but requires simple association learning. Their impaired performance of the cue task indicates that their deficiency was not limited to spatial cognition. These results suggested that structural abnormalities of brain cells in the hippocampus may be more responsible for the severe learning deficits of IER/Fs than are the seizures themselves. Epilepsia 2006;47(1).

December 2005
Epileptogenesis and Reduced Inward Rectifier Potassium Current in Tuberous Sclerosis Complex-1 Deficient Astrocytes
Laura A. Jansen, Erik J. Uhlmann, Peter B. Crino, David H. Gutmann, and Michael Wong

Individuals with Tuberous Sclerosis Complex (TSC) frequently suffer from intractable epilepsy. To gain insight into the causes of epilepsy in TSC, we previously developed a mouse model of TSC with inactivation of the Tsc1 gene selectively in brain astrocytes (Tsc1GFAPCKO mice). Astrocytes, the main category of glial cells in brain, are important supporting cells in the brain, and also have direct effects on brain physiology, development and repair. These mice develop progressive seizures, suggesting that astrocyte dysfunction may be involved in the development of epilepsy in TSC. Since one of the important functions of astrocytes is to restrain sudden elevations of extracellular potassium in the brain, which would lead to neuronal excitability, in this study we investigated the hypothesis that impairment of potassium uptake may contribute to the development of seizures in Tsc1GFAPCKO mice. Astrocytes allow potassium flow from the cell to are taken up from the extracellular space via a membrane channel called the Kir channel. Cultured astrocytes from Tsc1GFAPCKO mice exhibited reduced Kir potassium currents and decreased expression of specific Kir channel protein subunits. mRNA expression of the same Kir subunits was also reduced in astrocytes from Tsc1GFAPCKO mice. Furthermore, we showed that the impairment in Kir channel function was reversed with drugs (roscovitine and retinoic acid) that modulate cell signalling pathways implicated in TSC. Lastly, hippocampal slices from Tsc1GFAPCKO mice exhibited decreased astrocytic Kir currents, as well as increased susceptibility to potassium-induced seizure-like activity. In conclusion, impaired extracellular potassium uptake by astrocytes through Kir channels may contribute to increased neuronal excitability and the development of epilepsy in a mouse model of TSC. Epilepsia 2005;46(12).

Transcortical Cooling Inhibits Hippocampal-kindled Seizures in the Rat

Justin M. Burton, Gregory A. Peebles, Devin K. Binder, Steven M. Rothman, and Matthew D. Smyth

Surgery for epilepsy has a fairly high success rate for controlling seizures, but often the regions of brain causing the seizures are also located in areas that cannot be removed without causing neurologic deficits such as weakness, speech problems or memory deficits. A reversible method of suppressing brain activity might control seizures but preserve normal brain function. This paper describes an animal model of seizures (electrically “kindled” seizures) arising from the hippocampus, and evaluates the effect of focal brain cooling on controlling these induced seizures. Cooling was delivered using surface copper cooling coils circulating coolant of two temperatures: 8 and 16 degrees C. We found that focal brain cooling of the hippocampal target between 24 and 27 degrees C reliably suppressed kindled seizure activity in a temperature-dependent manner. Anticonvulsant activity occurred at temperatures well above those known to result in tissue injury or inhibition of normal neurologic function. These findings have important implications for the potential use of implantable cooling devices in humans with medication-resistant epilepsies in or near eloquent brain regions. Epilepsia 2005;46(12).

The Acute Anticonvulsant Effects of Deoxycorticosterone in Developing Rats: Role of Metabolites and Mineralocorticoid Receptor Responses

Heather E. Edwards, Sutha Vimal, and W. McIntyre Burnham

ACTH is a hormone used in the treatment of certain forms of serious childhood epilepsy. It has never been known how ACTH exerts its anticonvulsant effects. Recent studies from our laboratory have suggested that ACTH acts - at least in part - by increasing the release of an adrenal hormone called “DOC”. The present study was designed to investigate the various receptors in the brain that may be mediating DOC’s anticonvulsant actions. Our experiments suggest that DOC may be working through at least two different receptors, including: 1) mineralocorticoid receptors, and 2) GABAA receptors. Some of these actions seem to be mediated by DOC itself, whereas others seem to be mediated by DOC’s metabolites, DHDOC and THDOC. Epilepsia 2005;46(12).

Febrile Convulsions Induced by the Combination of Lipopolysaccharide and Low Dose Kainic Acid Enhance Seizure Susceptibility not Epileptogenesis in Rats

James G. Heida, G. Campbell Teskey, and Quentin J. Pittman

Febrile convulsions (FC) are seizures that occur as a result of fever. They affect 3-5% of children between 6 months and 5 years of age, making them the most common convulsive disorder in humans. One of the most critical questions in the study of FC is the effect of these convulsions on the latter development of temporal lobe epilepsy in adulthood. Yet the experimental and clinical data addressing this question are inconclusive. Thus we tested the hypothesis that FC during infancy affects development of seizures in adulthood. To address this we used a novel animal model of FC that incorporates true fever in which FC were induced in 14-day-old male rats. Eight – 10 weeks after the initial FC, animals were prepared for amygdala kindling. Amygdala kindling is a valid and reliable model of human temporal lobe epilepsy whereby progressively worsening seizures are caused by daily electrical stimulation of the amygdala with an invariant stimulus. We find that animals with a history of FC have lower seizure thresholds, longer duration seizures, and enhanced neural degeneration in the hippocampus, but do not show a facilitation of epileptogenesis (ie spread to other structures) compared to control rats. Our results indicate that FC during infancy, in this model, enhances the propensity for seizures but does not increase the progression to more severe seizures. Thus we conclude that FC during infancy affects local circuits in the amygdala and possibly the hippocampus but not circuits responsible for seizure generalization. Epilepsia 2005;46(12).

Causal Links between Brain Cytokines and Experimental Febrile Convulsions in the Rat

James G. Heida and Quentin J. Pittman

Febrile convulsions (FCs) are seizures that are caused by fever. They are the most common type of seizure in humans and occur exclusively in young children. Despite their prevalence and potential for causing long term changes in the brain, the mechanisms of FCs remain poorly understood. During fever, the activation of the immune system results in the production of molecules called cytokines in the brain. Some of these are pro-inflammatory (ie interleukin 1_(IL-1_) and others are anti-inflammatory (IL-1ra). Several studies conducted in humans have indicated a potential role for cytokines (particularly IL-1_ and IL-1ra) in the genesis of FCs. Therefore we tested the hypothesis that IL-1_ and IL-1ra affect the genesis of febrile convulsions. We induced fevers in infant rats that resulted in convulsions with fever in approximately 50% of animals tested. We then measured brain levels of IL-1_ and IL-1ra during a FC. We find that FC were associated with increased levels of IL-1_ in the hypothalamus and hippocampus, while IL-1ra does not change. Next we established a causal role for IL-1_ and IL-1ra in FC genesis. This was accomplished by direct application of IL-1_ and IL-1ra into the brain of animals treated in our model. These experiments showed that the pro-inflammatory cytokine IL-1_ dose dependently increased, while the anti-inflammatory cytokine IL-1ra decreased seizure frequency. We therefore conclude that alterations of cytokines (IL-1_ and IL-1ra) within the brain may be of causal significance in the pathophysiology of FCs. Epilepsia 2005;46(12).

