Extrasynaptic δGABAA receptors mediate resistance to migraine-like phenotype in rats.

BACKGROUND: GABA, a key inhibitory neurotransmitter, has synaptic and extrasynaptic receptors on the postsynaptic neuron. Background GABA, which spills over from the synaptic cleft, acts on extrasynaptic delta subunit containing GABAA receptors. The role of extrasynaptic GABAergic input in migraine is unknown. We investigated the susceptibility to valid migraine-provoking substances with clinically relevant behavioral readouts in Genetic Absence Epilepsy of Rats Strasbourg (GAERS), in which the GABAergic tonus was altered. Subsequently, we screened relevant GABAergic mechanisms in Wistar rats by pharmacological means to identify the mechanisms. METHODS: Wistar and GAERS rats were administered nitroglycerin (10 mg/kg) or levcromakalim (1 mg/kg). Mechanical allodynia and photophobia were assessed using von Frey monofilaments and a dark-light box. Effects of GAT-1 blocker tiagabine (5 mg/kg), GABAB receptor agonist baclofen (2 mg/kg), synaptic GABAA receptor agonist diazepam (1 mg/kg), extrasynaptic GABAA receptor agonists gaboxadol (4 mg/kg), and muscimol (0.75 mg/kg), T-type calcium channel blocker ethosuximide (100 mg/kg) or synaptic GABAA receptor antagonist flumazenil (15 mg/kg) on levcromakalim-induced migraine phenotype were screened. RESULTS: Unlike Wistar rats, GAERS exhibited no reduction in mechanical pain thresholds or light aversion following nitroglycerin or levcromakalim injection. Ethosuximide did not reverse the resistant phenotype in GAERS, excluding the role of T-type calcium channel dysfunction in this phenomenon. Tiagabine prevented levcromakalim-induced mechanical allodynia in Wistar rats, suggesting a key role in enhanced GABA spillover. Baclofen did not alleviate mechanical allodynia. Diazepam failed to mitigate levcromakalim-induced migraine phenotype. Additionally, the resistant phenotype in GAERS was not affected by flumazenil. Extrasynaptic GABAA receptor agonists gaboxadol and muscimol inhibited periorbital allodynia in Wistar rats. CONCLUSION: Our study introduced a rat strain resistant to migraine-provoking agents and signified a critical involvement of extrasynaptic δGABAergic receptors. Extrasynaptic δ GABAA receptors, by mediating constant background inhibition on the excitability of neurons, stand as a novel drug target with a therapeutic potential in migraine.

Pharmacokinetic characterization of favipiravir in patients with COVID-19.

This prospective observational study describes the pharmacokinetic characteristics of favipiravir in adult patients hospitalized for mild to moderate COVID-19 with a positive RT-PCR test. Favipiravir was administered for 5 days, with a loading dose of 3200 mg and a maintenance dose of 1200 mg/day. Serial blood samples were collected on Day 2 and Day 4 of the therapy. Laboratory findings of the patients (n = 21) and in-hospital mortality were recorded. Favipiravir concentrations exhibited substantial variability and a significant decrease during the treatment of COVID-19. The median favipiravir trough concentration (C0-trough ) on Day 2 was 21.26 (interquartile range [IQR], 8.37-30.78) µg/mL, whereas it decreased significantly to 1.61 (IQR, 0.00-6.41) µg/mL on Day 4, the area under the concentration-time curve decreased by 68.5%. Day 2 C0-trough of female patients was higher than male patients. Our findings indicate that favipiravir concentrations show significant variability during the treatment of COVID-19 and therapeutic drug monitoring may be necessary to maintain targeted concentrations.

Decreased Hyperpolarization-Activated Cyclic Nucleotide-Gated Channel 2 Activity in a Rat Model of Absence Epilepsy and the Effect of ZD7288, an Ih Inhibitor, on the Spike-and-Wave Discharges.

