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1.  Sequel of spontaneous seizures after kainic acid-induced status epilepticus and associated neuropathological changes in the subiculum and entorhinal cortex 
Neuropharmacology  2012;63(5):806-817.
Injection of the seaweed toxin kainic acid (KA) in rats induces a severe status epilepticus initiating complex neuropathological changes in limbic brain areas and subsequently spontaneous recurrent seizures. Although neuropathological changes have been intensively investigated in the hippocampus proper and the dentate gyrus in various seizure models, much less is known about changes in parahippocampal areas. We now established telemetric EEG recordings combined with continuous video monitoring to characterize the development of spontaneous seizures after KA-induced status epilepticus, and investigated associated neurodegenerative changes, astrocyte and microglia proliferation in the subiculum and other parahippocampal brain areas. The onset of spontaneous seizures was heterogeneous, with an average latency of 15 ± 1.4 days (range 3–36 days) to the initial status epilepticus. The frequency of late spontaneous seizures was higher in rats in which the initial status epilepticus was recurrent after its interruption with diazepam compared to rats in which this treatment was more efficient. Seizure-induced neuropathological changes were assessed in the subiculum by losses in NeuN-positive neurons and by Fluoro-Jade C staining of degenerating neurons. Neuronal loss was already prominent 24 h after KA injection and only modestly progressed at the later intervals. It was most severe in the proximal subiculum and in layer III of the medial entorhinal cortex and distinct Fluoro-Jade C labeling was observed there in 75% of rats even after 3 months. Glutamatergic neurons, labeled by in situ hybridization for the vesicular glutamate transporter 1 followed a similar pattern of cell losses, except for the medial entorhinal cortex and the proximal subiculum that appeared more vulnerable. Glutamate decarboxylase65 (GAD65) mRNA expressing neurons were generally less vulnerable than glutamate neurons. Reactive astrocytes and microglia were present after 24 h, however, became prominent only after 8 days and remained high after 30 days. In the proximal subiculum, parasubiculum and entorhinal cortex the number of microglia cells was highest after 30 days. Although numbers of reactive astrocytes and microglia were reduced again after 3 months, they were still present in most rats. The time course of astrocyte and microglia proliferation parallels that of epileptogenesis.
► The onset of spontaneous seizures was highly variable (3–36 days) with an average latency of 15 days. ► Massive neurodegeneration was already present 24 h after KA-induced seizures. ► Neurons of the proximal subiculum and EC layer III preferentially degenerate. ► Distribution of reactive gliosis roughly matches the pattern of neurodegeneration. ► Time course of reactive gliosis parallels that of epileptogenesis.
PMCID: PMC3409872  PMID: 22722023
Temporal lobe epilepsy; Subiculum; Entorhinal cortex; Animal epilepsy model; EEG; Epileptogenesis; DG, dentate gyrus; FJ-C, Fluoro Jade C; GAD65 and GAD67, glutamate decarboxylase 65 and 67; GFAP, glial fibrillary acidic protein; KA, kainic acid; NeuN, neuron-specific nuclear protein; SE, status epilepticus; Sub, subiculum; TLE, temporal lobe epilepsy; VGLUT1, vesicular glutamate transporter 1
2.  Chronic Cellular Hyperexcitability in Elderly Epileptic Rats with Spontaneous Seizures Induced by Kainic Acid Status Epilepticus while Young Adults 
Aging and Disease  2011;2(4):332-338.
Emerging data indicate that age-related brain changes alter seizure susceptibility, seizure-associated neurodegeneration, and responsiveness to AEDs. The present study assessed long-term animal survival in the Kainic Acid (KA) model along with in-vivo spontaneous seizure frequency, cellular hyperexcitability in CA1 in-vitro and in-vivo in subiculum, and responsiveness of in-vitro CA1 hyperexcitability to topiramate. Sprague-Dawley male rats were given KA to induce convulsive status epilepticus (KA-SE) at 2–3 months of age. The one-month mortality after KA-SE was 27%. One-month survivor rats had 37% sudden unexplained late mortality after KA-SE as compared to none in saline controls during their second year of life. In-vivo seizure frequency was examined prior to terminal experiments. The diurnal average seizure frequency in the KA-SE group at age 2 years was 1.06 ± 0.24 seizures/hour while no seizures were observed in the saline age-matched controls (p<0.001). In-vitro recordings of CA1 pyramidal neurons revealed that depolarizing current injection from −60 mV evoked an increased number of action potentials in the aged KA-SE group compared to controls (p<0.002). Topiramate exhibited dose-dependent inhibition of action potential firing evoked by current injections into CA1 pyramidal neurons of KA-SE rats. In subiculum, KA-SE rats had frequent interictal spikes associated with high frequency oscillations while only rare spontaneous EPSPs were recorded in saline controls. Our experiments revealed that the hippocampal formation of aged epileptic rats shares features of hyperexcitability previously described in young adult epileptic rats using the KA model.
PMCID: PMC3295074  PMID: 22396885
Topiramate; Aged; Epilepsy; Bursting; Hippocampal slice; SUDEP
3.  Sequel of spontaneous seizures after kainic acid-induced status epilepticus and associated neuropathological changes in the subiculum and entorhinal cortex 
Neuropharmacology  2012;63(5):806-817.
Injection of the seaweed toxin kainic acid (KA) in rats induces a severe status epilepticus initiating complex neuropathological changes in limbic brain areas and subsequently spontaneous recurrent seizures. Although neuropathological changes have been intensively investigated in the hippocampus proper and the dentate gyrus in various seizure models, much less is known about changes in parahippocampal areas. We now established telemetric EEG recordings combined with continuous video monitoring to characterize the development of spontaneous seizures after KA-induced status epilepticus, and investigated associated neurodegenerative changes, astrocyte and microglia proliferation in the subiculum and other parahippocampal brain areas. The onset of spontaneous seizures was heterogeneous, with an average latency of 15 ± 1.4 days (range 3–36 days) to the initial status epilepticus. The frequency of late spontaneous seizures was higher in rats in which the initial status epilepticus was recurrent after its interruption with diazepam compared to rats in which this treatment was more efficient. Seizure-induced neuropathological changes were assessed in the subiculum by losses in NeuN-positive neurons and by Fluoro-Jade C staining of degenerating neurons. Neuronal loss was already prominent 24 h after KA injection and only modestly progressed at the later intervals. It was most severe in the proximal subiculum and in layer III of the medial entorhinal cortex and distinct Fluoro-Jade C labeling was observed there in 75% of rats even after 3 months. Glutamatergic neurons, labeled by in situ hybridization for the vesicular glutamate transporter 1 followed a similar pattern of cell losses, except for the medial entorhinal cortex and the proximal subiculum that appeared more vulnerable. Glutamate decarboxylase65 (GAD65) mRNA expressing neurons were generally less vulnerable than glutamate neurons. Reactive astrocytes and microglia were present after 24 h, however, became prominent only after 8 days and remained high after 30 days. In the proximal subiculum, parasubiculum and entorhinal cortex the number of microglia cells was highest after 30 days. Although numbers of reactive astrocytes and microglia were reduced again after 3 months, they were still present in most rats. The time course of astrocyte and microglia proliferation parallels that of epileptogenesis.
