FMRI of brain activation in a genetic rat model of absence seizures

PURPOSE: EEG-triggered functional magnetic resonance imaging (fMRI) was used to identify areas of brain activation during spontaneous spike-and-wave discharges (SWDs) in an epileptic rat strain under awake conditions. METHODS: Spontaneous absence seizures from 10 WAG/Rij rats were imaged by using T2*-weighted echo planar imaging at 4.7 Tesla. fMRI of the blood-oxygenation-level-dependent (BOLD) signal was triggered based on EEG recordings during imaging. Images obtained during spontaneous SWDs were compared with baseline images. RESULTS: Significant positive BOLD signal changes were apparent in several areas of the cortex and several important nuclei of the thalamus. In addition, no negative BOLD signal was found in any brain area. CONCLUSIONS: We have shown that EEG-triggered BOLD fMRI can be used to detect cortical and thalamic activation related to the spontaneous SWDs that characterize absence seizures in awake WAG/Rij rats. These results draw an anatomic correlation between areas in which increased BOLD signal is found and those in which SWDs have been recorded. In addition, no negative BOLD signal was found to be associated with these spontaneous SWDs. We also demonstrated the technical feasibility of using EEG-triggered fMRI in a genetic rat model of absence seizure.     

Thalamic lesions in a genetic rat model of absence epilepsy: Dissociation between spike-wave discharges and sleep spindles

Recent findings have challenged the traditional view that the thalamus is the primary driving source of generalized spike-wave discharges (SWDs) characteristic for absence seizures, and indicate a leading role for the cortex instead. In light of this we investigated the effects of thalamic lesions on SWDs and sleep spindles in the WAG/Rij rat, a genetic model of absence epilepsy. EEG was recorded from neocortex and thalamus in freely moving rats, both before and after unilateral thalamic ibotenic acid lesions. Complete unilateral destruction of the reticular thalamic nucleus (RTN) combined with extensive destruction of the thalamocortical relay (TCR) nuclei, resulted in the bilateral abolishment of SWDs and ipsilateral abolishment of sleep spindles. A suppression of both types of thalamocortical oscillations was found when complete or extensive damage to the RTN was combined with minor to moderate damage to the TCR nuclei. Lesions that left the rostral pole of the RTN and part of the TCR nuclei intact, resulted in an ipsilateral suppression of sleep spindles, but a large increase of bilateral SWDs. These findings demonstrate that the thalamus in general and the RTN in particular are a prerequisite for both the typical bilateral 7-11 Hz SWDs and natural occurring sleep spindles in the WAG/Rij rat, but suggest that different intrathalamic subcircuits are involved in the two types of thalamocortical oscillations. Whereas the whole RTN appears to be critical for the generation of sleep spindles, the rostral pole of the RTN seems to be the most likely part that generates SWDs.     

A role for the preoptic sleep-promoting system in absence epilepsy

Absence epilepsy (AE) in humans and the genetic AE model in WAG/Rij rats are both associated with abnormalities in sleep architecture that suggest insufficiency of the sleep-promoting mechanisms. In this study we compared the functionality of sleep-active neuronal groups within two well-established sleep-promoting sites, the ventrolateral and median preoptic nuclei (VLPO and MnPN, respectively), in WAG/Rij and control rats. Neuronal activity was assessed using c-Fos immunoreactivity and chronic single-unit recording techniques. We found that WAG/Rij rats exhibited a lack of sleep-associated c-Fos activation of GABAergic MnPN and VLPO neurons, a lower percentage of MnPN and VLPO cells increasing discharge during sleep and reduced firing rates of MnPN sleep-active neurons, compared to non-epileptic rats. The role of sleep-promoting mechanisms in pathogenesis of absence seizures was assessed in non-epileptic rats using electrical stimulation and chemical manipulations restricted to the MnPN. We found that fractional activation of the sleep-promoting system in waking was sufficient to elicit absence-like seizures. Given that reciprocally interrelated sleep-promoting and arousal neuronal groups control thalamocortical excitability, we hypothesize that malfunctioning of sleep-promoting system results in impaired ascending control over thalamocortical rhythmogenic mechanisms during wake–sleep transitions thus favoring aberrant thalamocortical oscillations. Our findings suggest a pathological basis for AE-associated sleep abnormalities and a mechanism underlying association of absence seizures with wake–sleep transitions.     

