Nonepileptic Control Animals Research Articles

Elevated sterol regulatory elementary binding protein 1 and GluA2 levels in the hippocampal nuclear fraction of Genetic Absence Epilepsy Rats from Strasbourg

Studies in animal models and human tissues show that nuclear translocation of sterol regulatory element binding protein 1 (SREBP1) and glutamate A2 subunit (GluA2) of cell-surface AMPA receptor (AMPAR) trigger neuronal excitotoxicity-induced apoptosis in stroke. However, it is not known whether a similar type of underlying pathophysiology occurs in absence epilepsy. To explore this issue, we examined the levels of mature SREBP1, GluA2, glyceraldehyde 3-phosphate dehydrogenase (GAPDH), p53, and activated to total caspase 3 ratio in nuclear fractions (NF) of hippocampal homogenate from 8 to 10 week old male Genetic Absence Epilepsy Rats from Strasbourg (GAERS) and non-epileptic control (NEC) strains. Mature SREBP1 and GluA2 levels were elevated approximately two-fold in NFs of GAERS hippocampal homogenates compared to NEC animals. Significant increases in GAPDH (∼15-fold) and total caspase 3 (∼10-fold) levels were also found in NFs of GAERS hippocampal homogenates in comparison to the non-epileptic strain. Data from the current study suggest that absence epilepsy in GAERS is associated with nuclear translocation of mature SREBP1, GluA2 subunit of AMPARs, and recruitment of pro-cell death signaling proteins such as GAPDH and caspase 3. These changes may contribute to hippocampal neuronal/glial cell death in GAERS. Therefore, inhibiting the nuclear accumulation of mature SREBP1 and GluA2 translocation may reduce the pathophysiology of absence epilepsy.

Sociability impairments in Genetic Absence Epilepsy Rats from Strasbourg: Reversal by the T-type calcium channel antagonist Z944

Childhood absence epilepsy (CAE) is associated with interictal co-morbid symptoms including abnormalities in social behaviour. Genetic Absence Epilepsy Rats from Strasbourg (GAERS) is a model of CAE that exhibits physiological and behavioural alterations characteristic of the human disorder. However, it is unknown if GAERS display the social deficits often observed in CAE. Sociability in rodents is thought to be mediated by neural circuits densely populated with T-type calcium channels and GAERS contain a missense mutation in the Cav3.2 T-type calcium channel gene. Thus, the objective of this study was to examine the effects of the clinical stage pan-T-type calcium channel blocker, Z944, on sociability behaviour in male and female GAERS and non-epileptic control (NEC) animals. Female GAERS showed reduced sociability in a three-chamber sociability task whereas male GAERS, male NECs, and female NECs all showed a preference for the chamber containing a stranger rat. In drug trials, pre-treatment with 5mg/kg of Z944 normalized sociability in female GAERS. In contrast, female NECs showed impaired sociability following Z944 treatment. Dose-dependent decreases in locomotor activity were noted following Z944 treatment in both strains. Treatment with 10mg/kg of Z944 altered exploration such that only 8 of the 16 rats tested explored both sides of the testing chamber. In those that explored the chamber, significant preference for the stranger rat was observed in GAERS but not NECs. Overall, the data suggest that T-type calcium channels are critical in regulating sociability in both GAERS and NEC animals. Future research should focus on T-type calcium channels in the treatment of sociability deficits observed in disorders such as CAE.

Thalamocortical neurons display suppressed burst-firing due to an enhanced Ih current in a genetic model of absence epilepsy.