PMCID: PMC3409872  PMID: 22722023
Temporal lobe epilepsy; Subiculum; Entorhinal cortex; Animal epilepsy model; EEG; Epileptogenesis
4.  Development of later life spontaneous seizures in a rodent model of hypoxia induced neonatal seizures 
Epilepsia  2011;52(4):753-765.
To study the development of epilepsy following hypoxia-induced neonatal seizures in Long Evans rats and to establish the presence of spontaneous seizures in this model of early life seizures.
Long-Evans rat pups were subjected to hypoxia-induced neonatal seizures at postnatal day 10 (P10). Epidural cortical electroencephalography (EEG) and hippocampal depth electrodes were used to detect the presence of seizures in later adulthood (>P60). In addition, subdermal wire electrode recordings were used to monitor age at onset and progression of seizures in the juvenile period, at intervals between P10–P60. Timm staining was performed to evaluate mossy fiber sprouting in the hippocampi of P100 adult rats that had experienced neonatal seizures.
Key Findings
In recordings made from adult rats (P60–P180), the prevalence of epilepsy in cortical and hippocampal EEG recordings was 94.4% following early life hypoxic seizures. These spontaneous seizures were identified by characteristic spike and wave activity on EEG accompanied by behavioral arrest and facial automatisms (electroclinical seizures). Phenobarbital injection transiently abolished spontaneous seizures. EEG in the juvenile period (P10–60) showed that spontaneous seizures first occurred approximately 2 weeks after the initial episode of hypoxic seizures. Following this period, spontaneous seizure frequency and duration progressively increased with time. Furthermore, significantly increased sprouting of mossy fibers was observed in the CA3 pyramidal cell layer of the hippocampus in adult animals following hypoxia-induced neonatal seizures. Notably, Fluoro-Jade B staining confirmed that hypoxic seizures at P10 did not induce acute neuronal death.
The rodent model of hypoxia-induced neonatal seizures leads to the development of epilepsy in later life, accompanied by increased mossy fiber sprouting. In addition, this model appears to exhibit a seizure-free latent period, following which there is a progressive increase in the frequency of electroclinical seizures.
PMCID: PMC3071424  PMID: 21366558
Neonatal Seizures; electroencephalogram; epilepsy; infant; animal model
5.  Age-Dependent Changes in Intrinsic Neuronal Excitability in Subiculum after Status Epilepticus 
PLoS ONE  2015;10(3):e0119411.
Kainic acid-induced status epilepticus (KA-SE) in mature rats results in the development of spontaneous recurrent seizures and a pattern of cell death resembling hippocampal sclerosis in patients with temporal lobe epilepsy. In contrast, KA-SE in young animals before postnatal day (P) 18 is less likely to cause cell death or epilepsy. To investigate whether changes in neuronal excitability occur in the subiculum after KA-SE, we examined the age-dependent effects of SE on the bursting neurons of subiculum, the major output region of the hippocampus. Patch-clamp recordings were used to monitor bursting in pyramidal neurons in the subiculum of rat hippocampal slices. Neurons were studied either one or 2-3 weeks following injection of KA or saline (control) in immature (P15) or more mature (P30) rats, which differ in their sensitivity to KA as well as the long-term sequelae of the KA-SE. A significantly greater proportion of subicular pyramidal neurons from P15 rats were strong-bursting neurons and showed increased frequency-dependent bursting compared to P30 animals. Frequency-dependent burst firing was enhanced in P30, but not in P15 rats following KA-SE. The enhancement of bursting induced by KA-SE in more mature rats suggests that the frequency-dependent limitation of repetitive burst firing, which normally occurs in the subiculum, is compromised following SE. These changes could facilitate the initiation of spontaneous recurrent seizures or their spread from the hippocampus to other parts of the brain.
PMCID: PMC4361192  PMID: 25775210
6.  Parvalbumin interneurons and calretinin fibers arising from the thalamic nucleus reuniens degenerate in the subiculum after kainic acid-induced seizures 
Neuroscience  2011;189(1-2):316-329.
The subiculum is the major output area of the hippocampus. It is closely interconnected with the entorhinal cortex and other parahippocampal areas. In animal models of temporal lobe epilepsy (TLE) and in TLE patients it exerts increased network excitability and may crucially contribute to the propagation of limbic seizures. Using immunohistochemistry and in situ-hybridization we now investigated neuropathological changes affecting parvalbumin and calretinin containing neurons in the subiculum and other parahippocampal areas after kainic acid-induced status epilepticus. We observed prominent losses in parvalbumin containing interneurons in the subiculum and entorhinal cortex, and in the principal cell layers of the pre- and parasubiculum. Degeneration of parvalbumin-positive neurons was associated with significant precipitation of parvalbumin-immunoreactive debris 24 h after kainic acid injection. In the subiculum the superficial portion of the pyramidal cell layer was more severely affected than its deep part. In the entorhinal cortex, the deep layers were more severely affected than the superficial ones. The decrease in number of parvalbumin-positive neurons in the subiculum and entorhinal cortex correlated with the number of spontaneous seizures subsequently experienced by the rats. The loss of parvalbumin neurons thus may contribute to the development of spontaneous seizures. On the other hand, surviving parvalbumin neurons revealed markedly increased expression of parvalbumin mRNA notably in the pyramidal cell layer of the subiculum and in all layers of the entorhinal cortex. This indicates increased activity of these neurons aiming to compensate for the partial loss of this functionally important neuron population. Furthermore, calretinin-positive fibers terminating in the molecular layer of the subiculum, in sector CA1 of the hippocampus proper and in the entorhinal cortex degenerated together with their presumed perikarya in the thalamic nucleus reuniens. In addition, a significant loss of calretinin containing interneurons was observed in the subiculum. Notably, the loss in parvalbumin positive neurons in the subiculum equaled that in human TLE. It may result in marked impairment of feed-forward inhibition of the temporo-ammonic pathway and may significantly contribute to epileptogenesis. Similarly, the loss of calretinin-positive fiber tracts originating from the nucleus reuniens thalami significantly contributes to the rearrangement of neuronal circuitries in the subiculum and entorhinal cortex during epileptogenesis.