INTRODUCTION: Hyperpolarization-activated cyclic nucleotide-gated (HCN) channel currents of Ih and absence epilepsy seizures are associated, but studies reveal differential results. OBJECTIVE: In our study, we aimed to investigate the role of the HCN channels on the expression of spike-and-wave discharges (SWDs) using the Genetic Absence Epilepsy Rats from Strasbourg (GAERS) model. METHODS: HCN isoform levels from isolated brains of both naïve nonepileptic Wistar and GAERS groups were evaluated by enzyme-linked immunosorbent assay. ZD7288, an Ih inhibitor as well as an HCN channel antagonist, was administered intracerebroventricularly to the adult GAERS groups, and to evaluate their SWD activities, electroencephalography was recorded. The effect of ZD7288 on the cumulative total duration and number of SWDs and the mean duration of each SWD complex was evaluated. RESULTS: The HCN2 levels in the cortex and hippocampus of the GAERS group were lower compared to the naïve nonepileptic Wistar group (p < 0.05). ZD7288 increased the number of SWDs at the 20th and 120th min with the highest administered dose of 7 µg (p < 0.05). CONCLUSION: The Ih inhibitor ZD7288 increased the number of SWDs in a genetic absence epilepsy rat model, although this increase may not be significant due to the inconsistent time-dependent effects. In GAERS, the cortical and hippocampal HCN2 channel levels were significantly lower compared to the control group. Further studies are needed with higher doses of ZD7288 to determine if the effects will increase drastically.

Climate change and epilepsy: Insights from clinical and basic science studies.

Climate change is with us. As professionals who place value on evidence-based practice, climate change is something we cannot ignore. The current pandemic of the novel coronavirus, SARS-CoV-2, has demonstrated how global crises can arise suddenly and have a significant impact on public health. Global warming, a chronic process punctuated by acute episodes of extreme weather events, is an insidious global health crisis needing at least as much attention. Many neurological diseases are complex chronic conditions influenced at many levels by changes in the environment. This review aimed to collate and evaluate reports from clinical and basic science about the relationship between climate change and epilepsy. The keywords climate change, seasonal variation, temperature, humidity, thermoregulation, biorhythm, gene, circadian rhythm, heat, and weather were used to search the published evidence. A number of climatic variables are associated with increased seizure frequency in people with epilepsy. Climate change-induced increase in seizure precipitants such as fevers, stress, and sleep deprivation (e.g. as a result of more frequent extreme weather events) or vector-borne infections may trigger or exacerbate seizures, lead to deterioration of seizure control, and affect neurological, cerebrovascular, or cardiovascular comorbidities and risk of sudden unexpected death in epilepsy. Risks are likely to be modified by many factors, ranging from individual genetic variation and temperature-dependent channel function, to housing quality and global supply chains. According to the results of the limited number of experimental studies with animal models of seizures or epilepsy, different seizure types appear to have distinct susceptibility to seasonal influences. Increased body temperature, whether in the context of fever or not, has a critical role in seizure threshold and seizure-related brain damage. Links between climate change and epilepsy are likely to be multifactorial, complex, and often indirect, which makes predictions difficult. We need more data on possible climate-driven altered risks for seizures, epilepsy, and epileptogenesis, to identify underlying mechanisms at systems, cellular, and molecular levels for better understanding of the impact of climate change on epilepsy. Further focussed data would help us to develop evidence for mitigation methods to do more to protect people with epilepsy from the effects of climate change.

Atipamezole, a specific α2A antagonist, suppresses spike-and-wave discharges and alters Ca2⁺ /calmodulin-dependent protein kinase II in the thalamus of genetic absence epilepsy rats.