Burst-firing in distinct subsets of thalamic relay (TR) neurons is thought to be a key requirement for the propagation of absence seizures. However, in the well-regarded Genetic Absence Epilepsy Rats from Strasbourg (GAERS) model as yet there has been no link described between burst-firing in TR neurons and spike-and-wave discharges (SWDs). GAERS ventrobasal (VB) neurons are a specific subset of TR neurons that do not normally display burst-firing during absence seizures in the GAERS model, and here, we assessed the underlying relationship of VB burst-firing with Ih and T-type calcium currents between GAERS and non-epileptic control (NEC) animals. In response to 200-ms hyperpolarizing current injections, adult epileptic but not pre-epileptic GAERS VB neurons displayed suppressed burst-firing compared to NEC. In response to longer duration 1,000-ms hyperpolarizing current injections, both pre-epileptic and epileptic GAERS VB neurons required significantly more hyperpolarizing current injection to burst-fire than those of NEC animals. The current density of the Hyperpolarization and Cyclic Nucleotide-activated (HCN) current (Ih) was found to be increased in GAERS VB neurons, and the blockade of Ih relieved the suppressed burst-firing in both pre-epileptic P15–P20 and adult animals. In support, levels of HCN-1 and HCN-3 isoform channel proteins were increased in GAERS VB thalamic tissue. T-type calcium channel whole-cell currents were found to be decreased in P7–P9 GAERS VB neurons, and also noted was a decrease in CaV3.1 mRNA and protein levels in adults. Z944, a potent T-type calcium channel blocker with anti-epileptic properties, completely abolished hyperpolarization-induced VB burst-firing in both NEC and GAERS VB neurons.

Manganese-enhanced magnetic resonance imaging detects mossy fiber sprouting in the pilocarpine model of epilepsy.

Mossy fiber sprouting (MFS) is a frequent finding following status epilepticus (SE). The present study aimed to test the feasibility of using manganese-enhanced magnetic resonance imaging (MEMRI) to detect MFS in the chronic phase of the well-established pilocarpine (Pilo) rat model of temporal lobe epilepsy (TLE). To modulate MFS, cycloheximide (CHX), a protein synthesis inhibitor, was coadministered with Pilo in a subgroup of animals. In vivo MEMRI was performed 3 months after induction of SE and compared to the neo-Timm histologic labeling of zinc mossy fiber terminals in the dentate gyrus (DG). Chronically epileptic rats displaying MFS as detected by neo-Timm histology had a hyperintense MEMRI signal in the DG, whereas chronically epileptic animals that did not display MFS had minimal MEMRI signal enhancement compared to nonepileptic control animals. A strong correlation (r = 0.81, p < 0.001) was found between MEMRI signal enhancement and MFS. This study shows that MEMRI is an attractive noninvasive method for detection of mossy fiber sprouting in vivo and can be used as an evaluation tool in testing therapeutic approaches to manage chronic epilepsy.

Altered expression of brain monocarboxylate transporter 1 in models of temporal lobe epilepsy

Monocarboxylate transporter 1 (MCT1) facilitates the transport of monocarboxylate fuels (lactate, pyruvate and ketone bodies) and acidic drugs, such as valproic acid, across cell membranes. We recently reported that MCT1 is deficient on microvessels in the epileptogenic hippocampal formation in patients with medication-refractory temporal lobe epilepsy (TLE). To further define the role of MCT1 in the pathophysiology of TLE, we used immunohistochemistry and stereological analysis to localize and quantify the transporter in the hippocampal formation in three novel and highly relevant rat models of TLE and in nonepileptic control animals. One model utilizes methionine sulfoximine to induce brain glutamine synthetase deficiency and recurrent limbic seizures, while two models employ an episode of perforant pathway stimulation to cause epilepsy. MCT1 was lost on microvessels and upregulated on astrocytes in the hippocampal formation in all models of TLE. Notably, the loss of MCT1 on microvessels was not due to a reduction in microvessel density. The similarities in MCT1 expression among human subjects with TLE and several animal models of the disease strongly suggest a critical role of this molecule in the pathogenesis of TLE. We hypothesize that the downregulation of MCT1 may promote seizures via impaired uptake of ketone bodies and antiepileptic drugs by the epileptogenic brain. We also propose that the overexpression of MCT1 on astrocytes may lead to increased uptake or release of monocarboxylates by these cells, with important implications for brain metabolism and excitability. These hypotheses can now be rigorously tested in several animal models that replicate key features of human TLE.