Graphical Abstract
▶A subpopulation of PV neurons degenerates in subiculum and entorhinal cortex after KA seizures. ▶Surviving PV neurons exhibit increased PV mRNA expression. ▶The loss in PV neurons in subiculum and entorhinal cortex correlates to spontaneous seizures. ▶Degeneration of PV neurons in the subiculum may be related to seizure-induced loss of feed-forward inhibition. ▶CR-ir neurons in the N. reuniens thalami and their projections to the subiculum degenerate.
PMCID: PMC3152681  PMID: 21616128
status epilepticus; temporal lobe epilepsy; epileptogenesis; entorhinal cortex; epilepsy models; CR, calretinin; EC, entorhinal cortex; -ir, immunoreactive; KA, kainic acid; NeuN, neuron specific nuclear protein; O-LM, oriens-lacunosum moleculare; PV, parvalbumin; ROD, relative optical densities; SE, status epilepticus; TBS, tris-buffered saline; TLE, temporal lobe epilepsy
7.  Long-lasting pro-ictogenic effects induced in vivo by rat brain exposure to serum albumin in the absence of concomitant pathology 
Epilepsia  2012;53(11):1887-1897.
Dysfunction of the blood–brain barrier (BBB) is a common finding during seizures or following epileptogenic brain injuries, and experimentally induced BBB opening promotes seizures both in naive and epileptic animals. Brain albumin extravasation was reported to promote hyperexcitability by inducing astrocytes dysfunction. To provide in vivo evidence for a direct role of extravasated serum albumin in seizures independently on the pathologic context, we did the following: (1) quantified the amount of serum albumin extravasated in the rat brain parenchyma during status epilepticus (SE); (2) reproduced a similar concentration in the hippocampus by intracerebroventricular (i.c.v.) albumin injection in naive rats; (3) measured electroencephalography (EEG) activity in these rats, their susceptibility to kainic acid (KA)–induced seizures, and their hippocampal afterdischarge threshold (ADT).
Brain albumin concentration was measured in the rat hippocampus and other forebrain regions 2 and 24 h after SE by western blot analysis. Brain distribution of serum albumin or fluorescein isothiocyanate (FITC)-albumin was studied by immunohistochemistry and immunofluorescence, respectively. Naive rats were injected with rat albumin or FITC-albumin, i.c.v., to mimic the brain concentration attained after SE, or with dextran used as control. Inflammation was evaluated by immunohistochemistry by measuring glial induction of interleukin (IL)-1β. Western blot analysis was used to measure inward rectifying potassium channel subunit Kir4.1 protein levels in the hippocampus. Seizures were induced in rats by intrahippocampal injection of 80 ng KA and quantified by EEG analysis, 2 or 24 h after rat albumin or dextran administration. ADT was measured by electrical stimulation of the hippocampus 3 months after albumin injection. In these rats, EEG was continuously monitored for 2 weeks to search for spontaneous seizures.
Key Findings
The hippocampal serum albumin concentration 24 h post-SE was 0.76 ± 0.21 μm. Similar concentrations were measured in other forebrain regions, whereas no changes were found in cerebellum. The hippocampal albumin concentration was similarly reproduced in naive rats by i.c.v. administration of 500 μg/4 μl rat albumin: albumin was predominantly detected extracellularly 2 h after injection, whereas at 24 h it was visible inside pyramidal neurons and in only a few scattered chondroitin sulphate proteoglycan (NG2)-positive cells, but not in glial fibrillary acidic protein (GFAP)-positive astrocytes or CR-3 complement receptor (OX-42)-positive microglia. The presence of albumin in naive rat hippocampus was associated with induced IL-1β in GFAP-positive astrocytes and a concomitant tissue down-regulation of Kir4.1. Spiking activity was evoked by albumin in the hippocampus lasting for 2 h. When KA was intrahippocampally applied either 2 or 24 h after albumin injection, the number of total interictal spikes in 3 h EEG recording was significantly increased by twofold on average. Three months after albumin injection, neither albumin nor inflammation was detected in brain tissue; at this time, the ADT was reduced by 50% but no spontaneous seizures were observed.
Transient hippocampal exposure to albumin levels similar to those attained after prominent BBB breakdown resulted in increased seizure susceptibility and long-term reduction in seizure threshold, but it did not evoke spontaneous seizures. These effects may be mediated by albumin-induced astrocytes dysfunction and the associated induction of proinflammatory molecules.
PMCID: PMC3651831  PMID: 22984896
Afterdischarge; Astrocytes; Blood–brain barrier; Inflammation; Neurodegeneration; Seizures
8.  Low Frequency Stimulation of Hippocampal Commissures Reduces Seizures in Chronic Rat Model of Temporal Lobe Epilepsy 
Epilepsia  2011;53(1):147-156.
To investigate the effects of low frequency stimulation (LFS) of a fiber track for the suppression of spontaneous seizures described by Nissinen in a rat model of human temporal lobe epilepsy.
Stimulation electrodes were implanted into the ventral hippocampal commissure (VHC) in a rat post-status epilepticus (SE) model of human temporal lobe epilepsy (n = 7). Two recordings electrodes were placed in the CA3 regions bilaterally and neural data was recorded for a minimum of six weeks. LFS (60 minute train of 1Hz biphasic square wave pulses, each 0.1ms in duration and 200μA in amplitude, followed by 15 minutes of rest) was applied to the VHC for, two weeks, 24 hours a day.
Key Findings
The baseline mean seizure frequency of the study animals was 3.7 seizures per day. The seizures were significantly reduced by the application of LFS in every animal (n=7). By the end of the two-week period of stimulation, there was a significant 90% (<1 seizure/day) reduction of seizure frequencies (p < 0.05) and a 57% reduction during the period following LFS (p < 0.05) when compared to baseline. LFS also resulted in a significant reduction of hippocampal interictal spike frequency (71%, p < 0.05), during two weeks LFS session. The hippocampal histological analysis showed no significant difference between rats that received LFS and SE-induction and those that had only received SE-induction. None of the animals showed any symptomatic hemorrhage, infection or complication.
LFS applied at a frequency of 1Hz significantly reduced both the excitability of the neural tissue as well as the seizure frequency in a rat model of human temporal lobe epilepsy. The results support the hypothesis that LFS of fiber tracts can be an effective method for the suppression of spontaneous seizures in a temporal lobe model of epilepsy in rats and could be lead to the development of the new therapeutic modality for human patients with temporal lobe epilepsy.