November 2005
In Vivo Epileptogenicity of Focal Cortical Dysplasia: A Direct Cortical Paired Stimulation Study
Riki Matsumoto, Masako Kinoshita, Junya Taki, Takefumi Hitomi, Nobuhiro Mikuni, Hiroshi Shibasaki, Hidenao Fukuyama, Nobuo Hashimoto, and Akio Ikeda

Focal cortical dysplasia (FCD) is a developmental abnormality of cerebral cortical cell architecture characterized by disorganized cortical layers and by an excess of large, aberrant neurons. FCD increasingly has been recognized as a major cause for intractable partial epilepsy. Epileptic seizures arise based on imbalance between cortical inhibitory and excitatory mechanisms in the epileptic focus. Apart from studies using animal models or surgical specimens, little is known about imbalance between these two mechanisms within FCD to cause epilepsy in patients. A patient with FCD at the left foot primary sensorimotor area was studied for possible brain surgery to treat the epilepsy. A grid (sheet) of recording and stimulating electrodes was place on the brain as part of standard procedure to “map” the brain prior to removing the parts thought to be responsible for the seizures. Using these implanted electrodes placed on the surface of brain, we investigated the cortical inhibitory and excitatory mechanisms by applying paired electrical pulses directly to the cortex and recording cortical evoked responses from the surrounding cortex. Between seizures, cortical inhibition was abnormally enhanced in the focus compared with normal control cortex. While the patient was having the somatosensory aura (seizure warning), which later evolved to the left leg clonic seizure, the increased cortical inhibition was replaced by increased cortical excitability and decreased inhibition. It was demonstrated that the dynamic alternation of cortical inhibitory and excitatory mechanisms occurred toward increased cortical excitability during seizure generation in a patient with FCD. Epilepsia 2005;46(11).

In Situ Produced 7-Chlorokynurenate Has Different Effects on Evoked Responses in Rats with Limbic Epilepsy in Comparison to Naive Controls

Edward H. Bertram, DeXing Zhang, John M. Williamson, HuQui Wu, and Robert Schwarcz

There is growing evidence that neurons (brain cells) from seizure-generating brain regions respond differently to drugs, in comparison to the neurons from normal animals, an observation that may be important for finding new epilepsy treatments. One potential target for drug therapy has been neurotransmission by glutamate, the main excitatory neurotransmitter in the brain, but this approach has been greatly limited by the significant neurological and cognitive consequences of this type of treatment. There is some evidence that blocking the glycineB coagonist site on the NMDA component of the glutamate receptor could be an alternative approach in reducing excitatory neurotransmission without the side effects that are associated with direct blockade of the NMDA receptor. We examined the potential of the glycineB site antagonist 7-Cl-kynurenic acid (7-Cl-KYNA) to benefit the seizure-like response in hippocampus of the rat. Hippocampus is the most seizure-prone structure in the brain.. We gave the prodrug 4-chloro-kynurenine (4-Cl-KYN), which is converted by the glial (supporting) cells to 7-Cl-KYNA, the active drug. Synaptic responses from hippocampal neurons in epileptic and naive rats were obtained for two hours before and for five hours after the administration of the drug. The two groups had significantly different responses to the drug. In the epileptic animals there was a significant reduction in the amplitude of the response as well as in the epilepsy-like features (number of population spikes); whereas, in the naive animals there were minimal changes, primarily an enhancement of response amplitudes. The tissue concentration of the drug was the same in both groups. These findings suggest that antagonism of the glycineB site on the NMDA receptor may be a target for antiepileptic drug development. The findings also emphasize the unique pharmacology of epilepsy. Epilepsia 2005;46(11).

Brain Inflammation in Epilepsy: Experimental and Clinical Evidence

Annamaria Vezzani and Tiziana Granata

In recent years, there has been increasing evidence that immune/inflammatory reactions occur in brain in various central nervous system (CNS) diseases, including autoimmune, neurodegenerative and epileptic disorders. Furthermore, inflammatory processes such as the production of pro-inflammatory cytokines and related molecules, have been described in blood, CSF and brain after seizures induced in experimental models and in clinical cases of epilepsy. Although little is known about the role of inflammation in epilepsy, it has been hypothesized that activation of the inflammatory reactions in brain may mediate some of the molecular, structural and functional changes occurring during and after seizure activity. Whether inflammatory reactions taking place in epileptic tissue is beneficial or noxious to brain is still an open and intriging question that needs to be addressed by further investigations. However, studies in experimental models of seizures show that inflammatory reactions in brain enhance neuronal excitability, impair cell survival and permit the entry into the brain of molecules and cells that are otherwise excluded. Moreover, some anti-inflammatory treatments reduce seizures in experimental models, and in some instances, in clinical cases of epilepsy. This evidence raises the possibility that inflammation may be a common factor contributing, or predisposing, to the occurrence of seizures and cell death, in various forms of epilepsy of different etiologies. The elucidation of this aspect may open new perspectives for the pharmacological treatment of seizures. Epilepsia 2005;46(11).

October 2005
Effects of Nefiracetam, a Novel Pyrrolidone-type Nootropic Agent on the Amygdala-kindled Seizures in Rats
Yutaka Kitano, Chika Komiyama, Mitsuhiro Makino, Yoshio Kasai, Kiyoshi Takasuna, Masakazu Kinoshita, Osamu Yamazaki, Akira Takazawa, Toshio Yamauchi, and Shinobu Sakurada

Nefiracetam is a novel pyrrolidone-type nootropic agent, and it has been reported to possess various pharmacological effects as well as cognition-enhancing effects. The present study focused on the effects of nefiracetam in amygdala-kindled seizures and its potential for antiepileptic therapy. Effects of nefiracetam on fully amygdala-kindled seizures and development of amygdala-kindled seizures were investigated in rats and compared with those of levetiracetam (Keppra), a pyrrolidone-type antiepileptic drug. In fully amygdala-kindled rats, nefiracetam (25, 50 and 100 mg/kg, p.o.) decreased after-discharge induction, after-discharge duration, seizure stage and motor seizure duration in a dose-dependent manner. Levetiracetam (25, 50 and 100 mg/kg, p.o.) had no effects on after-discharge induction and slightly decreased after-discharge duration, while it markedly decreased seizure stage and motor seizure duration. In contrast to the results in fully amygdala-kindled rats, nefiracetam (25 and 50 mg/kg/day, p.o.) had little or no effects on the development of amygdala-kindled seizures. As well as fully amygdala-kindled seizures, levetiracetam (50 mg/kg/day, p.o.) markedly inhibited the development of behavioral seizures without reducing daily after-discharge duration. Although nefiracetam possesses potent anticonvulsant effects on fully amygdala-kindled seizures, it has little or no effects on the development of amygdala-kindled seizures. Levetiracetam shows marked anticonvulsant effects on both phases of kindling. In fully amygdala-kindled rats, nefiracetam inhibits both electroencephalographic and behavioral seizures, whereas levetiracetam inhibits only behavioral seizures. This double dissociation suggests that nefiracetam has distinct anticonvulsant spectrum and mechanisms from those of levetiracetam in spite of the structural similarity. Nefiracetam possesses a potential for antiepileptic therapy. Epilepsia 2005;46(10).

Expression of Multidrug Transporters MRP1, MRP2 and BCRP Shortly after Status Epilepticus, During the Latent Period, and in Chronic Epileptic Rats

Erwin A. van Vliet, Sandra Redeker, Eleonora Aronica, Peter M. Edelbroek, and Jan A. Gorter

Overexpression of proteins that transport a variety of anti-epileptic drugs (multidrug transporters) may decrease drug levels in the brain, thereby contributing to pharmacological intractable epilepsy. However, it is not known whether overexpression is due to an initial insult, or evolves more gradually because of recurrent spontaneous seizures. To study this, we used a rat model of temporal lobe epilepsy, in which an initial insult (status epilepticus) was followed by recurrent spontaneous seizures. We examined the expression of multidrug resistance-associated proteins MRP1 and MRP2 and breast cancer resistance protein (BCRP) during epileptogenesis by immunocytochemistry and western blot analysis. In addition, we determined whether these transporters affected the distribution of the anti-epileptic drug phenytoin in the brain by HPLC analysis in the presence and absence of the MRP inhibitor probenecid. Shortly after SE, MRP1 MRP2 and BCRP were overexpressed in astrocytes within several limbic structures, including hippocampus. In chronic epileptic rats, these proteins were overexpressed in the parahippocampal cortex, specifically in blood vessels and astrocytes surrounding these vessels. Overexpression was related to the occurrence of SE and mainly present in rats with a high seizure frequency. Brain phenytoin levels were significantly lower in epileptic rats compared to control rats, but pharmacological inhibition of MRPs increased the phenytoin levels. These data suggest that administration of anti-epileptic drugs in combination with specific inhibitors for multidrug transporters can increase brain concentrations of anti-epileptic drugs and may be a promising therapeutic strategy in patients with pharmacological intractable epilepsy. Epilepsia 2005;46(10).