Comparison of the antiepileptogenic effects of an early long‐term treatment with ethosuximide or levetiracetam in a genetic animal model of absence epilepsy

Purpose: Epilepsy is a heterogeneous syndrome characterized by recurrent, spontaneous seizures; continuous medication is, therefore, necessary, even after the seizures have long been suppressed with antiepileptic drug (AED) treatments. The most disturbing issue is the inability of AEDs to provide a persistent cure, because these compounds generally suppress the occurrence of epileptic seizures without necessarily having antiepileptogenic properties. The aim of our experiments was to determine, in the WAG/Rij model of absence epilepsy, if early long‐term treatment with some established antiabsence drugs might prevent the development of seizures, and whether such an effect could be sustained. Methods: WAG/Rij rats were treated for ～3.5 months (starting at 1.5 months of age, before seizure onset) with either ethosuximide (ETH; drug of choice for absence epilepsy) or levetiracetam (LEV; a broad‐spectrum AED with antiabsence and antiepileptogenic properties). Results: We have demonstrated that both drugs are able to reduce the development of absence seizures, exhibiting antiepileptogenic effects in this specific animal model. Discussion: These findings suggest that absence epilepsy in this strain of rats very likely follows an epileptogenic process during life and that early therapeutic intervention is possible, thereby opening a new area of research for absence epilepsy and AED treatment strategies.     

Metabotropic glutamate receptors in the thalamocortical network: strategic targets for the treatment of absence epilepsy

Metabotropic glutamate (mGlu) receptors are positioned at synapses of the thalamocortical network that underlie the development of spike-and-wave discharges (SWDs) associated with absence epilepsy. The modulatory role of individual mGlu receptor subtypes on excitatory and inhibitory synaptic transmission in the cortico-thalamo-cortical circuitry makes subtype-selective mGlu receptor ligands potential candidates as novel antiabsence drugs. Some of these compounds are under clinical development for the treatment of numerous neurologic and psychiatric disorders, and might be soon available for clinical studies in patients with absence seizures refractory to conventional medications. Herein we review the growing evidence that links mGlu receptors to the pathophysiology of pathologic SWDs moving from the anatomic localization and function of distinct mGlu receptor subtypes in the cortico-thalamo-cortical network to in vivo studies in mouse and rat models of absence epilepsy.     

Reduced GABAB receptor subunit expression and paired-pulse depression in a genetic model of absence seizures

Neocortical networks play a major role in the genesis of generalized spike-and-wave (SW) discharges associated with absence seizures in humans and in animal models, including genetically predisposed WAG/Rij rats. Here, we tested the hypothesis that alterations in GABA(B) receptors contribute to neocortical hyperexcitability in these animals. By using Real-Time PCR we found that mRNA levels for most GABA(B(1)) subunits are diminished in epileptic WAG/Rij neocortex as compared with age-matched non-epileptic controls (NEC), whereas GABA(B(2)) mRNA is unchanged. Next, we investigated the cellular distribution of GABA(B(1)) and GABA(B(2)) subunits by confocal microscopy and discovered that GABA(B(1)) subunits fail to localize in the distal dendrites of WAG/Rij neocortical pyramidal cells. Intracellular recordings from neocortical cells in an in vitro slice preparation demonstrated reduced paired-pulse depression of pharmacologically isolated excitatory and inhibitory responses in epileptic WAG/Rij rats as compared with NECs; moreover, paired-pulse depression in NEC slices was diminished by a GABA(B) receptor antagonist to a greater extent than in WAG/Rij rats further suggesting GABA(B) receptor dysfunction. In conclusion, our data identify changes in GABA(B) receptor subunit expression and distribution along with decreased paired-pulse depression in epileptic WAG/Rij rat neocortex. We propose that these alterations may contribute to neocortical hyperexcitability and thus to SW generation in absence epilepsy.     