PMCID: PMC3568386  PMID: 22150779
DBS; LFS; VHC; TLE; temporal lobe epilepsy; brain stimulation; amygdala
9.  Therapeutic effect of low frequency electric stimulation on the epileptogenic focus in amygdale-kindled rats 
This study was to examine the therapeutic effect of low-frequency electric stimulation (LFS) on the epileptogenic focus in amygdale-kindled rats, and to find out the optimal stimulus parameters. A microelectrode was implanted into the right amygdale of adult male rats. After fully kindling, LFS was delivered to the right amygdale (through the electrode) between seizures to induce stimulus trains (10 repetitive sequences). Next, we undertook controlled experiment in order to exclude the influence of seizure induction intervals on seizure. Fully kindled rats experienced trials for 4 days, the intervals of the repetitive seizure inducing stimulation was randomized for 5 min, 10 min, 15 min, and 20 min respectively. Finally, we applied an orthogonal design to test the 4 factors of parameters (frequency, pulse duration, current intensity and persistence time), in order to find out the best stimulus parameters. Results showed that compared to control group, the stage-4 seizure induction rate decreased dramatically in LFS group and animals in experiment group were more likely to be non-responsive to seizure-inducing stimuli. There were no statistical differences in the different seizure induction intervals. Significant differences were observed in different stimulus frequencies and stimulus train persistence times on the stage-4 seizure induction rate. These findings indicated that 1 Hz LFS is the best. Training for 5 min is more efficacious in controlling seizure. Thus, our results suggest that LFS applied directly to the site of seizure was effective and further studies are required to explore the most effective parameters for developing implanted stimulator.
PMCID: PMC4276176  PMID: 25550918
Kindling; low-frequency electric stimulation; best parameters; orthogonal design
10.  Effects of Anterior Thalamic Nucleus Deep Brain Stimulation in Chronic Epileptic Rats 
PLoS ONE  2014;9(6):e97618.
Deep brain stimulation (DBS) has been investigated for the treatment of epilepsy. In rodents, an increase in the latency for the development of seizures and status epilepticus (SE) has been reported in different animal models but the consequences of delivering stimulation to chronic epileptic animals have not been extensively addressed. We study the effects of anterior thalamic nucleus (AN) stimulation at different current intensities in rats rendered epileptic following pilocarpine (Pilo) administration. Four months after Pilo-induced SE, chronic epileptic rats were bilaterally implanted with AN electrodes or had sham-surgery. Stimulation was delivered for 6 h/day, 5 days/week at 130 Hz, 90 µsec. and either 100 µA or 500 µA. The frequency of spontaneous recurrent seizures in animals receiving stimulation was compared to that recorded in the preoperative period and in rats given sham treatment. To investigate the effects of DBS on hippocampal excitability, brain slices from animals receiving AN DBS or sham surgery were studied with electrophysiology. We found that rats treated with AN DBS at 100 µA had a 52% non-significant reduction in the frequency of seizures as compared to sham-treated controls and 61% less seizures than at baseline. Animals given DBS at 500 µA had 5.1 times more seizures than controls and a 2.8 fold increase in seizure rate as compared to preoperative values. In non-stimulated controls, the average frequency of seizures before and after surgery remained unaltered. In vitro recordings have shown that slices from animals previously given DBS at 100 µA had a longer latency for the development of epileptiform activity, shorter and smaller DC shifts, and a smaller spike amplitude compared to non-stimulated controls. In contrast, a higher spike amplitude was recorded in slices from animals given AN DBS at 500 µA.
PMCID: PMC4043725  PMID: 24892420
11.  Synaptic reorganization in subiculum and CA3 after early-life status epilepticus in the kainic acid rat model 
Epilepsy research  2006;73(2):156-165.
The immature rat brain is highly susceptible to seizures, but has a resistance to pathological changes induced by seizures as compared to adult rats. However, prolonged seizures during early-life enhance cellular injury and hyperexcitability induced by convulsive insults later in adulthood. The mechanisms underlying these phenomena are not understood. In adult models, the CA1 axons reorganize their projections to subiculum. Seizure induced plasticity in this pathway has not been investigated in immature seizure models, and may contribute to the vulnerability to later seizures.
On postnatal day 15, rats experienced convulsive status epilepticus with kainic acid (KA). Seizure induced plasticity was examined with Timm histochemistry and iontophoretic injections of sodium selenite, a retrograde tracer. Cellular injury was evaluated with Fluoro-Jade B histochemistry.
Retrograde tracing experiments determined a 67% larger dorsoventral extent of retrograde labeling in the CA1 pyramidal region after tracer injections in subiculum. The synaptic reorganization of the CA1 projection to subiculum was noted in the absence of overt neuronal injury in subiculum or CA1. In contrast, mossy fiber sprouting was detected into the stratum oriens of CA3 with limited neuronal injury to CA3 pyramidal neurons. No mossy fiber sprouting into the inner molecular layer of the dentate gyrus, or CA1 sprouting into the stratum moleculare of CA1 were noted.
The results indicate that the developing brain has distinct mechanisms of seizure induced reorganization as compared to the adult brain. Our experiments show that the concept of “resistance of the immature brain to excitotoxicity” is considerably more complicated than generally believed. Morphological plasticity in the immature brain appears more extensive in distal, but not proximal, projections of hippocampal pathways, and across hippocampal lamellae. The abnormal connectivity between hippocampal lamellae might play a role in the increased susceptibility to injury and hyperexcitability associated with later convulsive insults.
PMCID: PMC1876715  PMID: 17070016
sprouting; seizures; immature; retrograde tracing; Zinc
12.  Seizure suppression by EEG-guided repetitive transcranial magnetic stimulation in the rat 
To test the anticonvulsive potential of a range of repetitive transcranial magnetic stimulation (rTMS) frequencies by novel methods for simultaneous EEG and rTMS in a rat seizure model.
Seizures were triggered by intraperitoneal kainic acid (KA; 10 mg/kg). Rats (n = 21) were divided into three groups in which individual seizures were treated with rTMS trains at one of three frequencies: 0.25, 0.5 or 0.75 Hz. EEG was continuously viewed by an operator who identified each seizure onset. Consecutive seizures in each animal were (1) treated with active rTMS, (2) treated with sham rTMS, or (3) were untreated. EEG was re-analyzed post hoc by visual inspection, and seizure durations were compared within and between treatment groups.
KA-induced seizures were abbreviated by 0.75 Hz (P = 0.019) and 0.5 Hz (P = 0.033) active EEG-guided rTMS. In contrast, neither active 0.25 Hz rTMS nor the control conditions affected seizure duration (P > 0.2).