OBJECTIVE: The role of α2A adrenergic receptors (α2A ARs) in absence epilepsy is not well characterized. Therefore, we investigated the outcomes of the specific antagonism of α2A ARs on the spike-and-wave discharges (SWDs) in genetic absence epilepsy rats from Strasbourg (GAERSs), together with its influence on the behavior and second messenger systems, which may point to the mechanisms to which a possible SWD modulation can be related. METHODS: Atipamezole, an α2A AR antagonist, was administered intracerebroventricularly to the adult GAERSs, and electroencephalography (EEG) was conducted. The cumulative duration and number of SWDs, and the mean duration of each SWD complex were counted. The relative power of the EEG frequency bands and behavioral activity after the acute application of two doses (12 and 31 µg/5 µL) of atipamezole were evaluated. The levels of cyclic adenosine monophosphate and calcium/calmodulin-dependent kinase II (CaMKII) were measured in the cortex, thalamus, and hippocampus of naive Wistar rats and GAERSs, administered with artificial cerebrospinal fluid (aCSF) as a vehicle, or either acute or chronic atipamezole (12 µg), the latter being administered for 5 consecutive days. RESULTS: Atipamezole significantly suppressed SWDs dose-dependently, without affecting the relative power values of EEG frequency spectrum. The stereotypic activity was significantly lower in both naive Wistar rats and GAERSs receiving the highest dose (31 µg) of atipamezole compared to GAERSs receiving aCSF. In GAERSs, CaMKII levels were found to be higher in the thalamus after the acute and chronic application of SWD-suppressing doses of atipamezole (12 and 31 µg) compared to aCSF. SIGNIFICANCE: This study emphasizes the α2 AR-related modulation of absence epilepsy and particularly the significance of α2 AR antagonism in suppressing SWDs. Atipamezole's SWD-suppressive actions may be through CaMKII-mediated second messenger systems in the thalamus.

Effect of U-92032, T-Type Ca2+ Channel Blocker, on Rats with Genetic Absence Epilepsy.

INTRODUCTION: Absence epilepsy is associated with diffuse spike-and-wave discharges (SWD) on the electroencephalogram (EEG). Recent studies have demonstrated that the primary somatosensory cortex is also implicated in the generation of the SWDs. OBJECTIVE: This study investigated the effects of systemic and local administrations of U-92032 into the brain of Genetic Absence Epilepsy Rats from Strasbourg (GAERS). METHODS: GAERS animals underwent stereotaxic surgery for the placement of EEG recording electrodes and guide cannulas for U-92032 administration into the lateral ventricle (intracerebroventricular [i.c.v.]), upper lips area (S1Ulp) or barrel field area (S1B) of primary somatosensory cortex. Following 7 days of recovery, electrical activity was recorded continuously for 1 h before and 6 h after intraperitoneal (0.25; 1; 5 mg/kg i.p.) or local U-92032 or dimethyl sulfoxide (DMSO) injections. RESULTS: No changes were detected in the cumulative duration, mean duration, and number of SWDs following i.p. U-92032 injections. Local i.c.v. injections of U-92032 caused a significant decrease in the cumulative duration (i.c.v., 50 and 100 nmol/L), mean duration (i.c.v., 50, 100, and 250 nmol/L), and the number (i.c.v., 250 nmol/L) of SWDs compared to DMSO groups. Intra-cortical (S1Ulp and S1B) U-92032 injections caused a significant decrease in all 3 parameters compared to DMSO groups, as well. CONCLUSION: Intra-cortical injection of U-92032 caused almost complete removal of SWDs in GAERS and i.c.v. administration resulted in a significant reduction. However, systemic i.p. administration did not cause a significant change with the applied -doses.

Ultrastructural GABA immunogold labeling in the substantia nigra pars reticulata of kindled genetic absence epilepsy rats.