Quinine, A Blocker of Neuronal Cx36 Channels, Suppresses Seizure Activity in Rat Neocortex in Vivo

Zita Gajda, Zoltán Szupera, Gábor Blazsóand, and Magdolna Szente

There is growing evidence that, besides the chemical synapses, direct coupling via gap junction (GJ) provides a second major pathway, contributing to normal and abnormal neuronal synchronization. GJs are intercellular channels composed of connexins (Cx). Cx36, the major neuronal Cx, is present predominantly in GABAergic interneurons. In this study EEG recording was performed on anesthetized adult rats, in which either quinine - a selective blocker of Cx36 - or the broad-spectrum GJ blockers carbenoxolone and octanol were applied locally, prior to the induction or at already active epileptic foci induced by 4-aminopyridine. Pretreatment with quinine slightly reduces epileptogenesis and the expression of seizure discharges, while application of quinine after 25-30 spontaneously occurring seizures has an anticonvulsive effect and modifies the manifestation of the 1-18 Hz seizure discharges. The summated ictal activity decreased, because of the significant reduction in the duration of the seizures. The amplitudes of the seizure discharges of all the patterns decreased, with the exception of those with frequencies of 11-12 Hz, and a new discharge pattern appeared with frequencies above 15 Hz at the initiation of seizures. The blockade of Cx36 channels and the global blockade of the GJ by carbenoxolone result in qualitatively different modifications in ictogenesis. Our findings indicate that the GJ communication via Cx36 channels is differently involved in the synchronization of the activities of the networks generating seizure discharges with different frequencies. Additionally, we conclude that both neuronal and glial GJ communication contribute to the manifestation and propagation of seizures in the adult rat neocortex. Epilepsia 2005;46(10).

Analysis of Chronic Seizure Onsets after Intrahippocampal Kainic Acid Injection in Freely Moving Rats

Anatol Bragin, Avetis Azizyan, Joyel Almajano, Charles L. Wilson, and Jerome Engel, Jr.

The goal of this study was to analyze the transition period between interictal and ictal activity in freely moving rats with recurrent spontaneous seizures after unilateral intrahippocampal kainic acid (KA) injection. Pairs of tungsten electrodes (50 µm O/D) were implanted bilaterally under anesthesia at symmetrical points in the dentate gyrus (DG) and CA1 regions of anterior and posterior hippocampi and entorhinal cortex of adult Wistar rats. Stimulating electrodes were placed in the right angular bundle and KA was injected into the right posterior CA3 area of hippocampus after 1 week of baseline EEG recording. Beginning 24 hours after injection, electrographic activity was recorded with video monitoring for seizures every day for 8 hrs/day for 60 days. Seventy percent of seizures started locally in the DG ipsilateral to injection with an increase in frequency of interictal EEG spikes (hypersynchronous type, HYP) and 26% of seizures started with a decrease of EEG amplitude with parallel increase in frequency (low voltage fast type, LVF). During HYP seizures a significant increase was observed in amplitude of beta-gamma range frequencies, ripple frequency and fast ripple (FR) frequency, while during LVF seizure there was an increase only in the beta-gamma range. In all cases but one, an EEG wave preceded ripple and FR oscillations. Prior to seizure onset, the amplitude of dentate gyrus evoked responses to single pulses decreased, while the amplitude of the response to the second pulse delivered at 30 ms interval increased. If ripple and FR oscillations indicate the seizure generating neuronal substrate, these areas must be small and widespread so that the probability of recording from them directly is very low. The decreased response to electrical stimulation prior to seizures could indicate protective inhibitory mechanism which contain or prevent seizure occurrence. The presence of decreased paired pulse suppression could indicate a network predisposition to follow an external input with a certain frequency. Epilepsia 2005;46(10).

Prolonged Exposure to Lead Lowers the Threshold of Pentylenetetrazole-induced Seizures in Rats

Oscar Arrieta, Guadalupe Palencia, Guadalupe García-Arenas, Daniela Morales-Espinosa, Norma Hernández-Pedro, and Julio Sotelo

Epilepsy is more prevalent in developing than in industrialized countries due to a higher incidence of some diseases and adverse socioeconomic factors, among them, is the chronic exposure to environmental pollutants. Diverse circumstances, such as malnutrition and exposure to volatile solvents lower the threshold for experimental epilepsy. The aim of this work was to study the effects of chronic exposure to lead on the threshold of experimental seizures induced by the seizure-producing drug, pentylenetetrazole (PTZ). Chronic exposure to environmental toxicants is a growing problem of public health. Lead is a component of many industrial products; humans are exposed through various routes. Although all age groups are susceptible, children are more vulnerable due to a greater gastrointestinal absorption of lead, which exerts higher toxicity in the developing nervous system We allocated randomly 120 male rats into four groups; controls, and 3 lead treatment groups who received lead acetate in the drinking water for a period of 30 days at different concentrations. After exposure, a trial of PTZ-induced seizures was conducted in all groups and blood contents of lead were determined. Blood lead contents increased in a dose-dependent manner. Time elapsed to develop the first myoclonic jerk and the tonic-clonic seizure was less in all lead-exposed groups than in controls. Also, the required doses of PTZ to induce myoclonic jerks and tonic-clonic seizures were lower in lead- exposed rats than in controls. We found a reduction in the threshold for seizures in rats whose blood contents of lead were similar to those of humans from some areas of urban centers with high levels of air pollution. Epilepsia 2005;46(10).

Gender Differences in Febrile Seizure-induced Proliferation and Survival in the Rat Dentate Gyrus

Evi M.P. Lemmens, Tim Lubbers, Olaf E.M.G. Schijns, Emile A.M. Beuls, and Govert Hoogland

Febrile seizures are fever-associated early-life seizures that are thought to play a role in the development of epilepsy. In recent years it has become clear that nerve cells continue to divide during adulthood in some brain regions (e.g. hippocampus). Several studies have reported that seizures can increase cell division, also known as proliferation, but most of them only use adult male rodents. Because it is known that the effect of seizures on proliferation depends on age and sex hormones, our goal was to study gender differences in proliferation shortly after early-life febrile seizures and in adults. Febrile seizures were mimicked by increasing the body temperature of ten-day-old male and female rats to 42°C. Afterwards, the animals were injected with bromodeoxyuridine, a substance used to label newborn cells. One or six weeks later, the animals were sacrificed and the number of newborn cells in the hippocampus was compared between animals that had experienced seizures and seizure-free controls. We found that one week after treatment, females had less newborn cells than males and this was not affected by seizures. Six weeks later, male and female controls had the same amount of newborn cells; seizures still had no effect on proliferation in females whereas males with seizures had 25% more newborn cells than males without seizures. Apparently, early-life seizures cause a different response on proliferation in males and females later in life. These findings suggest that gender-based differences seen in epilepsy patients might be related to gender differences in proliferation after seizures. Epilepsia 2005;46(10).

A Pilot Study on Brain-to-plasma Partition of 10,11-dyhydro-10-hydroxy-5Hdibenzo(b,f)azepine-5-carboxamide and MDR1 Brain Expression in Epileptic Patients Not Responding to Oxcarbazepine

Nicola Marchi, Giovanna Guiso, Massimo Rizzi, Susanne Pirker, Klaus Novak, Thomas Czech, Christoph Baumgartner, Damir Janigro, Silvio Caccia, and Annamaria Vezzani

Refractory epilepsy is defined by failure to respond to treatment with several appropriate antiepileptic drugs (AED) at their maximal tolerated doses. Pharmacoresistance occurs in a wide variety of epilepsy types or syndromes and involves a broad range of AED with different mechanisms of action suggesting that the causes underlying pharmacoresistance are likely to be non-specific. In this respect, one of the proposed mechanisms of pharmacoresistance concerns the role of multidrug transport proteins, initially identified for their ability to confer resistance of cancer cells to chemotherapeutics. These transporters that include permeability glycoprotein and the family of multidrug resistance proteins, actively extrude a broad variety of clinically relevant drugs, including AED, from the cells. The current hypothesis is that their overexpression in cerebrovascular endothelium of epileptic tissue reduce the brain uptake of AED, resulting in sub-therapeutic concentrations at their neuronal targets.