We demonstrate that EEG-guided rTMS can suppress seizures in the rat KA epilepsy model, and that the effect is frequency dependent, with 0.75 and 0.5 Hz rTMS being superior to 0.25 Hz rTMS.
These data support the use of rat seizure models in translational research aimed at evaluation and development of effective rTMS anticonvulsive protocols. We also offer a proof of principle that real-time analysis of EEG can be used to guide rTMS to suppress individual seizures.
PMCID: PMC2668608  PMID: 18977170
Transcranial magnetic stimulation; Seizure; Rat; EEG
13.  Oral administration of fructose-1,6-diphosphate has anticonvulsant activity 
Neuroscience letters  2008;446(2-3):75-77.
Recently it has been shown that fructose-1,6-diphosphate (FDP) has dose-dependent anticonvulsant activity in rat models of acute generalized motor seizures induced with chemical convulsants. This present study asked whether FDP also has activity in an epileptic brain after oral administration and activity against non-convulsive seizures. Animals (n=14) were administered pilocarpine to induce status epilepticus. Several weeks later, these animals had spontaneous seizures and a baseline rate of seizure frequency was determined over a 6 day period. Animals were then continued without treatment (n=8) or 0.5% FDP was added to the drinking water (n=6). In animals treated with FDP the seizures completely stopped after 7 days. Removal of FDP from the water resulted in the return of seizure activity in 4 of the 6 animals by 16 days of observation. To induce non-convulsive seizures, animals (n=6) received a single injection of γ-butyrolactone (GBL, 100 mg/kg ip). All animals had spike-wave activity recorded in the cortex within minutes after GBL administration. Administration of a single injection of FDP (500 g/kg ip) had no effect on the baseline cortical activity, nor on the spike-wave activity induced by GBL (n=5). These experiments suggest that oral administration of FDP may have utility in the treatment of partial or generalized convulsive seizure disorders, but not absence seizures.
PMCID: PMC2633033  PMID: 18832008
epilepsy; pilocarpine; seizures; gamma-butyrolactone
14.  Noninvasive transcranial focal stimulation via tripolar concentric ring electrodes lessens behavioral seizure activity of recurrent pentylenetetrazole administrations in rats 
Epilepsy affects approximately one percent of the world population. Antiepileptic drugs are ineffective in approximately 30% of patients and have side effects. We have been developing a noninvasive transcranial focal electrical stimulation with our novel tripolar concentric ring electrodes as an alternative/complementary therapy for seizure control. In this study we demonstrate the effect of focal stimulation on behavioral seizure activity induced by two successive pentylenetetrazole administrations in rats. Seizure onset latency, time of the first behavioral change, duration of seizure, and maximal seizure severity score were studied and compared for focal stimulation treated (n = 9) and control groups (n = 10). First, we demonstrate that no significant difference was found in behavioral activity for focal stimulation treated and control groups after the first pentylenetetrazole administration. Next, comparing first and second pentylenetetrazole administrations, we demonstrate there was a significant change in behavioral activity (time of the first behavioral change) in both groups that was not related to focal stimulation. Finally, we demonstrate focal stimulation provoking a significant change in seizure onset latency, duration of seizure, and maximal seizure severity score. We believe that these results, combined with our previous reports, suggest that transcranial focal stimulation may have an anticonvulsant effect.
PMCID: PMC3601189  PMID: 22692938
Epilepsy; pentylenetetrazole; noninvasive transcranial focal electrical stimulation; tripolar concentric ring electrode; seizure
15.  Role of subdural electrocorticography in prediction of long-term seizure outcome in epilepsy surgery 
Brain  2009;132(4):1038-1047.
Since prediction of long-term seizure outcome using preoperative diagnostic modalities remains suboptimal in epilepsy surgery, we evaluated whether interictal spike frequency measures obtained from extraoperative subdural electrocorticography (ECoG) recording could predict long-term seizure outcome. This study included 61 young patients (age 0.4–23.0 years), who underwent extraoperative ECoG recording prior to cortical resection for alleviation of uncontrolled focal seizures. Patient age, frequency of preoperative seizures, neuroimaging findings, ictal and interictal ECoG measures were preoperatively obtained. The seizure outcome was prospectively measured [follow-up period: 2.5–6.4 years (mean 4.6 years)]. Univariate and multivariate logistic regression analyses determined how well preoperative demographic and diagnostic measures predicted long-term seizure outcome. Following the initial cortical resection, Engel Class I, II, III and IV outcomes were noted in 35, 6, 12 and 7 patients, respectively. One child died due to disseminated intravascular coagulation associated with pseudomonas sepsis 2 days after surgery. Univariate regression analyses revealed that incomplete removal of seizure onset zone, higher interictal spike-frequency in the preserved cortex and incomplete removal of cortical abnormalities on neuroimaging were associated with a greater risk of failing to obtain Class I outcome. Multivariate logistic regression analysis revealed that incomplete removal of seizure onset zone was the only independent predictor of failure to obtain Class I outcome. The goodness of regression model fit and the predictive ability of regression model were greatest in the full regression model incorporating both ictal and interictal measures [R2 0.44; Area under the receiver operating characteristic (ROC) curve: 0.81], slightly smaller in the reduced model incorporating ictal but not interictal measures (R2 0.40; Area under the ROC curve: 0.79) and slightly smaller again in the reduced model incorporating interictal but not ictal measures (R2 0.27; Area under the ROC curve: 0.77). Seizure onset zone and interictal spike frequency measures on subdural ECoG recording may both be useful in predicting the long-term seizure outcome of epilepsy surgery. Yet, the additive clinical impact of interictal spike frequency measures to predict long-term surgical outcome may be modest in the presence of ictal ECoG and neuroimaging data.
PMCID: PMC2668945  PMID: 19286694
clinical neurophysiology; paediatric epilepsy surgery; intracranial electroencephalography (EEG); irritative zone
16.  Montelukast Inhibits Pentylenetetrazol-Induced Seizures in Rats 
Montelukast is an antiinflammatory drug with an antioxidant property. In this study, we aimed to reveal whether montelukast has a preventive effect against seizures and post-seizure oxidative stress in pentylenetetrazol (PTZ)-induced seizures in rats.
Of the 48 male Sprague-Dawley rats used in the study, 24 were assigned to EEG recordings (group A) and 24 were assigned to behavioral studies (group B). In group A, the electrodes were implanted on dura over the left frontal cortex for EEG recording. After 10 days, in group A, i.p. saline, 25, 50, or 100 mg/kg montelukast+35 mg/kg PTZ was administered to the rats. EEG was recorded and spike percentage was evaluated. In group B, i.p. saline, 25, 50, or 100 mg/kg montelukast+70 mg/kg PTZ was administered to the rats. Racine’s Convulsion Scale (RCS) and onset times of first myoclonic jerk (FMJ) was used to evaluate the seizures. Malondialdehyde (MDA) and superoxide dismutase (SOD) levels were determined in the brain tissue of animals.