Chromosomal mapping of genetic loci controlling absence epilepsy phenotypes in the WAG/Rij rat

PURPOSE: The WAG/Rij rat is among the most appropriate models for the study of spontaneous childhood absence epilepsy, without complex neurologic disorders that are associated with some mouse models for absence epilepsy. Previous studies have allowed the identification of distinct types of spike-wave discharges (SWDs) characterizing seizures in this strain. The purpose of this study was to investigate the genetic basis of electroencephalographic (EEG) properties of SWDs. METHODS: An intercross was derived from WAG/Rij and ACI inbred strains that are known to differ substantially in the number of SWDs. Phenotypic analyses based on 23-h EEG recording in all progenies allowed the quantification of type I and type II SWD phenotypes. A genome-wide scan was performed with 145 microsatellite markers, which were used to test for evidence of genetic linkage to SWD quantitative phenotypes. RESULTS: We were able to map quantitative trait loci independently, controlling type I and type II SWD variables to rat chromosomes 5 and 9. Strongest linkages were obtained for D5Mgh15 and total duration of type II SWD (lod, 3.64) and for D9Rat103 and the average duration of type I SWD (lod, 3.91). These loci were denoted T2swd/wag and T1swd/wag, respectively. CONCLUSIONS: The independent genetic control of type I and type II SWDs underlines the complexity of the molecular mechanisms participating in SWDs. The identification of these genetic loci represents an important step in our fundamental knowledge of the architecture of SWDs and may provide new insights for resolving the genetic heterogeneity of absence epilepsy.     

Upholding WAG/Rij rats as a model of absence epileptogenesis: Hidden mechanisms and a new theory on seizure development

The WAG/Rij rat model has recently gathered attention as a suitable animal model of absence epileptogenesis. This latter term has a broad definition encompassing any possible cause that determines the development of spontaneous seizures; however, most of, if not all, preclinical knowledge on epileptogenesis is confined to the study of post-brain insult models such as traumatic brain injury or post-status epilepticus models. WAG/Rij rats, but also synapsin 2 knockout, Kv7 current–deficient mice represent the first examples of genetic models where an efficacious antiepileptogenic treatment (ethosuximide) was started before seizure onset. In this review, we have critically reconsidered all articles published regarding WAG/Rij rats, from the perspective that the period before SWD onset is considered as the latent period. In our new theory on seizure development, it is proposed that genes might be considered as the initial ‘insult’ responsible for all plastic changes underpinning the development of spontaneous seizures. According to this idea, in WAG/Rij rats, genetic predisposition would lead to the development of abnormal bilateral cortical epileptic foci, which would then non-genetically stimulate the rest of the brain to rearrange networks in order to phenotypically develop seizures similarly to what happens during electrical kindling.     

Increased persistent sodium current determines cortical hyperexcitability in a genetic model of amyotrophic lateral sclerosis