In this pilot study, we address the contribution of the multidrug transporter P-glycoprotein (P-gp) in pharmacoresistance to oxcarbazepine (OXCBZ). Steady state plasma and brain concentrations of 10-OHCBZ, the active metabolite of OXCBZ, were determined intraoperatively in 11 pharmacoresistant patients. We found that the brain concentrations of 10-OHCBZ did not reflect its plasma levels suggesting that this drug does not cross the blood-brain barrier by simple diffusion. In vitro uptake studies demonstrated that 10-OHCBZ acts as a substrate of P-gp. In addition, we showed that the brain concentrations of 10-OHCBZ were lower when the corresponding tissue level of P-gp were higher.

In summary, P-gp may play a role in pharmacoresistance to OXCBZ by determining the attainment of insufficient concentrations of its active metabolite at neuronal targets. Pharmacological blockade of multidrug transport proteins or the design of AED that do not act as substrates of these proteins may be envisaged as means for optimizing seizure control in drug-refractory epilepsy. Epilepsia 2005;46(10).

September 2005
Preclinical Profiling and Safety Studies of ABT-769: A Compound with Potential for Broad-Spectrum Antiepileptic Activity
William J. Giardina, Michael J. Dart, Richard R. Harris, Robert S. Bitner, Richard J. Radek, Gerard B. Fox, Sanjay R. Chemburkar, Kennan C. Marsh, Jeffrey F. Waring, Julia Y Hui, Jinhua Chen, Peter Curzon, George K. Grayson, Victoria A. Komater, Yiyin Ku, Mark Lockwood, Holly M. Miner, Arthur L. Nikkel, Jia Bao Pan, Yu-Ming Pu, Lei Wang, Youssef Bennani, Niklaus Durmuller, Robert Jolly, Sylvain Roux, James P. Sullivan, and Michael W. Decker

The search for new antiepileptic drugs is driven by the need for drugs with broad-spectrum efficacy and with safety and tolerability better than current agents. This study identified a new compound, ABT-769, that meets these criteria in animal models. In the mouse, ABT-769 protected against convulsive seizures induced by electroshock and by the chemical convulsant pentylenetetrazol. In the rat, ABT-769 suppressed focal seizures produced by repeated brain stimulation. ABT-769 also reduced absence-like seizures in a rat model of non-convulsive epilepsy, in which spontaneous, abnormal EEG patterns resemble those observed in human absence seizures and are not accompanied by overt convulsions. Thus, ABT-769 was effective against both convulsive and non-convulsive seizures in rodents. No tolerance was evident after repeated dosing. Moreover, ABT-769 did not impair learning and memory in the mouse at anticonvulsant doses. Similarly, the compound did not impair motor coordination at anticonvulsant doses in the mouse, although it did disrupt coordination at higher doses. ABT-769 did not alter the oxidation of fatty acids in mitochondria in rat liver, and it did not produce neural tube defects in mice exposed to the compound in utero. Thus, ABT-769 is a potent antiseizure agent in animal models of convulsive and nonconvulsive epilepsy and has a favorable safety profile. ABT-769 has an efficacy profile in these models like that of the broad-spectrum antiepileptic drug, valproic acid. In contrast, its profile in animal models is clearly different from those of several other antiepileptic drugs, including carbamazepine, phenytoin, lamotrigine, topiramate, vigabatrin and tiagabine. Epilepsia 2005;46(9).

Long-lasting Antiepileptic Effects of Levetiracetam Against Epileptic Seizures in the Spontaneously Epileptic Rat (SER): Differentiation of Levetiracetam from Conventional Antiepileptic Drugs

Cai Ji-qun, Kumatoshi Ishihara, Takashi Nagayama, Tadao Serikawa, and Masashi Sasa

Levetiracetam is a novel anti-epileptic drug with broad?@spectrum activity in several animal models mimicking both partial and generalized epilepsy. There is evidence to suggest that levetiracetam also possesses anti-epileptogenic properties and has a novel mechanism of action. The purpose of this study was to investigate the time-course of seizure protection by levetiracetam compared to that of phenytoin, phenobarbital, valproate and carbamazepine in the Spontaneously Epileptic Rat which is a double mutant (tm/tm, zi/zi) showing both tonic convulsions and absence-like seizures. Both seizure types were inhibited following single administration of levetiracetam at 80 and 160 mg/kg i.p., but not significantly at 40 mg/kg i.p.. The number of tonic convulsions and absence-like seizures were significantly reduced to 39.1 % and 38.4 % respectively compared with pre-values on the last day after 5 days of administration (80 mg/kg/day i.p.). Furthermore, significant inhibition of tonic convulsions and absence-like seizures were detected up to 3 days and 8 days, respectively, after the last administration of levetiracetam. These results demonstrates long-lasting seizure protection by levetiracetam after cessation of the treatment. Phenytoin, phenobarbital, valproate and carbamazepine inhibited tonic convulsions more potently by 5 day administration compared with levetiracetam in SER. However, long-lasting protection as with levetiracetam, was not observed with any of these drugs except for phenytoin and carbamazepine, both of which showed moderately prolonged anti-seizure effects. The long-lasting effect of levetiracetam suggests that the drug may possesses an anti-epileptogenic effect distinct from conventional anti-epileptic drugs. Epilepsia 2005;46(9).

Increased Expression of Ferritin, an Iron Storage Protein, in Specific Regions of the Parahippocampal Cortex of Epileptic Rats

Jan A. Gorter, Ana R.M. Mesquita, Erwin A. van Vliet, Fernando H. Lopes da Silva, and Eleonora Aronica

Iron accumulation in the brain has been associated with neurodegenerative disorders, including epilepsy. Ferritin, an iron storage protein, is one of the genes that show overexpression prior to the chronic epileptic phase. In this study we used ferritin as indicator for disturbed iron homeostasis in order to get insight whether this could play a role in the pathogenesis of temporal lobe epilepsy. Using immuno-cytochemistry, we studied the regional and cellular distribution of ferritin protein in an animal model for temporal lobe epilepsy in which spontaneous seizures develop a few weeks after electrically- induced status epilepticus (SE). Increased ferritin expression was observed in regions known to be vulnerable to cell death, mainly in reactive microglial cells of epileptic rats. Ferritin expression after SE was initially high, especially throughout the hippocampus, but decreased over time. In the chronic epileptic phase it was still upregulated in regions where extensive cell loss occurs during the early acute and latent period. Within the parahippocampal region, the most persistent ferritin overexpression was present in microglial cells in layer III of the medial entorhinal area. The upregulation was most extensive in rats that had developed a progressive form of epilepsy with frequent seizures (~5-10 seizures per day). The fact that ferritin upregulation is still present in specific limbic regions in chronic epileptic rats, when neuronal loss is absent or minimal, suggests a role of iron in the pathogenesis and progression of epilepsy. Epilepsia 2005;46(9).

Brainstem Seizure Severity Regulates Forebrain Seizure Expression in the Audiogenic Kindling Model

Michelle A. Merrill, Richard W. Clough, Phillip C. Jobe, and Ronald A. Browning

Generalized convulsive seizures can be clonic or tonic-clonic depending on whether they begin in the forebrain or brainstem, respectively. Sound-induced or “audiogenic” seizures in genetically susceptible rat strains originate from the brainstem. However, daily repetition of audiogenic seizures results in seizure spread into the forebrain and “kindling” of the forebrain seizure network. This results in the behavioral manifestations of forebrain-evoked convulsions (i.e. facial and forelimb clonus) and is referred to as “audiogenic kindling”. It has previously been shown that one substrain of Genetically Epilepsy-Prone Rat, the GEPR-9, that display the most severe audiogenic seizure (i.e., tonic extensor convulsions) in response to a loud sound, fails to display a forebrain convulsion following audiogenic kindling. The present study tested the hypothesis that the forebrain networks of GEPR-9s were, in fact, kindled to seize, but that the clonic convulsions are not be expressed due to continued brainstem activity that accompanies the more severe brainstem seizures (e.g. ongoing epileptic discharge). This hypothesis is supported by the present study, in which suppression of the brainstem seizure with either low dose phenytoin or lesions of the midbrain leads to the expression of forebrain convulsions in GEPR-9s after audiogenic kindling. Similarly, rats that display only moderate audiogenic seizures (GEPR-3s) and have distinct forebrain clonus when kindled, became resistant to forebrain clonus when the severity of their brainstem seizures is increased by pharmacological treatment. These findings show that brainstem seizure severity can modulate forebrain seizure expression. Epilepsia 2005;46(9).