Genetic Absence Epilepsy Rats from Strasbourg (GAERS) is a well-known animal model of absence epilepsy and they are resistant to electrical kindling stimulations. The present study aimed to examine possible differences in gamma-aminobutyric acid (GABA) levels and synapse counts in the substantia nigra pars reticulata anterior (SNRa) and posterior (SNRp) regions between GAERS and Wistar rats receiving kindling stimulations. Animals in the kindling group either received six stimulations in the amygdala and had grade 2 seizures or they were kindled, having grade five seizures. Rats were decapitated one hour after the last stimulation. SNR regions were obtained after vibratome sectioning of the brain tissue. GABA immunoreactivity was detected by immunogold method and synapses were counted. Sections were observed by transmission electron microscope and analyzed by Image J program. GABA density in the SNRa region of fully kindled GAERS and Wistar groups increased significantly compared to that of their corresponding grade 2 groups. The number of synapses increased significantly in kindled and grade 2 GAERS groups, compared to kindled and grade 2 Wistar groups, respectively, in the SNRa region. GABA density in the SNRp region of kindled GAERS group increased significantly compared to that of GAERS grade 2 group. In the SNRp region, both kindled and grade 2 GAERS groups were found to have increased number of synapses compared to that of GAERS control group. We concluded that both SNRa and SNRp regions may be important in modulating resistance of GAERS to kindling stimulations.

Ultrastructural investigation of synaptic alterations in the rat hippocampus after irradiation and hyperthermia.

This study aimed to investigate ultrastructural synaptic alterations in rat hippocampus after in utero exposure to irradiation (IR) and postnatal exposure to hyperthermia (HT). There were four groups in each of the time points (3rd and 6th months). IR group: Pregnant rats were exposed to radiation on the 17th gestational day. HT group: Hyperthermia was applied to the rat pups on the 10th day after their birth. IR+HT group: Both IR and HT were applied at the same time periods. Control group: No IR or HT was applied. Rat pups were sacrificed after 3 and 6 months. Thin sections from the dentate gyrus (DG) and the CA3 of hippocampus were evaluated for synapse numbers by electron microscopy. Synapses were counted, and statistical analysis was performed. Abnormalities in myelin sheath, mossy terminals and neuropil were observed in the CA3 and DG of all groups. The synapses in the CA3 region were significantly increased in the IR-3rd month, IR-6th month, and IR+HT-3rd month groups vs control group. Synapses were significantly increased in the DG of HT-3rd month group. A trend for an increase in synapse numbers was seen in the CA3 and DG. Increased number of synapses in the rat hippocampus may be due to mossy fiber sprouting, possibly caused by in utero irradiation and/or postnatal hyperthermia.

Suppression of Hyperpolarization-Activated Cyclic Nucleotide-Gated Channel Function in Thalamocortical Neurons Prevents Genetically Determined and Pharmacologically Induced Absence Seizures.

Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels and the Ih current they generate contribute to the pathophysiological mechanisms of absence seizures (ASs), but their precise role in neocortical and thalamic neuronal populations, the main components of the network underlying AS generation, remains controversial. In diverse genetic AS models, Ih amplitude is smaller in neocortical neurons and either larger or unchanged in thalamocortical (TC) neurons compared with nonepileptic strains. A lower expression of neocortical HCN subtype 1 channels is present in genetic AS-prone rats, and HCN subtype 2 knock-out mice exhibit ASs. Furthermore, whereas many studies have characterized Ih contribution to "absence-like" paroxysmal activity in vitro, no data are available on the specific role of cortical and thalamic HCN channels in behavioral seizures. Here, we show that the pharmacological block of HCN channels with the antagonist ZD7288 applied via reverse microdialysis in the ventrobasal thalamus (VB) of freely moving male Genetic Absence Epilepsy Rats from Strasbourg decreases TC neuron firing and abolishes spontaneous ASs. A similar effect is observed on γ-hydroxybutyric acid-elicited ASs in normal male Wistar rats. Moreover, thalamic knockdown of HCN channels via virally delivered shRNA into the VB of male Stargazer mice, another genetic AS model, decreases spontaneous ASs and Ih-dependent electrophysiological properties of VB TC neurons. These findings provide the first evidence that block of TC neuron HCN channels prevents ASs and suggest that any potential anti-absence therapy that targets HCN channels should carefully consider the opposite role for cortical and thalamic Ih in the modulation of absence seizures.SIGNIFICANCE STATEMENT Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels play critical roles in the fine-tuning of cellular and network excitability and have been suggested to be a key element of the pathophysiological mechanism underlying absence seizures. However, the precise contribution of HCN channels in neocortical and thalamic neuronal populations to these nonconvulsive seizures is still controversial. In the present study, pharmacological block and genetic suppression of HCN channels in thalamocortical neurons in the ventrobasal thalamic nucleus leads to a marked reduction in absence seizures in one pharmacological and two genetic rodent models of absence seizures. These results provide the first evidence that block of TC neuron HCN channels prevents absence seizures.