Cortical hyperexcitability has been observed in Amyotrophic Lateral Sclerosis (ALS) patients. Familial ALS accounts for 10% of all cases and mutations of the Cu,Zn superoxide dismutase (SOD1) gene have been identified in about 20% of the familial cases. The aim of this study was to investigate whether in a mouse model of ALS the cortical neurons developed hyperexcitability due to intrinsic properties of the single cell. We first examined the passive membrane properties and the pattern of repetitive firing in cultured cortical neurons from Control mice and transgenic mice expressing high levels of the human mutated protein (Gly⁹³→Ala, G93A). The former did not display significantly differing values between Control and G93A cortical neurons. However, the threshold potential and time of the first action potential decreased significantly and the firing frequency increased significantly in the G93A compared to Control neurons. The analysis of the voltage-dependent sodium currents revealed that the fast transient sodium current was unaffected by the SOD1 mutation whereas the persistent sodium current was significantly higher in the mutated neurons. Finally, Riluzole, a selective blocker of the persistent sodium current at low concentrations, decreased the firing frequency in G93A neurons, strongly indicating an involvement of this current in the observed hyperexcitability. These are the first data that demonstrate an intrinsic hyperexcitability in the G93A cortical neurons due to a higher current density of the persistent sodium current in the mutated neurons and open up new prospects of understanding ALS disease etiopathology.     

Enhancement of persistent sodium current by internal fluorescence in isolated hippocampal neurons

Following up on an earlier chance observation, voltage-dependent whole-cell currents were recorded from isolated hippocampal neurons filled with the fluorescent dyes Fluo-3 and Fura-red, that were intermittently excited by 488 nm laser light. In the absence of any ion channel blocking drugs, in most cells depolarizing voltage steps initially evoked only the 'Hodgkin-Huxley' type early, fast inward surge followed by sustained outward current. Over 5-20 min of intermittent electrical stimulation and laser-excited fluorescence pulses, a voltage-dependent, slowly inactivating inward current also appeared and grew, while sustained outward current diminished. When K(+) currents were blocked, a small persistent inward current was usually detectable immediately, and then it increased in amplitude. This current was blocked by tetrodotoxin (TTX) and it had current-voltage (I-V) characteristics of a persistent sodium current, I(Na,P). In cells not filled with dye but illuminated by laser, and in cells with dye but not illuminated, I(Na,P) remained small. There was a more than 12-fold difference in the maximal amplitude of I(Na, P) of fluorescent compared to non-fluorescent cells. Once induced, I(Na,P) decreased very slowly. Fluorescence increased the duration but not the amplitude of the transient Na(+) current, I(Na,T). With membrane potential clamped to a constant voltage, the laser-induced fluorescence did not evoke a membrane current. It is not certain whether fluorescence-induced I(Na,P) potentiation is related to photodynamic action.     

Enhanced inactivation and acceleration of activation of the sodium channel associated with epilepsy in man

Generalized epilepsy with febrile seizures-plus (GEFS+) is a benign Mendelian syndrome characterized by childhood-onset febrile and afebrile seizures. Three point mutations within two voltage-gated sodium channel genes have been identified so far: in GEFS+ type 1 a mutation in the beta1-subunit gene SCN1B, and in GEFS+ type 2 two mutations within the neuronal alpha-subunit gene SCN1A. Functional expression of the SCN1B and one of the SCN1A mutations revealed defects in fast channel inactivation which are in line with previous findings on myotonia causing mutations in SCN4A, the skeletal muscle sodium channel alpha-subunit gene, all showing an impaired fast inactivation. We now studied the second GEFS+ mutation (T875M in SCN1A), using the highly homologous SCN4A gene (mutation T685M). Unexpectedly, the experiments revealed a pronounced enhancement of both fast and slow inactivation and a defect of channel activation for T685M compared to wild-type channels. Steady-state fast and slow inactivation curves were shifted in the hyperpolarizing direction, entry into slow inactivation was threefold accelerated, recovery from slow inactivation was slowed by threefold and the time course of activation was slightly but significantly accelerated. In contrast to other disease-causing mutations in SCN1A, SCN1B and SCN4A, the only mechanism that could explain hyperexcitability of the cell membrane would be the acceleration of activation. Because the enhancement of slow inactivation was the most obvious alteration in gating found for T685M, this might be the disease-causing mechanism for that mutation. In this case, the occurrence of epileptic seizures could be explained by a decrease of excitability of inhibitory neurons.     