Animals treated with 50 or 100 mg/kg montelukast had significantly lower RCS and significantly increased FMJ onset time compared to the saline-treated animals. Moreover, groups given 25, 50, or 100 mg/kg montelukast had significantly lower MDA and higher SOD levels compared to the saline-treated group. The differences were more pronounced in the 100 mg/kg montelukast-pretreated group (p<0.001).
Montelukast showed anticonvulsant action and led to amelioration of oxidative stress markers in PTZ-induced seizures in rats.
PMCID: PMC4384514  PMID: 25803241
Malondialdehyde; Oxidative Stress; Pentylenetetrazole; Seizures; Superoxide Dismutase
17.  An Excitatory Loop with Astrocytes Contributes to Drive Neurons to Seizure Threshold 
PLoS Biology  2010;8(4):e1000352.
Studies in rodent brain slices suggest that seizures in focal epilepsies are sustained and propagated by the reciprocal interaction between neurons and astroglial cells
Seizures in focal epilepsies are sustained by a highly synchronous neuronal discharge that arises at restricted brain sites and subsequently spreads to large portions of the brain. Despite intense experimental research in this field, the earlier cellular events that initiate and sustain a focal seizure are still not well defined. Their identification is central to understand the pathophysiology of focal epilepsies and to develop new pharmacological therapies for drug-resistant forms of epilepsy. The prominent involvement of astrocytes in ictogenesis was recently proposed. We test here whether a cooperation between astrocytes and neurons is a prerequisite to support ictal (seizure-like) and interictal epileptiform events. Simultaneous patch-clamp recording and Ca2+ imaging techniques were performed in a new in vitro model of focal seizures induced by local applications of N-methyl-D-aspartic acid (NMDA) in rat entorhinal cortex slices. We found that a Ca2+ elevation in astrocytes correlates with both the initial development and the maintenance of a focal, seizure-like discharge. A delayed astrocyte activation during ictal discharges was also observed in other models (including the whole in vitro isolated guinea pig brain) in which the site of generation of seizure activity cannot be precisely monitored. In contrast, interictal discharges were not associated with Ca2+ changes in astrocytes. Selective inhibition or stimulation of astrocyte Ca2+ signalling blocked or enhanced, respectively, ictal discharges, but did not affect interictal discharge generation. Our data reveal that neurons engage astrocytes in a recurrent excitatory loop (possibly involving gliotransmission) that promotes seizure ignition and sustains the ictal discharge. This neuron–astrocyte interaction may represent a novel target to develop effective therapeutic strategies to control seizures.
Author Summary
In focal epilepsy, seizures are generated by a localized, synchronous neuronal electrical discharge that may spread to large portions of the brain. Despite intense experimental research in this field, a key question relevant to the human epilepsy condition remains completely unanswered: what are the cellular events that lead to the onset of a seizure in the first place? In various in vitro models of seizures using rodent brain slices, we simultaneously recorded neuronal firing and Ca2+ signals both from neurons and from astrocytes, the principal population of glial cells in the brain. We found that activation of astrocytes by neuronal activity and signalling from astrocytes back to neurons contribute to the initiation of a focal seizure. This reciprocal excitatory loop between neurons and astrocytes represents a new mechanism in the pathophysiology of epilepsy that should be considered by those aiming to develop more effective therapies for epilepsies that are not controlled by currently available treatments.
PMCID: PMC2854117  PMID: 20405049
18.  The mTOR Inhibitor Rapamycin Has Limited Acute Anticonvulsant Effects in Mice 
PLoS ONE  2012;7(9):e45156.
The mammalian target of rapamycin (mTOR) pathway integrates signals from different nutrient sources, including amino acids and glucose. Compounds that inhibit mTOR kinase activity such as rapamycin and everolimus can suppress seizures in some chronic animal models and in patients with tuberous sclerosis. However, it is not known whether mTOR inhibitors exert acute anticonvulsant effects in addition to their longer term antiepileptogenic effects. To gain insights into how rapamycin suppresses seizures, we investigated the anticonvulsant activity of rapamycin using acute seizure tests in mice.
Following intraperitoneal injection of rapamycin, normal four-week-old male NIH Swiss mice were evaluated for susceptibility to a battery of acute seizure tests similar to those currently used to screen potential therapeutics by the US NIH Anticonvulsant Screening Program. To assess the short term effects of rapamycin, mice were seizure tested in ≤6 hours of a single dose of rapamycin, and for longer term effects of rapamycin, mice were tested after 3 or more daily doses of rapamycin.
The only seizure test where short-term rapamycin treatment protected mice was against tonic hindlimb extension in the MES threshold test, though this protection waned with longer rapamycin treatment. Longer term rapamycin treatment protected against kainic acid-induced seizure activity, but only at late times after seizure onset. Rapamycin was not protective in the 6 Hz or PTZ seizure tests after short or longer rapamycin treatment times. In contrast to other metabolism-based therapies that protect in acute seizure tests, rapamycin has limited acute anticonvulsant effects in normal mice.
The efficacy of rapamycin as an acute anticonvulsant agent may be limited. Furthermore, the combined pattern of acute seizure test results places rapamycin in a third category distinct from both fasting and the ketogenic diet, and which is more similar to drugs acting on sodium channels.
PMCID: PMC3440313  PMID: 22984623
19.  Electrical, Molecular and Behavioral Effects of Interictal Spiking in the Rat 
Neurobiology of Disease  2012;47(1):92-101.
Epilepsy is a disease characterized by chronic seizures, but is associated with significant comorbidities between seizures including cognitive impairments, hyperactivity, and depression. To study this interictal state, we characterized the electrical, molecular, and behavior effects of chronic, neocortical interictal spiking in rats.
A single injection of tetanus toxin into somatosensory cortex generated chronic interictal spiking measured by long-term video EEG monitoring and was correlated with motor activity. The cortical pattern of biomarker activation and the effects of blocking MAPK signaling on interictal spiking and behavior were determined.
Interictal spiking in this model increases in frequency, size, and becomes repetitive over time, but is rarely associated with seizures. Interictal spiking was sufficient to produce the same molecular and cellular pattern of layer 2/3-specific CREB activation and plasticity gene induction as is seen in the human interictal state (Beaumont 2011, submitted). Increasing spike frequency was associated with hyperactivity, demonstrated by increased ambulatory activity and preferential circling toward the spiking hemisphere. Loud noises induced epileptic discharges, identical to spontaneous discharges. Treatment with a selective MAPK inhibitor prevented layer 2/3 CREB activation, reduced the frequency of epileptic discharges, and normalized behavioral abnormalities, but had no effect on seizures induced by electrical kindling.