Assessment of seizure susceptibility in pilocarpine epileptic and nonepileptic Wistar rats and of seizure reinduction with pentylenetetrazole and electroshock models

Pentylenetetrazole (PTZ) and maximal electroshock (MES) models are often used to induce seizures in nonepileptic control animals or naive animals. Despite being widely used to screen antiepileptic drugs (AEDs), both models have so far failed to detect potentially useful AEDs for treating drug-resistant epilepsies. Here we investigated whether the acute induction of MES and PTZ seizures in epileptic rats might yield a distinct screening profile for AEDs. Status epilepticus (SE) was induced in adult male Wistar rats by intraperitoneal pilocarpine injection (Pilo, 320 mg/kg, i.p.). One month later, controls or naive animals (Cont) that did not develop SE postpilocarpine (N-Epi) and pilocarpine-epileptic rats (Epi) received one of the following: phenobarbital (PB, 40 mg/kg), phenytoin (PHT, 50 mg/kg), or valproic acid (VPA, 400 mg/kg). Thirty min later the animals were challenged with either subcutaneous MES or PTZ (50 mg/kg, s.c.). VPA, PB, and PHT were able to prevent MES in all groups tested (Cont, N-Epi, and Epi groups), whereas for the PTZ model, only the Cont group (naive animals) had seizure control with the same AEDs. In addition, Epi and N-Epi groups when challenged with PTZ exhibited a higher incidence of severe seizures (scores IV-IX) and SE (p < 0.05, Fisher's exact test). Our findings suggest that the induction of acute seizures with PTZ, but not with MES, in animals pretreated with pilocarpine (regardless of SE induction) might constitute an effective and valuable method to screen AEDs and to study mechanisms involved in pharmacoresistant temporal lobe epilepsy (TLE).

Cell Cycle Reentry and Cell Proliferation as Candidates for the Seizure Predispositions in the Hippocampus of EL Mouse Brain

We have recently found that there was DNA fragmentation without cell loss in the hippocampus in EL mice, an epileptic mutant. Neurotrophic factors are also expressed at high levels during the early developmental stages. In the present study, we used EL mice to examine how altered cyclin and the corresponding cyclin dependent kinase (CDK) family are related to cell proliferation during development and during epileptogenesis. Developmental changes of cyclin family and corresponding CDK family (cyclin D/CDK-4, cyclin E/CDK-2, cyclin A/CDK-2, cyclin A/CDK-1, cyclin B/CDK-1) were examined by Western blotting in the hippocampus of EL mice and in nonepileptic control animals (DDY mice). In addition, we attempted to quantify cell proliferation during this period. The developmental changes in cell proliferation were determined by using systemic injections of Bromo-deoxyUridine (BrdU) to label dividing cells. As compared with the control DDY mice, EL mice show an upregulation of cell cycle specific Cyclins/CDKs during early developmental stages suggesting that reentry into the cell cycle is enhanced prior to the onset of seizure activity, possibly due to the abundance of neurotrophic factors. These results show that Cyclins/CDKs are activated during early stages of development in an epileptic animal, before the mouse exhibits seizures. These results suggest that reentry of cells into the cell cycle, with consequent cell proliferation in the hippocampus, contribute to the seizure predispositions of EL mice.

Neocortical Hyperexcitability in a Genetic Model of Absence Seizures and Its Reduction by Levetiracetam