Modulation of CaM Kinase II Activity is Coincident with Induction of SE in the Rat Pilocarpine Model

Michael W. Singleton, William H. Holbert II, Anh Tuyet Lee, James M. Bracey, and Severn B. Churn

Status epilepticus (SE), a life-threatening medical emergency, is defined as continuous seizure activity, or recurrent seizure activity without the patient regaining consciousness, for greater than 30 minutes. However, more recent reports state the importance of treating seizures early, within 5 minutes. This is important because as seizure activity progresses in SE, the ability to terminate seizure activity with current medications is lost. Therefore, understanding the early cellular effects of prolonged seizure activity is imperative to understanding the sequence of events through which SE patients lose the ability to respond to medical treatments.

The purpose of this study was to characterize the early SE-induced changes in CaM kinase II activity; a neuronally enriched enzyme that has been shown to modulate neuronal excitation. Seizure progression in the pilocarpine model of SE was characterized both behaviorally and electrographically to carefully assess seizure progression. At specific time points, brain tissue was harvested and tested for CaM kinase II activity, and protein expression levels in both whole tissue homogenates and in subcellular fractions.

The data demonstrated that inhibition of CaM kinase II activity was co-incident with the induction of SE. Examination of subcellular fractions demonstrated a significant increase in synaptic kinase activity, followed by a continual decline as SE progresses. The results suggest that modulation of CaM kinase II activity is involved in the early cellular responses to SE induction. In addition, the data suggests that CaM kinase II activity is differentially modulated in specific subcellular regions in both the hippocampus and cortex. Epilepsia 2005;46(9).

Consequences of the Chronic Caffeine Administration and its Withdrawal over Pilocarpine- and Kainate-induced Seizures in Rats

Marcelo Q. Hoexter, Pedro S. Rosa, Sergio Tufik, and Luiz E. Mello

o investigate the consequences of caffeine consumption over epileptic seizures we used the pilocarpine and the kainate models of epilepsy. We hypothesized that chronic caffeine consumption or its withdrawal would alter adenosine levels and hence alter seizure susceptibility. Chronic administration of a 0.1% caffeine solution on the drinking water of the adult, male Wistar rats over a 2 week period did not alter the threshold for the induction of seizures by a sub-convulsant dose of pilocarpine (200 mg/kg, i.p.) or kainic acid (8 mg/kg, i.p.). Similarly, challenging another group of animals with the same doses of pilocarpine or kainate 12 hs after the withdrawal of the same caffeine administration protocol did not lead to any significant changes in seizures. To investigate whether caffeine administration and withdrawal could influence the occurrence of chronically spontaneous rather than acutely induced seizures we used the pilocarpine model of epilepsy. Here again we were not able to find any significant difference in seizure profile that could stem from either caffeine administration or of its withdrawal. Despite the extensive laboratory evidence on the convulsant properties of xanthine derivatives in animal models of epilepsy such strong evidence is lacking in the clinical settings. Our current findings with the administration of caffeine at doses similar to those of daily life both support and confirm the clinical sense. Epilepsia 2005;46(9).

August 2005
Suppression of Kindled Seizures by Paracrine Adenosine Release from Stem Cell-derived Brain Implants
Martin Güttinger, Denise Fedele, Peter Koch, Vivianne Padrun, William F. Pralong, Oliver Brüstle, and Detlev Boison

Stem cells and their derivatives have emerged as a promising tool for cell-based drug delivery due to (i) their unique ability to differentiate into various somatic cell types, (ii) the virtually unlimited donor source for transplantation, and (iii) the advantage of being amenable to a wide spectrum of genetic manipulations. Embryonic stem (ES) cells have been engineered to release adenosine, an endogenous antiepileptic substance of the brain. This has been achieved by a genetic disruption of the gene coding for adenosine kinase (ADK), the major adenosine-removing enzyme. Lack of ADK did not compromise the potential of the cells to differentiate into specialized cell types. The aim of the present study was to investigate the potential of adenosine releasing ES cell-derived brain implants for seizure suppression. To isolate local adenosine-mediated effects from effects caused by integration of the transplanted cells into preexisting networks, ES cell-derived embryoid bodies and glial precursor cells were encapsulated into semipermeable polymer membranes and grafted into the lateral brain ventricle of kindled rats, a widely used model of temporal lobe epilepsy. While seizure activity in kindled rats with normal control implants remained unaltered, rats with adenosine-releasing ES cell-derived implants displayed transient protection from convulsive seizures and a profound reduction of seizure activity in recordings of electrical brain activity. We thereby provide a proof-of-principle that adenosine releasing ES cell-derived brain implants can suppress seizure activity by a local, paracrine mode of action. ADK-deficient stem cells therefore represent a potential tool for the treatment of epileptic disorders. Epilepsia 2005;46(8).

Separation of Anti-epileptogenic and Anti-seizure Effects of Levetiracetam in the Spontaneously Epileptic Rat

Hai-Dun Yan, Cai Ji-qun, Kumatoshi Ishihara, Takashi Nagayama, Tadao Serikawa, and Masashi Sasa

Levetiracetam is an anti-epileptic drug introduced in 2000. The long-lasting anti-seizure effects of levetiracetam have been observed in the Spontaneously Epileptic Rat (SER) that expresses both tonic and absence-like seizures. These effects may at least partly reflect its anti-epileptogenic effects. This study aimed to differentiate levetiracetam’s anti-seizure and potential anti-epileptogenic effects by administrating levetiracetam continuously to SER at 80 mg/kg/day (i.p.) prior to the appearance of any seizure (from postnatal 5th to 8th week). The period of observation for tonic and absence-like seizures was from the postnatal 5th to the 13th week, and week 12 and 13, respectively. Development of tonic convulsions was significantly inhibited in the levetiracetam-group, compared with the control-group by the middle of the 9th week. A significant reduction of tonic convulsions was also observed in the levetiracetam-group until the 13th week (5 weeks after termination of the administration). In the 12th week, the absence-like seizures were significantly lower in the levetiracetam-group, compared with the control-group. This study demonstrates a significant inhibition of seizures following chronic treatment with levetiracetam prior to the developmental expression of seizure activity in SERs. It is suggested that this effect is due to an anti-epileptogenic effect, not the anti-seizure effect of levetiracetam, since half of the drug disappears from blood every 2 to 3 hours after a single dose and termination of chronic administration. Furthermore, the inhibition of seizure expression in SERs was still apparent 5 weeks after termination of levetiracetam treatment. These results further suggest that levetiracetam possess not only anti-seizure effects but also anti-epileptogenic properties. Epilepsia 2005;46(8).