These results provide insights into the development of interictal epileptic spiking, their relationship to behavior, and suggest that interictal and ictal activities utilize distinct molecular pathways. This model, that parallels recent observations in humans, will be useful to develop therapeutics against interictal spiking and its behavioral comorbidities.
PMCID: PMC3358592  PMID: 22472188
20.  Increased GABA-ergic Inhibition in the Midline Thalamus Affects Signaling and Seizure Spread in the Hippocampus-Prefrontal Cortex Pathway 
Epilepsia  2011;52(3):523-530.
The midline thalamus is an important component of the circuitry in limbic seizures, but it is unclear how synaptic modulation of the thalamus affects that circuitry. In this study, we wished to understand how synaptic modulation of the thalamus can affect inter-regional signaling and seizure spread in the limbic network.
We examined the effect of GABA modulation of the mediodorsal (MD) region of the thalamus on responses in the prefrontal cortex (PFC) by stimulation of the subiculum (SB). Muscimol, a GABA-A agonist, was injected into the MD, and the effect on local responses to subiculum stimulation were examined. Evoked potentials were induced in the MD and the PFC by low frequency stimulation of the SB, and seizures were generated in the subiculum by repeated 20 Hz stimulations. The effect of muscimol in the MD on the evoked potentials and seizures was measured.
Key Findings
Thalamic responses to stimulation of the subiculum were reduced in the presence of muscimol. Reduction of the amplitudes of evoked potentials in the MD resulted in an attenuation of the late, thalamic components of the responses in the PFC, as well as of seizure durations.
Activation of GABA- A receptors in the midline thalamus not only causes changes within the thalamus, but has broader effects on the limbic network. This work provides further evidence that synaptic modulation within the midline thalamus alters system excitability more broadly and reduces seizure activity.
PMCID: PMC3058300  PMID: 21204829
GABA; mediodorsal nucleus; subiculum; prefrontal cortex; seizures; limbic system
21.  Temporal lobe epilepsy after experimental prolonged febrile seizures: prospective analysis 
Brain : a journal of neurology  2006;129(Pt 4):911-922.
Experimental prolonged febrile seizures (FS) lead to structural and molecular changes that promote hippocampal hyperexcitability and reduce seizure threshold to further convulsants. However, whether these seizures provoke later-onset epilepsy, as has been suspected in humans, has remained unclear. Previously, intermittent EEGs with behavioural observations for motor seizures failed to demonstrate spontaneous seizures in adult rats subjected to experimental prolonged FS during infancy. Because limbic seizures may be behaviourally subtle, here we determined the presence of spontaneous limbic seizures using chronic video monitoring with concurrent hippocampal and cortical EEGs, in adult rats (starting around 3 months of age) that had sustained experimental FS on postnatal day 10. These subjects were compared with groups that had undergone hyperthermia but in whom seizures had been prevented (hyperthermic controls), as well as with normothermic controls. Only events that fulfilled both EEG and behavioural criteria, i.e. electro-clinical events, were considered spontaneous seizures. EEGs (over 400 recorded hours) were normal in all normothermic and hyperthermic control rats, and none of these animals developed spontaneous seizures. In contrast, prolonged early-life FS evoked spontaneous electro-clinical seizures in 6 out of 17 experimental rats (35.2%). These seizures consisted of sudden freezing (altered consciousness) and typical limbic automatisms that were coupled with polyspike/sharp-wave trains with increasing amplitude and slowing frequency on EEG. In addition, interictal epileptiform discharges were recorded in 15 (88.2%) of the experimental FS group and in none of the controls. The large majority of hippocampally-recorded seizures were heralded by diminished amplitude of cortical EEG, that commenced half a minute prior to the hippocampal ictus and persisted after seizure termination. This suggests a substantial perturbation of normal cortical neuronal activity by these limbic spontaneous seizures. In summary, prolonged experimental FS lead to later-onset limbic (temporal lobe) epilepsy in a significant proportion of rats, and to interictal epileptifom EEG abnormalities in most others, and thus represent a model that may be useful to study the relationship between FS and human temporal lobe epilepsy.
PMCID: PMC3100674  PMID: 16446281
prolonged febrile seizures; temporal lobe epilepsy; video-EEG; rat; prospective study
22.  Vagus nerve stimulation vs. corpus callosotomy in the treatment of Lennox–Gastaut syndrome: A meta-analysis 
Lennox–Gastaut syndrome (LGS) is an epileptogenic disorder that arises in childhood and is typically characterized by multiple seizure types, slow spike-and-wave complexes on EEG and cognitive impairment. If medical treatment fails, patients can proceed to one of two palliative surgeries, vagus nerve stimulation (VNS) or corpus callosotomy (CC). Their relative seizure control rates in LGS have not been well studied. The purpose of this paper is to compare seizure reduction rates between VNS and CC in LGS using meta-analyses of published data.
A systematic search of Pubmed, Ovidsp, and Cochrane was performed to find articles that met the following criteria: (1) prospective or retrospective study, (2) at least one patient diagnosed with Lennox–Gastaut syndrome, and (3) well-defined measure of seizure frequency reduction. Seizure reduction rates were divided into seizure subtypes, as well as total seizures, and categorized as 100%, >75%, and >50%. Patient groups were compared using chi-square tests for categorical variables and t-test for continuous measures. Pooled proportions with 95% confidence interval (95% CI) of seizure outcomes were estimated for total seizures and seizure subtypes using random effects methods.
17 VNS and 9 CC studies met the criteria for inclusion. CC had a significantly better outcome than VNS for >50% atonic seizure reduction (80.0% [67.0–90.0%] vs. 54.1% [32.1–75.4%], p < 0.05) and for >75% atonic seizure reduction (70.0% [48.05–87.0%] vs. 26.3% [5.8–54.7%], p < 0.05). All other seizure types, as well as total number of seizures, showed no statistically significant difference between VNS and CC.
CC may be more beneficial for LGS patients whose predominant disabling seizure type is atonic. For all other seizure types, VNS offers comparable rates to CC.