To study the effect of the antiepileptic drug levetiracetam (LEV) on the patterns of intrinsic optical signals (IOSs) generated by slices of the somatosensory cortex obtained from 3- and 6-month-old WAG/Rij and age-matched, nonepileptic control (NEC) rats. WAG/Rij and NEC animals were anesthetized with enfluorane and decapitated. Brains were quickly removed, and neocortical slices were cut coronally with a vibratome, transferred to a submerged tissue chamber, and superfused with oxygenated artificial cerebrospinal fluid (aCSF). Slices were illuminated with a dark-field condensor and examined with a x2.5 objective; images were processed with a real time digital video image-enhancement system. Images were acquired before (background) and during electrical stimulation with a temporal resolution of 10 images/s and were displayed in pseudocolors. Extracellular stimuli (200 micros; <4 V) were delivered through bipolar stainless steel electrodes placed in the white matter. IOSs recorded in NEC slices bathed in control aCSF became less intense and of reduced size with age (p < 0.05); this trend was not seen in WAG/Rij slices. Age-dependent decreases in IOS intensity and area size were also seen in NEC slices superfused with aCSF containing the convulsant 4-aminopyridine (4-AP, 5 microM); in contrast, significant increases in both parameters occurred with age in 4-AP-treated WAG/Rij slices (p < 0.05). Under any of these conditions, the IOS intensity and area size slices were larger in WAG/Rij than in NEC slices. LEV (50-500 microM) application to WAG/Rij slices caused dose-dependent IOS reductions that were evident both in control and in 4-AP-containing aCSF and were more pronounced in 6-month-old tissue. These data demonstrate age-dependent IOS modifications in NEC and WAG/Rij rat slices and identify a clear pattern of hyperexcitability that occurs in 6-month-old WAG/Rij neocortical tissue, an age when absence seizures occur in all animals. The ability of LEV to reduce these patterns of network hyperexcitability supports the potential use of this new antiepileptic drug in primary generalized epileptic disorders.

Ketamine Prevents Learning Impairment When Administered Immediately after Status Epilepticus Onset

Permanent cognitive impairment is common following status epilepticus (SE) in both humans and animals. We examined the effect of the NMDA antagonist ketamine administered after SE onset on two forms of associative learning in the rat: conditioned taste aversion and fear-conditioned analgesia. Following the onset of lithium/pilocarpine-induced SE, rats were administered either ketamine (100 mg/kg) or acepromazine (25 mg/kg). Acepromazine-treated animals show marked deficits in both learning measures at 1 month after SE. In contrast, ketamine-treated and nonepileptic control animals did not differ in performance for either task. Although studies have shown that ketamine is ineffective at controlling electrographic seizures early in SE, these results are consistent with previous studies showing that ketamine can preserve learning proficiency if administered shortly after seizure onset. As a clinically available drug, ketamine may prove useful in the treatment of SE when combined with conventional antiepileptic strategies.

GABA release and uptake measured in crude synaptosomes from Genetic Absence Epilepsy Rats from Strasbourg (GAERS)

GABA release and uptake were examined in Genetic Absence Epilepsy Rats from Strasbourg and in non-epileptic control animals, using crude synaptosomes prepared from the cerebral cortex and thalamus. Uptake of [ 3H]GABA over time was reduced in thalamic synaptosomes from epileptic rats, compared to controls. The affinity of the uptake process in thalamic synaptosomes was lower in epileptic animals. NNC-711, a ligand for the GAT-1 uptake protein, reduced synaptosomal uptake by more than 95%; β-alanine, an inhibitor selective for the uptake proteins GAT-2 and -3, did not significantly reduce synaptosomal uptake. Autoradiography studies using [ 3H]tiagabine, a ligand selective for GAT-1, revealed no differences between the strains in either affinity or levels of binding. Ethanolamine O-sulphate (100 μM), a selective inhibitor of GABA-transaminase, did not affect uptake levels. Aminooxyacetic acid (10–100 μM), an inhibitor of GABA-transaminase and, to a lesser extent, glutamate decarboxylase, caused an increase in measured uptake in both thalamic and cortical synaptosomes, in both strains. We found no difference in in vitro basal or KCl-stimulated endogenous GABA release between epileptic and control rats. These results indicate that GABA uptake in the thalamus of Genetic Absence Epilepsy Rats from Strasbourg was reduced, compared to control animals. The lower uptake affinity in the epileptic animals probably contributed to the reduction in uptake over time. Uptake appeared to be mediated primarily by the ‘neuronal’ transporter GAT-1. Autoradiography studies revealed no differences in the number or affinity of this uptake protein. It is therefore possible that altered functional modulation of GAT-1 caused the decrease in uptake shown in the epileptic animals. Inhibition of GABA-transaminase activity had no effect on measured GABA uptake, whereas a reduction in glutamate decarboxylase activity may have affected measured uptake levels.