Cav 3.2 T-type calcium channel mutation influences kindling-induced thalamic neuronal firing patterns in Genetic Absence Epilepsy Rats From Strasbourg.

OBJECTIVE: Recent data indicate that amygdala kindling leads to significant changes in interictal neuronal firing patterns of thalamic reticular nucleus (TRN) neurons by decreasing the spontaneous firing rate and increasing burst firing in nonepileptic control (NEC) rats. Genetic Absence Epilepsy Rats From Strasbourg (GAERS) were resistant to these kindling-induced firing changes in TRN neurons, and are also resistant to the progression of kindling. We investigated whether a homozygous, missense, single nucleotide mutation (R1584P) in the Cav 3.2 T-type Ca2+ channel gene, which has been correlated with the expression of absence seizures in GAERS, influenced kindling progression and TRN firing patterns. METHODS: Double-crossed (GAERS vs NEC; F2) rats that were homozygous for the Cav 3.2 mutation (PP) and those negative for the mutation (RR) were implanted with a stimulating electrode in the amygdala. Rats received a total of 30 kindling stimulations at their afterdischarge threshold current twice daily, and kindling progression was evaluated. Thereafter, the extracellular neuronal activity of TRN neurons was recorded in vivo under neuroleptanesthesia to investigate the influence of Cav 3.2 mutation on TRN firing patterns. RESULTS: We found that the R1584P mutation did not affect kindling progression in F2 crosses (P = 0.78). However, it influenced kindling-induced neuronal firing of TRN neurons. After 30 stimulations, RR rats exhibited a lower firing rate and a higher percentage of burst firing compared to PP rats. The decrease in firing frequency was correlated with the increase in the amount of burst firing in RR rats (R2  = 0.497). SIGNIFICANCE: Our findings suggest that mutation in Cav 3.2 T-type Ca2+ channels may play a role in the resistance to kindling-induced changes in TRN neurons to a low-frequency and high-percentage bursting pattern seen in association with the convulsive stages of amygdala kindling, but is not in itself enough to explain the resistance to kindling progression observed in GAERS.

Prelimbic Cortex Deep Brain Stimulation Reduces Binge Size in a Chronic Binge Eating Rat Model.

BACKGROUND: Binge eating (BE) involves the consumption of a large amount of food in a short period of time and a loss of control during the binge episode. It is a key feature of the major subtypes of eating disorders like bulimia nervosa, BE disorder, anorexia nervosa binge/purge type. Alterations in the mesocorticolimbic pathway play a crucial role in its pathophysiology. OBJECTIVES: We hypothesized that BE rats receiving deep brain stimulation (DBS) in the prelimbic cortex, a functional analog of the dorsolateral prefrontal cortex in humans, would have a reduced binge size compared with those receiving sham stimulation. METHODS: Eight male Sprague-Dawley rats were implanted with a DBS electrode in the left prelimbic cortex. A protocol which included limited access to a "sweet-fat" diet was used to achieve a chronic BE state in the rats. After reaching a stable binge size, each rat had undergone sham, low-frequency stimulation (60 Hz), and high-frequency (130 Hz) stimulation for 3 sessions each, and 2 consecutive treatments were separated by at least 2 empty sessions to allow a washout of the effects. A one-way ANOVA was used for the data analysis. RESULTS: Low-frequency (60 Hz) stimulation of the prelimbic cortex significantly reduced the binge size compared to the sham stimulation (p < 0.0001). High-frequency DBS (130 Hz) had no significant influence on this behavior when compared to sham stimulation (p = 0.9). CONCLUSIONS: This study suggests that low-frequency prelimbic cortex stimulation in BE would be useful for correcting prefrontal hypofunction which is strongly associated with BE and addiction pathogenesis.