Time Course and Mapping of Cerebral Perfusion During Amygdala Secondary Generalized Seizures

Serge Chassagnon, Anne Pereira de Vasconcelos, Arielle Ferrandon, Estelle Koning, Christian Marescaux, and Astrid Nehlig

About 30% of focal epilepsies remain drug-resistant despite modern anti-epileptic drugs thus requiring surgery. Electrophysiological and neuroimaging methods contribute to the optimal definition of the epileptogenic zone before surgical removal. In humans, imaging of cerebral blood flow (CBF) during seizures is widely used to study the lateralization and location of the epileptic focus. Although it is advisable to inject the tracer as close as possible to seizure onset controlled by video-EEG monitoring, the influence of the tracer injection time on spatial distribution and extent of ictal CBF remains unclear. We used a rat model of temporal lobe epilepsy to follow seizure-induced, time-dependent changes in CBF. Rats were repetitively stimulated in the left amygdala until they experienced secondary generalized limbic (temporal lobe) seizures. CBF was measured by [14C]iodoantipyrine autoradiography bilaterally in 35 regions. The tracer was injected at 30 s before seizure induction (early ictal), at the time of stimulation (ictal), 60 s after stimulation (late ictal), at the end of the electrical afterdischarge (early postictal) and 6 min after stimulation (late postictal). This study shows a rapid spread of activation from the stimulated amygdala bilaterally to numerous limbic, cortical and subcortical structures. The unilateral site of origin of seizures led to minor asymmetrical and lateralized CBF changes, merely at early ictal and late postictal times, whereas intermediate tracer injections induced widespread bilateral changes. Only late postictal measurements allowed us to identify significant focal changes, namely in hippocampus, which is known to play a critical role in the spread of limbic seizures. Epilepsia 2005;46(8).

Increased High Frequency Oscillations Precede in vitro Low Mg 2+ Seizures

Houman Khosravani, C. Robert Pinnegar, J. Ross Mitchell, Berj L. Bardakjian, Paolo Federico, and Peter L. Carlen

Seizures are due to abnormal brain activity in the form of hypersynchronous, hyperexcitable discharges of many brain cells. One way in which epileptic seizure activity is monitored is through electroencephalographic (EEGs) recordings. Recently, fast brain wave activity (faster than 100 Hz) termed high frequency oscillations (HFOs) have been observed and characterized in EEGs from epileptic patients. HFOs have been observed in various experimental models of epilepsy and have been observed in interictal discharges (abnormal very brief bursts of seizure activity seen in between seizures that are not perceived by the patients themselves) in intracranial EEG recordings. HFOs are robust markers of epileptogenicity in brain circuits. Although their presence is established, their changes over time are not well understood. Given the relation between HFOs and seizures, we used an experimental rat seizure model to describe the evolution of HFOs over time leading up to a seizure. We observed a significant increasing trend for HFOs (100-300 Hz) leading up to seizure onset. Characterization of HFOs in humans may lead to better seizure localization and offer insight into being able to possibly anticipate a coming seizure so that it may be averted using available therapeutic modalities. Epilepsia 2005;46(8).

July 2005
Kynurenateand 7-Chlorokynurenate Formation in Chronically Epileptic Rats
Hui-Qiu Wu, Arash Rassoulpour, Jeffrey H. Goodman, Helen E. Scharfman, Edward H. Bertram, and Robert Schwarcz

Uncontrolled epilepsy remains a significant health concern and requires new approaches to therapy. One of the more popular current paths is to develop drugs that block the so-called NMDA (N-methyl-D-aspartate) receptor, a subtype of receptors for the major excitatory neurotransmitter glutamate. Unfortunately, conventional NMDA receptor antagonists have adverse cognitive and behavioral effects, which have prevented the development of clinically useful compounds. These side effects are greatly reduced when the glycine co-agonist (“glycineB“) site of the NMDA receptor is targeted. In the present study, the authors tested chronically epileptic rats for their ability to produce the potent and specific glycineB site antagonist 7-chlorokynurenic acid (7-Cl-KYNA) from its pro-drug 4-chlorokynurenine (4-Cl-KYN). In contrast to 7-Cl-KYNA, which has known anti-epileptic properties in experimental animals, 4-Cl-KYN enters the brain readily after peripheral administration. The pro-drug is then enzymatically converted in glial cells (astrocytes), which release 7-Cl-KYNA to inhibit NMDA receptor function. This approach is especially advantageous since astrocytes are selectively activated in limbic regions of the epileptic brain, i.e. in areas where NMDA receptor blockade is most desirable.

Using microdialysis, which allows continuous sampling of chemicals in the living animal, and brain tissue slices in vitro, the study demonstrates enhanced de novo formation of 7-Cl-KYNA from 4-Cl-KYN in the entorhinal cortex and the hippocampus, two brain areas that are known to play a central role in the pathophysiology of epilepsy, and traces this finding to activated astrocytes. These results bode well for the use of 4-Cl-KYN in the treatment of chronic seizure disorders. Epilepsia 2005;46(7).

Prolonged Infusion of Cycloheximide Does Not Block Mossy Fiber Sprouting in a Model of Temporal Lobe Epilepsy

Izumi Toyoda and Paul S. Buckmaster

Hippocampus is a temporal lobe structure that is involved in many people with complex partial epilepsy. One theory of how epilepsy develops after injury involves fibers from surviving neurons in the subregion called the dentate gyrus, to sprout extra connections to surviving neurons. This attempted repair process can in theory lead to positive feedback, excess excitation and resultant seizures. Therefore, in an animal model system of temporal lobe epilepsy, we blocked protein synthesis, which would be required to make the new connections. We implanted minipumps and cannulae in rats to deliver cycloheximide (a protein blocker) to one dentate gyrus and control vehicle to the other. After 2 days of infusion rats were treated with pilocarpine to induce status epilepticus. Pumps were removed 3 days later. Cycloheximide-treated hippocampi displayed staining for more new fibers, and more tissue damage around the infusion site than vehicle-treated hippocampi. Prolonged infusion of cycloheximide, spanning the period of pilocarpine treatment, did not block mossy fiber sprouting. This finding suggests that protein-dependent mechanisms around the time of an epileptogenic injury are not necessary for the eventual development of synaptic reorganization. Epilepsia 2005;46(7).

Volumetric Magnetic Resonance Imaging of Functionally Relevant Structural Alterations in Chronic Epilepsy after Pilocarpine-induced Status Epilepticus in Rats

Heiko G. Niessen, Frank Angenstein, Stefan Vielhaber, Christian Frisch, Alexei Kudin, Christian E. Elger, Hans-Jochen Heinze, Henning Scheich, and Wolfram S. Kunz

To study the development and a potential treatment of human temporal lobe epilepsy (TLE) in greater detail, several animal models were generated in recent years. For our study, we used the drug pilocarpine to evoke epileptic seizures in rats. In this model, the seizure activity observed in humans is represented by the rat’s later phase of chronic epilepsy. To what extent brain damage was induced by epileptic seizures and whether structural damage implies functional impairment (e.g., in cognitive function) are important questions that need to be answered. Using non-invasive magnetic resonance imaging (MRI), we evaluated structural alterations in various parts of the rat brain, e.g., hippocampus and cortex, during the chronic phase of epilepsy. Our analysis showed epilepsy-induced changes in the size of hippocampus, putamen, cortex, thalamus, and the combined area of perirhinal, entorhinal, and piriform cortices. Furthermore, compared with controls, pilocarpine-treated animals performed significantly worse in the Morris water maze (a standard test for spatial learning) with a highly negative correlation between hippocampal damage and their performance. This observation indicates that even a damaged and reduced hippocampus is still involved in memory formation in epileptic animals. Finally, our study also demonstrates that non-invasive MRI can quickly and efficiently quantify the degree of damage in various brain structures. Since the progression of epileptic damage in functionally relevant structures of the brain can be monitored over time, this method may also allow for the validation of therapeutic approaches. Epilepsia 2005;46(7).

Morphological and Neurochemical Abnormalities in the Auditory Brainstem of the Genetically Epilepsy-prone Hamster (GPG/Vall)

Verónica Fuentes-Santamaría, Raquel Cantos, Juan Carlos Alvarado, Natividad García-Atarés, and Dolores E. López

Epilepsy is a common neurological disorder that affects about 50 million people in the world. Anatomical alterations of different brain structures, as well as disturbances of calcium regulatory mechanisms as a consequence of recurrent seizures, have been shown to affect neuronal viability and excitability in different models of epilepsy. Animal models of inherited epilepsy are essential to understand the origin of epileptic seizures, because they represent an abnormal nervous system and reproduce the mechanisms of hyperexcitability that occur in nature. The present study was performed to evaluate morphological and biochemical alterations in the auditory system of the genetically epilepsy-prone hamster (GPG/Vall). This strain of hamsters suffers from generalized tonic-clonic seizures that are triggered by exposure to intense auditory stimulation. Our results demonstrate morphological alterations in the cochlea as well as a decrease in both the volume and neuronal size of different auditory nuclei. These alterations are also accompanied by an increase in levels of parvalbumin, a calcium-binding protein that has been suggested to help regulate intracellular calcium concentration within neurons in the central nervous system. In conclusion, the present data are consistent with a series of atrophic changes along the auditory system in an animal model of inherited epilepsy. In addition, they show an increase in parvalbumin immunostaining that may reflect a protective mechanism to prevent cell death in auditory nuclei. Epilepsia 2005;46(7).