Low-voltage-activated calcium channel subunit expression in a genetic model of absence epilepsy in the rat

The Genetic Absence Epilepsy Rats from Strasbourg (GAERS) are an inbred strain of rats that display many of the characteristics of human absence epilepsy. In these rats, reciprocal thalamocortical projections play a critical role in the generation of spike-and-wave discharges that characterize absence seizures. When compared to those of the non-epileptic control strain, juvenile animals of the GAERS strain reportedly possess higher-amplitude T-type calcium currents in neurons of the thalamic reticular nucleus (nRt). We hypothesized that differences in calcium currents seen between GAERS and controls result from differences in expression of genes for low-voltage-activated calcium channels. Quantitative in situ hybridization was used to compare expression of alpha1G, alpha1H, alpha1I, and alpha1E calcium channel subunit mRNAs from adult and juvenile animals of the two strains. We found higher levels of alpha1H mRNA expression in nRt neurons of juvenile animals (34.9+/-2. 3 vs. 28.4+/-1.8 grains/10(3) pixels, p<0.05), perhaps accounting in part for earlier reports of elevated T-type current amplitude in those cells. In adult GAERS animals, we found elevated levels of alpha1G mRNA in neurons of the ventral posterior thalamic relay nuclei (64.8+/-3.5 vs. 53.5+/-1.7 grains/10(3) pixels, p<0.05), as well as higher levels of alpha1H mRNA in nRt neurons (32.6+/-0.8 vs. 28.2+/-1.6 grains/10(3) pixels, p<0.05). These results suggest that the epileptic phenotype apparent in adult GAERS may result in part from these significant, albeit small ( approximately 15-25%), elevations in T-type calcium channel mRNA levels.     

Inhibition of persistent sodium current fraction and voltage-gated L-type calcium current by propofol in cortical neurons: implications for its antiepileptic activity

PURPOSE: Although it is widely used in clinical practice, the mechanisms of action of 2,6-di-isopropylphenol (propofol) are not completely understood. We examined the electrophysiologic effects of propofol on an in vitro model of epileptic activity obtained from a slice preparation. METHODS: The effects of propofol were tested both on membrane properties and on epileptiform events consisting of long-lasting, paroxysmal depolarization shifts (PDSs) induced by reducing the magnesium concentration from the solution and by adding bicuculline and 4-aminopyridine.These results were integrated with a patch-clamp analysis of Na(+) and high-voltage activated (HVA) calcium (Ca(2+)) currents from isolated cortical neurons. RESULTS: In bicuculline, to avoid any interference by gamma-aminobutyric acid (GABA)-A receptors, propofol (3-100 microM) did not cause significant changes in the current-evoked, sodium (Na(+))-dependent action-potential discharge. However, propofol reduced both the duration and the number of spikes of PDSs recorded from cortical neurons. Interestingly, relatively low concentrations of propofol [half-maximal inhibitory concentration (IC(50)), 3.9 microM) consistently inhibited the "persistent" fraction of Na(+) currents, whereas even high doses (< or =300 microM) had negligible effects on the "fast" component of Na(+) currents. HVA Ca(2+) currents were significantly reduced by propofol, and the pharmacologic analysis of this effect showed that propofol selectively reduced L-type HVA Ca(2+) currents, without affecting N or P/Q-type channels. CONCLUSIONS: These results suggest that propofol modulates neuronal excitability by selectively suppressing persistent Na(+) currents and L-type HVA Ca(2+) conductances in cortical neurons. These effects might cooperate with the opening of GABA-A-gated chloride channels, to achieve depression of cortical activity during both anesthesia and status epilepticus.     