Influence of reduced oxygen availability on cerebral metabolic changes during bicuculline-induced seizures in rats.

The objective of the present work was to study cerebral energy metabolism at threshold levels of hypoxia, i.e., degrees of hypoxia that abolish cerebral electrical activity, in the "normal" and in the epileptic brain. Seizures were induced by intravenous bicuculline and cerebral oxygen availability was reduced by a combination of lowered PO2 and reduced blood pressure to give a transformation of the burst suppression pattern to either one with single spikes or overt EEG flattening. Nonepileptic control animals were exposed to degrees of hypoxia that gave either a markedly depressed EEG pattern with sparse slow waves or EEG flattening. Epileptic and nonepileptic groups proved comparable in terms of calculated oxygen availability and cerebral oxygen consumption at the threshold of "transmission failure." At levels of hypoxia that markedly attenuated or completely abolished seizure discharge, the cerebral metabolic changes were more marked than in comparable nonepileptic animals. These changes comprised an imminent severe perturbation of cerebral cortical phosphorylation potential, a pronounced lactic acidosis with a precipitous redox change, and a marked accumulation of ammonia. The more labile energy balance of the epileptic brain may indicate that the "seizure state" either increases cellular energy demands in spite of the electrical silence or reduces the efficiency of ATP production at the prevailing oxygen availability. It is conceivable that energy failure elicited by complicating hypoxia can aggravate or precipitate brain cell damage in epilepsy.

Influence of systemic factors on experimental epileptic brain injury. Structural changes accompanying bicuculline-induced seizures in rats following manipulations of tissue oxygenation or alpha-tocopherol levels.

A previous study from the laboratory showed that status epilepticus induced by bicuculline administration to ventilated rats produced astrocytic swelling and nerve cell changes ("type 1 and 2 injury") particularly in layers 3 and 5 of the neocortex (Söderfeldt et al. 1981). The type 1 injured neurons were characterized by condensation of cyto- and karyoplasm and the less common type 2 cells were characterized by swelling of endoplasmic reticulum including the nuclear envelope. In the present study we explored whether changes in cerebral oxygen availability altered the extent or character of the cellular alterations. Animals with 2 h of status epilepticus were made either hyperoxic (administration of 100% O2), hypoxic (arterial pO2 50 mm Hg) or hypotensive (arterial blood pressure of either 70-75 or 50 mm Hg). Furthermore, we explored whether "oxidative" damage occurred by manipulating tissue levels of alpha-tocopherol, a known free radical scavenger. Non-epileptic control animals exposed to comparable degrees of hypoxia or hypotension showed no or minimal structural alterations. In the epileptic animals the results were as follows. Hyperoxia did not change the quality or extent of the structural alterations previously observed in normoxic epileptic animals. Neither administration nor deficiency of vitamin E did modify this pattern of alterations. In hypoxia the extent of cell damage was the same or somewhat larger than in normoxic, epileptic animals. In addition, neurons often showed cytoplasmic microvacuoles due to swelling of mitochondria. The hypoxic animals also showed swelling of astrocytic nuclei with clumped chromatin. Changes similar to those observed in hypoxic animals also appeared in moderate hypotension (mean arterial blood pressure 50 mm Hg), whereas mild hypotension (70-75 mm Hg) did not change the character of the tissue injury from that seen in hyperoxic or normoxic epileptic rats. The present results demonstrate that the neuronal cell damage that can be observed when the brain is fixed by perfusion after status epilepticus of 2 h duration is not exaggerated by hyperoxia or vitamin E deficiency nor is it ameliorated by a moderate restriction in cerebral oxygen supply or by vitamin E administration. If anything, hypoxia (or moderate hypotension) appears to increase the extent of damage and it clearly alters its ultrastructural characteristics. However, although the results fail to support the notion that epileptic cell damage is "oxidative", definite conclusions must await information on the cell damage that remains upon arrest of the epileptic activity.