June 2005
Anticonvulsant Properties of the Novel Nootropic Agent Nefiracetam in Seizure Models of Mice and Rats
Yutaka Kitano, Chika Komiyama, Mitsuhiro Makino, Kiyoshi Takasuna, Akira Takazawa, and Shinobu Sakurada

Nefiracetam is a possible antiepileptic drug that may possess cognition-enhancing effects. It is structurally similar to the existing antiepileptic drug, levetiracetam (Keppra). In the present study, we investigated the anticonvulsant profile of nefiracetam in experimental seizure models of mice and rats, compared with levetiracetamand other standard antiepileptic drugs (zonisamide, phenytoin, carbamazepine, valproic acid, diazepam and ethosuximide). With reference to standard programs for evaluating potential antiepileptic drugs, the study included the traditional maximal electroshock seizure and subcutaneous chemoconvulsant (pentylenetetrazole, bicuculline, picrotoxin, strychnine or N-methyl-D-aspartate) seizure tests and two threshold models (the increasing-current electroshock seizure test and intravenous pentylenetetrazole seizure threshold test). Neurotoxic activities were examined by rotorod test (ability of the rat to balance on a rotating rod) and traction test. Nefiracetam inhibited electroshock-induced seizures at non-toxic doses, while it had no effect on seizures chemically induced by pentylenetetrazole, bicuculline, picrotoxin, strychnine or N-methyl-D-aspartate. The anticonvulsant spectrum of nefiracetam paralleled that of zonisamide, phenytoin and carbamazepine. The anticonvulsant efficacy of nefiracetam was comparable with that of zonisamide and less potent than that of phenytoin, carbamazepine and diazepam. However, the safety margin of nefiracetam was superior to that of zonisamide, carbamazepine, valproic acid and diazepam. Levetiracetam showed only slight anticonvulsant effects in threshold models and it was not effective in conventional screening models. These results suggest that nefiracetam has distinct anticonvulsant spectrum and mechanisms from those of levetiracetam in spite of the structural similarity. Nefiracetam presents an orally active and safe anticonvulsant drug and it possesses a potential for antiepileptic therapy. Epilepsia 2005;46(6).

Anticonvulsant Activity of Androsterone and Etiocholanolone

Rafal M. Kaminski, Herbert Marini, Won-Joo Kim, and Michael A. Rogawski

Men with epilepsy often have impaired sexual function and loss of libido associated with reduced levels of androgens, including testosterone, the principal male sex hormone. The deficiency of androgens is believed to be an unwanted side effect of antiepileptic medications and also may result from suppression by recurrent seizures of the hormonal system that controls androgen production in the testes. The major breakdown products (“metabolites”) of testosterone in the body are androsterone and etiocholanolone. These substances have been assumed to be largely inactive by-products formed from testosterone to facilitate its excretion in the urine. In this study, we found that androsterone and etiocholanolone can prevent the occurrence of epileptic seizures. The ability of chemical substances to protect against seizures can be assessed in animal models in which seizures are induced by chemicals (“chemoconvulsants”) or by electrical stimulation. In addition, anticonvulsant substances can be evaluated in brain slice preparations exposed to chemoconvulsants; effective anticonvulsants suppress seizure-like discharges recorded with extracellular microelectrodes. We found that androsterone injections protected mice from seizures induced by various chemoconvulsants and by electrical stimulation. Etiocholanolone was also protective, but higher doses were required. The two steroids also suppressed seizure-like discharges in slices of the rat hippocampus. Our results lead to the hypothesis that men suffering from epilepsy who have low testosterone levels may experience poor seizure control as a result of reduced amounts of the anticonvulsant testosterone metabolites androsterone and etiocholanolone. Uncontrolled seizures may set in motion a vicious cycle whereby testosterone and its anticonvulsant metabolites are suppressed, leading to worsening seizures. Epilepsia 2005;46(6).

In Vivo Modulatory Action of Extracellular Glutamate on the Anticonvulsant Effects of Hippocampal Dopamine and Serotonin

Ralph Clinckers, Sarah Gheuens, Ilse Smolders, Alfred Meurs, Guy Ebinger, and Yvette Michotte

Hippocampus, a sub-region of the temporal lobe, is a key brain structure for production of seizures. Seizures in hippocampus alter brain neurotransmitters, including dopamine and serotonin. Our recent work (Clinckers et al., J. Neurochem. 2004; 89:834-43) demonstrated that the elevation of hippocampal dopamine or serotonin levels offered complete protection against focal hippocampal seizures induced by the convulsant drug, pilocarpine. These seizures did not influence hippocampal levels of glutamate, the most important excitatory neurotransmitter in brain. However, at high concentrations of dopamine and serotonin the anticonvulsant effects were gone and seizures even could be worse than with baseline levels of dopamine and serotonin. This pro-convulsant effect always was accompanied by significant glutamate increases in tissue of hippocampus. In the present study, the role of hippocampal glutamate levels in seizures either made better or worse by dopamine and serotonin was investigated. Co-administration of anticonvulsant dopamine and serotonin with glutamate, prior to pilocarpine perfusion, abolished all anticonvulsant effects. Glutamate addition after pilocarpine administration did not affect seizure protection by dopamine or serotonin. No convulsions wore observed during glutamate administration alone. These results indicate that increases of extracellular glutamate concentrations do not necessarily induce seizures in themselves, but can produce seizures by modulating the anticonvulsant effects exerted by dopamine and serotonin in hippocampus. Epilepsia 2005;46(6).

Acute Effects of Kainic Acid on CA1 Hippocampal Interneurons Differentially Vulnerable to Excitotoxicity

Nathalie Sanon, Lionel Carmant, Martine Emond, Patrice Congar, and JeanClaude Lacaille

Kainic acid (KA, a neurotoxin) is commonly used as an experimental model of temporal lobe epilepsy (TLE), the most common form of epilepsy that is difficult to control with medicines. KA injections in rats mimic the clinical features and pathological findings of human TLE. This includes a selective vulnerability of interneurons in a region of the hippocampus important for the control of information outflow: the CA1 oriens?alveus layers. This selective vulnerability is present in both the mature animal but also in the immature animal, which does not exhibit the extensive hippocampal cell loss of the mature animal. We wanted to define the neurochemical subgroups of vulnerable interneurons and to investigate if this selective vulnerability was due to a direct toxic effect of KA. Our results showed that specific subgroups of interneurons in the vulnerable region are decreased in our model, but that the acute effects of KA are similar on interneurons located in the vulnerable versus toxicity-resistant regions. We conclude that the selective vulnerability of oriens?alveus interneurons is mediated through different glutamate receptor subtypes on different classes of interneurons. One type called the metabotropic glutamate receptor, may be especially important. Epilepsia 2005;46(6).