WONOEP appraisal: Biomarkers of epilepsy-associated comorbidities.

Neurologic and psychiatric comorbidities are common in patients with epilepsy. Diagnostic, predictive, and pharmacodynamic biomarkers of such comorbidities do not exist. They may share pathogenetic mechanisms with epileptogenesis/ictogenesis, and as such are an unmet clinical need. The objectives of the subgroup on biomarkers of comorbidities at the XIII Workshop on the Neurobiology of Epilepsy (WONOEP) were to present the state-of-the-art recent research findings in the field that highlighting potential biomarkers for comorbidities in epilepsy. We review recent progress in the field, including molecular, imaging, and genetic biomarkers of comorbidities as discussed during the WONOEP meeting on August 31-September 4, 2015, in Heybeliada Island (Istanbul, Turkey). We further highlight new directions and concepts from studies on comorbidities and potential new biomarkers for the prediction, diagnosis, and treatment of epilepsy-associated comorbidities. The activation of various molecular signaling pathways such as the "Janus Kinase/Signal Transducer and Activator of Transcription," "mammalian Target of Rapamycin," and oxidative stress have been shown to correlate with the presence and severity of subsequent cognitive abnormalities. Furthermore, dysfunction in serotonergic transmission, hyperactivity of the hypothalamic-pituitary-adrenocortical axis, the role of the inflammatory cytokines, and the contributions of genetic factors have all recently been regarded as relevant for understanding epilepsy-associated depression and cognitive deficits. Recent evidence supports the utility of imaging studies as potential biomarkers. The role of such biomarker may be far beyond the diagnosis of comorbidities, as accumulating clinical data indicate that comorbidities can predict epilepsy outcomes. Future research is required to reveal whether molecular changes in specific signaling pathways or advanced imaging techniques could be detected in the clinical settings and correlate with epilepsy-associated comorbidities. A reliable biomarker will allow a more accurate diagnosis and improved treatment of epilepsy-associated comorbidities.

The role of the substantia nigra pars reticulata in kindling resistance in rats with genetic absence epilepsy.

OBJECTIVE: Genetic Absence Epilepsy Rats from Strasbourg (GAERS) show a resistance to secondary generalization of focal limbic seizures evoked by kindling. The substantia nigra pars reticulata (SNR) is involved in the propagation and modulation of seizures in kindling. We first examined the role of the SNRanterior and SNRposterior subregions in the resistance to the development of kindling in GAERS. Subsequently, to determine whether kindling resistance relates to differential sensitivity of γ-aminobutyric acid γ-aminobutyric acid (GABA)ergic or dopaminergic SNR neurons to kindling, we studied the effects of kindling-inducing stimulations on parvalbumin (PRV; GABAergic neuron marker) or tyrosine hydroxylase (TH; dopaminergic neuron marker) immunoreactivity (ir), respectively, in GAERS and in nonepileptic control (NEC) Wistar rats that lack kindling resistance. METHODS: Adult male GAERS were implanted with a stimulation electrode in the amygdala, and bilateral injection cannulas for lidocaine or saline injection (30 min before each kindling stimulation until the animals reached three stage 5 seizures or the 22 stimulations) into the SNRanterior or SNRposterior . In another experiment, PRV-ir in SNRanterior and SNRposterior and TH-ir in SNRposterior only were densitometrically compared in GAERS-SHAM, NEC-SHAM GAERS-STIM, and NEC-STIM animals (6 kindling stimulations). RESULTS: Bilateral SNRposterior infusions of lidocaine eliminated the kindling resistance and resulted in stage 5 generalized motor seizures in all kindled rats. Bilateral lidocaine infusions in the SNRanterior failed to alter the kindling resistance in GAERS. PRV-ir in the SNRposterior was unaltered in GAERS-STIM but increased in NEC-STIM group. Cellular TH-ir in the SNRposterior significantly increased by kindling stimulations in both NEC-STIM and GAERS-STIM groups. SIGNIFICANCE: The kindling resistance in GAERS is mediated by the SNRposterior in a lidocaine-sensitive manner. The insensitivity to kindling stimulation of PRV-ir in SNRposterior of GAERS but not NEC rats, implicate GABAergic SNRposterior neurons in kindling resistance. In contrast, the observed stimulation-specific increase in TH-ir in the SNRposterior is unrelated to kindling resistance.