PMCID: PMC3655762  PMID: 23068970
Lennox–Gastaut syndrome; Vagus nerve stimulation; Corpus callosotomy; Seizure frequency; Atonic seizures
23.  Changes in the expression of GABAA receptor subunit mRNAs in parahippocampal areas after kainic acid induced seizures 
The parahippocampal areas including the subiculum, pre- and parasubiculum, and notably the entorhinal cortex (EC) are intimately involved in the generation of limbic seizures in temporal lobe epilepsy. We investigated changes in the expression of 10 major GABAA receptor subunit mRNAs in subfields of the ventral hippocampus, ventral subiculum, EC, and perirhinal cortex (PRC) at different intervals (1, 8, 30, and 90 days) after kainic acid (KA)-induced status epilepticus priming epileptogenesis in the rat. The most pronounced and ubiquitous changes were a transient (24 h after KA only) down-regulation of γ2 mRNA and lasting decreases in subunit α5, β3, and δ mRNAs that were prominent in all hippocampal and parahippocampal areas. In the subiculum similarly as in sectors CA1 and CA3, levels of subunit α1, α2, α4, and γ2 mRNAs decreased transiently (1 day after KA-induced status epilepticus). They were followed by increased expression of subunit α1 and α3 mRNAs in the dentate gyrus (DG) and sectors CA1 and CA3, and subunit α1 also in the EC layer II (30 and 90 days after KA). We also observed sustained overexpression of subunits α4 and γ2 in the subiculum and in the Ammon’s horn. Subunit γ2 mRNA was also increased in sector CA1 at the late intervals after KA. Taken together, our results suggest distinct regulation of mRNA expression for individual GABAA receptor subunits. Especially striking was the wide-spread down-regulation of the often peri- or extrasynaptically located subunits α5 and δ. These subunits are often associated with tonic inhibition. Their decrease could be related to decreased tonic inhibition or may merely reflect compensatory changes. In contrast, expression of subunit α4 that may also mediate tonic inhibition when associated with the δ-subunit was significantly upregulated in the DG and in the proximal subiculum at late intervals. Thus, concomitant up-regulation of subunit γ2, α1 and α4 mRNAs (and loss in δ-subunits) ultimately indicates significant rearrangement of GABAA receptor composition after KA-induced seizures.
PMCID: PMC3776158  PMID: 24065890
epilepsy; tonic inhibition; GABAA-receptor; temporal lobe epilepsy; subiculum; entorhinal cortex; epileptogenesis
Annals of neurology  2009;65(4):435-447.
Conventional anticonvulsants reduce neuronal excitability through effects on ion channels and synaptic function. Anticonvulsant mechanisms of the ketogenic diet remain incompletely understood. Since carbohydrates are restricted in patients on the ketogenic diet, we evaluated the effects of limiting carbohydrate availability by reducing glycolysis using the glycolytic inhibitor 2-deoxy-D-glucose (2DG) in experimental models of seizures and epilepsy.
Acute anticonvulsant actions of 2DG were assessed in vitro in rat hippocampal slices perfused with 7.5mM [K+]o, 4-aminopyridine (4-AP), or bicuculline and in vivo against seizures evoked by 6 Hz stimulation in mice, audiogenic stimulation in Fring’s mice, and maximal electroshock and subcutaneous Metrazol in rats. Chronic antiepileptic effects of 2DG were evaluated in rats kindled from olfactory bulb or perforant path.
2DG (10mM) reduced interictal epileptiform bursts induced by high [K+]o, 4-AP and bicuculline, and electrographic seizures induced by high [K+]o in CA3 of hippocampus. 2DG reduced seizures evoked by 6 Hz stimulation in mice (ED50 = 79.7 mg/kg) and audiogenic stimulation in Fring’s mice (ED50 = 206.4 mg/kg). 2DG exerted chronic antiepileptic action by increasing afterdischarge thresholds in perforant path (but not olfactory bulb) kindling and caused a 2-fold slowing in progression of kindled seizures at both stimulation sites. 2DG did not protect against maximal electroshock or Metrazol seizures.
The glycolytic inhibitor 2DG exerts acute anticonvulsant and chronic antiepileptic actions and has a novel pattern of effectiveness in preclinical screening models. These results identify metabolic regulation as a potential therapeutic target for seizure suppression and modification of epileptogenesis.
PMCID: PMC2910719  PMID: 19399874
seizures; kindling; glycolysis; 2-deoxy-D-glucose; ketogenic diet; NRSF; CtBP
25.  Impaired cognition in rats with cortical dysplasia: additional impact of early-life seizures 
Brain  2011;134(6):1684-1693.
One of the most common and serious co-morbidities in patients with epilepsy is cognitive impairment. While early-life seizures are considered a major cause for cognitive impairment, it is not known whether it is the seizures, the underlying neurological substrate or a combination that has the largest impact on eventual learning and memory. Teasing out the effects of seizures from pre-existing neurological disorder is critical in developing therapeutic strategies. We therefore investigated the additional cognitive effects of seizures on rodents with malformations of cortical development induced with methylazoxymethanol acetate. Pregnant rats were injected with saline or methylazoxymethanol acetate at embryonic Day 15 or 17 to induce differing malformation severity. From the day of birth to 9 days of age, half the pups received 50 flurothyl-induced seizures. All rats underwent testing in the Morris water maze to test spatial memory at 25 days of age (immediate post-weaning) or during adolescence at 45 days of age. Post-weaning rats had severe spatial cognitive deficits in the water maze and seizures worsened performance. In contrast, in animals tested during adolescence, there was no longer an additional adverse effect of seizures. We also investigated whether the severity of the structural abnormality and seizures impacted brain weight, cortical thickness, hippocampal area and cell dispersion area. The mean brain weight in control animals was greater than in rats exposed to methylazoxymethanol acetate at embryonic Day 17, which was greater than rats exposed to methylazoxymethanol acetate at embryonic Day 15. Rats exposed to methylazoxymethanol acetate at embryonic Day 15 had a thinner cortical mantle compared with rats exposed at embryonic Day 17 and control animals. The hippocampal area was similar in rats exposed at embryonic Days 15 and 17 but was smaller compared with controls. Methylazoxymethanol at embryonic Day 17 caused dispersion of the CA1–4 cell layers in the hippocampus, whereas methylazoxymethanol at embryonic Day 15 caused focal nodules in or above the CA1 layer, but the CA1–4 layers were intact and similar to control. Early-life seizures did not have a significant impact on any of these parameters. These observations indicate that the major factor responsible for the cognitive impairment in the rats with cortical dysplasia was the underlying brain substrate, not seizures. These findings have significant implications for the understanding of cognitive impairments in childhood epilepsy and suggest that early aggressive therapy of seizures alone may not be an adequate strategy for minimizing cognitive effects.
PMCID: PMC3102240  PMID: 21602270
epilepsy; cognitive impairment; cortical dysplasia; cortical malformations; spatial cognition

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