The 5-HT1A agonist 8-OH-DPAT increases the number of spike-wave discharges in a genetic rat model of absence epilepsy

The effects of the 5-HT_1A receptor agonist 8-hydroxy-2-(di-n-propylamino)-tetralin (8-OH-DPAT) on the epileptiform activity has been investigated in adult WAG/RIJ rats. Either intraperitoneal (0.1–0.5 mg/kg) or intracerebroventricular (2–20 μg/rat) administration of 8-OH-DPAT caused marked, dose-dependent increases in the number and mean cumulative duration of spike-wave discharges. These effects were attenuated by NAN-190, a 5-HT_1A receptor antagonist. These data indicate that serotonergic system regulates the epileptiform activity in this genetic model of human absence epilepsy.     

Early molecular and behavioral response to lipopolysaccharide in the WAG/Rij rat model of absence epilepsy and depressive-like behavior,involves interplay between AMPK,AKT/mTOR pathways and neuroinflammatory cytokine release

The mammalian target of rapamycin (mTOR) pathway has been recently indicated as a suitable drug target for the prevention of epileptogenesis. The mTOR pathway is known for its involvement in the control of the immune system. Since neuroinflammation is recognized as a major contributor to epileptogenesis, we wished to examine whether the neuroprotective effects of mTOR modulation could involve a suppression of the neuroinflammatory process in epileptic brain. We have investigated the early molecular mechanisms involved in the effects of intracerebral administration of the lipopolysaccharide (LPS) in the WAG/Rij rat model of absence epilepsy, in relation to seizure generation and depressive-like behavior; we also tested whether the effects of LPS could be modulated by treatment with rapamycin (RAP), a specific mTOR inhibitor. We determined, in specific rat brain areas, levels of p-mTOR/p-p70S6K and also p-AKT/p-AMPK as downstream or upstream indicators of mTOR activity and tested the effects of LPS and RAP co-administration. Changes in the brain levels of pro-inflammatory cytokines IL-1β and TNF-α and their relative mRNA expression levels were measured, and the involvement of nuclear factor-κB (NF-κB) was also examined in vitro. We confirmed that RAP inhibits the aggravation of absence seizures and depressive-like/sickness behavior induced by LPS in the WAG/Rij rats through the activation of mTOR and show that this effect is correlated with the ability of RAP to dampen and delay LPS increases in neuroinflammatory cytokines IL-1β and TNF-α, most likely through inhibition of the activation of NF-κB. Our results suggest that such a mechanism could contribute to the antiseizure, antiepileptogenic and behavioral effects of RAP and further highlight the potential therapeutic usefulness of mTOR inhibition in the management of human epilepsy and other neurological disorders. Furthermore, we show that LPS-dependent neuroinflammatory effects are also mediated by a complex interplay between AKT, AMPK and mTOR with specificity to selective brain areas. In conclusion, neuroinflammation appears to be a highly coordinated phenomenon, where timing of intervention may be carefully evaluated in order to identify the best suitable target.     

Increased persistent sodium currents in rat entorhinal cortex layer V neurons in a post-status epilepticus model of temporal lobe epilepsy

PURPOSE: Spontaneous seizures in rats emerge several weeks after induction of status epilepticus with pharmacologic treatment or electrical stimulation, providing an animal model for human temporal lobe epilepsy. In this study, we investigated whether status epilepticus caused changes in the function of voltage-gated sodium channels in entorhinal cortex layer V neurons, a cellular group important for the genesis of limbic seizures. METHODS: We induced status epilepticus in rats, by using lithium-pilocarpine, and then 2-12 weeks later, used whole-cell voltage-clamp to examine voltage-activated sodium currents of acutely dissociated layer V neurons. RESULTS: Transient sodium currents of entorhinal cortex layer V neurons isolated from 9- to 12-week post-status epilepticus rats were similar to currents in age-matched controls; however, low-threshold persistent sodium currents were significantly larger. This increase in persistent activity was not seen 2-3 weeks after pilocarpine treatment; thus it occurred after a delay comparable to the delay in the appearance of spontaneous seizures. CONCLUSIONS: Increased persistent currents are expected to accentuate neuronal excitability and thus may contribute to the genesis of spontaneous seizures after status epilepticus.