Localisation of Breast Cancer Resistance Protein (BCRP) in Micro-vessel Endothelium of Human Control and Epileptic Brain

Eleonora Aronica, Jan A. Gorter, Sandra Redeker, Erwin A. van Vliet, Marja Ramkema, George L. Scheffer, Richerd J Scheper, Paul van der Valk, Siger Leenstra, J.C. Baayen, Wim G.M. Spliet, and Dirk Troost

Resistance to pharmacological treatment with a broad range of anti-epileptic drugs is a crucial clinical problem in human epilepsy. Past work has identified defects in a family of proteins, whose job is to transport a wide range of drugs across cell membranes as possibly being involved in resistance to antiepileptic drugs. Excessive amounts of multi-drug transporter proteins in capillary endothelium, which represents the selective cellular barrier controlling drug delivery into the brain, has been proposed as a major mechanism responsible for drug resistance in epilepsy. Breast cancer resistance protein (BCRP) is one the group of multidrug resistant related proteins. In order to investigate the cellular distribution and levels of BCRP in pathological brain tissue, we applied immune-staining procedures to a large number of neoplastic and non-neoplastic brain tissue specimens removed during surgery on patients with uncontrolled epilepsy. Using protein measurements, we could demonstrate the presence of BCRP in both normal and epileptic human brain tissue. In contrast to two other drug transporters, P-glycoprotein and multidrug resistance-associated protein 2, BCRP expression levels did not change in tissue from patients with hippocampal epilepsy, compared to normal control hippocampus (surgically removed for reasons other than epilepsy). No BCRP immunoreactivity was observed in glial (the brain’s supporting cells) or neuronal cells. However, BCRP levels were increased at the inner cell surface of newly formed capillaries in tumor brain tissue. The higher drug transporter levels observed in astrocytoma tumors was related to the higher blood vessel density within the tumor tissue. These results suggest that the strong BCRP expression in the microvasculature of epileptogenic brain tumors could critically influence the bioavailability of drugs within the tumor and thereby contribute to drug resistance. Epilepsia 2005;46(6).

Spontaneous Epileptic Rats Show Changes in Sleep Architecture and Hypothalamic Pathology

Jesper F. Bastlund, Poul Jennum, Paul Mohapel, Silke Penschuck, and William P. Watson

In many patients with epilepsy, the occurrence of seizures is strongly influenced by the sleep-wake cycle. Sleep disturbances are also common among people with epilepsy. Seizures can affect the objective and subjective quality of sleep. The goal of the present study was to investigate the relation between seizures and sleep regulation in spontaneous epileptic rats, an experimental model of temporal lobe epilepsy (TLE). Furthermore, we investigated whether epileptic rats lost neuronal cells in the hypothalamus, a brain region important for normal sleep regulation. We demonstrated that spontaneous epileptic rats showed clinical features of TLE, such as spontaneous seizures and neuronal cell loss. Interestingly, epileptic rats showed disturbances in sleep architecture with a high percentage of the seizures occurring during sleep. Neuronal cell loss in the hypothalamus might contribute to these disturbances. We conclude that there is a close relation between seizures and alterations in sleep architecture in epileptic rats. Changes in sleep architecture as a result of chronic epilepsy or treatment with antiepileptic drugs may therefore contribute to the intractability of seizures in patients, thereby worsening the epileptic condition. A better understanding of the relationship between the sleep-wake cycle and epilepsy, resulting from further study, may ultimately improve the treatment of patients. Epilepsia 2005;46(6).

igabatrin in Low Doses Selectively Suppresses the Clonic Component of Audiogenically Kindled Seizures in Rats

Ludmilla V. Vinogradova, Galina D. Kuznetsova, A.B. Shatskova, and Clementina M. van Rijn

In this study we investigated the effect of the anti-epileptic drug vigabatrin (trade name Sabril) on sound-induced seizures in an animal model for epilepsy. The rats studied, get seizures when they are exposed to a sudden intense sound stimulus: a loud, high-pitched sound (60 dB, 10-80 kHz), generated by rattling a bunch of keys. The first time the animals hear it, they react by having a tonic (stiffening) spasm, which is often preceded by a short phase of wild running. But if the stimulus is applied repeatedly, eventually a third behavioral component arises: clonic convulsions, consisting of rhythmical jerking of the limbs. Vigabatrin is an anti-epileptic drug that increases the concentration of GABA, which is the most important inhibitory neurotransmitter in the brain. We found that vigabatrin did not change the latency or the duration of the running and tonic seizures, but that it did suppress the clonic convulsions in all the rat strains we used. We hypothesize that this selective anticonvulsive effect of vigabatrin results from different sensitivities of brainstem and forebrain epileptic networks to the GABA enhancement. Our research shows that it is possible to suppress just a single component in a complex of epileptic behavioral phenomena and we hope that this finding may contribute to a rational choice of anti-epileptic drugs in the clinic. Epilepsia 2005;46(6).

May 2005
Time-dependent Effect of Kainate-induced Seizures on Glutamate Receptor GLUR5, GLUR6 and GLUR7 mRNA and Protein Expression in Rat Hippocampus
Gautam Ullal, Margaret Fahnestock, and Ronald Racine

Neurons communicate with each other via chemical neurotransmitters that react with protein receptors. The most common of the excitatory neurotransmitters is glutamate, which reacts with a variety of different receptor subtypes. One of these subtypes (called the kainate receptor, because it responds to the excitatory drug, kainate), itself comes in at least 3 different forms (referred to as glutamate receptor (or GluR) 5, 6 and 7). It has been suggested that GluR 5 and GluR 6 play an important role in human epilepsy. The role of GluR7 in epilepsy is not yet understood. In this experiment, we monitored the messenger ribonucleic acid (mRNA) and protein expression of GluR5, GluR6 and GluR7 in rat hippocampus, a structure often implicated in epileptic activity, 72 hours, 90 days and 180 days following a strong and prolonged seizure induced by kainate injections. An increase in GluR5 expression, which may render the system more excitable, was seen at 72 hours and remained elevated until 180 days, so the change is very long lasting. The GluR7 measures showed only a decrease at 90 days following seizures. There was no change in the GluR6 measures at any of the time points following the seizure. These results suggest a rather complex array of seizure-induced changes in these receptor subtypes, but the increase in GluR5 expression may be related to a long-lasting increase in seizure susceptibility that develops following an initial, prolonged seizure. Epilepsia 2005;46(5).

April 2005
Electrical and Chemical Long-term Depression Do Not Attenuate Low-Mg 2+induced Epileptiform Activity in the Entorhinal Cortex
Jörg Solger, Uwe Heinemann, and Joachim Behr

Electrical and magnetic stimulation at low (slow) frequencies of brain cortex has been shown to reduce susceptibility to seizures in laboratory models of epilepsy and in humans. A phenomenon called long-term depression (LTD) of electrical activity may be part of the underlying mechanism. We investigated the effect of LTD in laboratory rats and mice on seizure-like activity produced by alteration of the ions or chemicals bathing the brain slices. Rats and mice were sacrificed painlessly, and testing was done on small brain slices taken from seizure-prone regions of brain. We found that LTD did not prevent the seizure-like activity, and therefore probably is not the mechanism for the effect of low-frequency stimulation on the tendency to have seizures. Other possible mechanisms should be investigated. Epilepsia 2005;46(4).

Hippocampal Melatonin Receptors Modulate Seizure Threshold

Lee S. Stewart and L. Stan Leung

The hormone melatonin has been reported to exhibit anti-epileptic properties in clinical trials. However, recent animal studies have demonstrated that melatonin can have opposite effects on brain function, depending on the dose and timing of melatonin administration. In other words, while high pharmacological doses are able to decrease brain excitability and suppress seizures, smaller doses of melatonin (administered at night when melatonin levels in the brain are highest) similar in amount to what is produced by the brain can actually increase the excitability of neurons making them more susceptible to seizure activity. In the present study, we used an animal model of epilepsy to study the effects of melatonin on seizure development. We made two important observations: 1) seizures induced by the drug pilocarpine occurred with a shorter latency at night (when brain melatonin levels are highest) than during the day, and 2) when small doses of drug that block melatonin receptors are injected directly into the hippocampus, an area of the brain important for the development and spread of seizures, then seizures during the night were delayed. Furthermore, this effect was reversed by a drug that blocks the activity of GABA, the major inhibitory neurotransmitter in the brain, suggesting that melatonin may decrease GABA receptor function in the hippocampus. Although we did not study the effects of melatonin directly, our data suggest that endogenous melatonin may enhance brain excitability and contribute to the development of epileptic seizures. This process may be involved with certain forms of nocturnal epilepsy, and may raise a caution for persons with epilepsy who take melatonin. Epilepsia 2005;46(4).