The effect of polymorphic metabolism enzymes on serum phenytoin level.

Phenytoin has a widespread use in epilepsy treatment and is mainly metabolized by hepatic cytochrome P450 enzymes (CYP). We have investigated CYP2C9*2, CYP2C9*3, CYP2C19*2 and CYP2C19*3 allelic variants in a Turkish population of patients on phenytoin therapy. Patients on phenytoin therapy (n = 102) for the prevention of epileptic seizures were included. Polymorphic alleles were analyzed by restriction fragment length polymorphism method. Serum concentrations of phenytoin were measured by fluorescence polarization immune assay method. The most frequent genotype was detected for CYP2C9 wild-type alleles (78.43 %), whereas CYP2C19*2/*2 (5.88 %) was the least frequent genotype group. According to the classification made with both enzyme polymorphisms, CYP2C9*1/*1-CYP2C19*1/*1 (G1: 41.17 %) genotype group was the most frequent whereas CYP2C9*1/*2-CYP2C19*1/*3 (G7: 0.98 %) was the least frequent one. The highest mean phenytoin level (27.95 ± 1.85 µg/ml) was detected in the G8 genotype group (CYP2C9*1/*3-CYP2C19*2/*3) and the G1 genotype group showed the lowest mean phenytoin level (7.43 ± 0.73 µg/ml). The mean serum concentration of phenytoin of the polymorphic patients with epilepsy was higher than that for the wild-type alleles both in the monotherapy and polytherapy patients. These results show the importance of the genetic polymorphism analysis of the main metabolizing enzyme groups of phenytoin for the dose adjustment.

Animal models of absence epilepsies: what do they model and do sex and sex hormones matter?

While epidemiological data suggest a female prevalence in human childhood- and adolescence-onset typical absence epilepsy syndromes, the sex difference is less clear in adult-onset syndromes. In addition, although there are more females than males diagnosed with typical absence epilepsy syndromes, there is a paucity of studies on sex differences in seizure frequency and semiology in patients diagnosed with any absence epilepsy syndrome. Moreover, it is unknown if there are sex differences in the prevalence or expression of atypical absence epilepsy syndromes. Surprisingly, most studies of animal models of absence epilepsy either did not investigate sex differences, or failed to find sex-dependent effects. However, various rodent models for atypical syndromes such as the AY9944 model (prepubertal females show a higher incidence than prepubertal males), BN model (also with a higher prevalence in males) and the Gabra1 deletion mouse in the C57BL/6J strain offer unique possibilities for the investigation of the mechanisms involved in sex differences. Although the mechanistic bases for the sex differences in humans or these three models are not yet known, studies of the effects of sex hormones on seizures have offered some possibilities. The sex hormones progesterone, estradiol and testosterone exert diametrically opposite effects in genetic absence epilepsy and pharmacologically-evoked convulsive types of epilepsy models. In addition, acute pharmacological effects of progesterone on absence seizures during proestrus are opposite to those seen during pregnancy. 17ß-Estradiol has anti-absence seizure effects, but it is only active in atypical absence models. It is speculated that the pro-absence action of progesterone, and perhaps also the delayed pro-absence action of testosterone, are mediated through the neurosteroid allopregnanolone and its structural and functional homolog, androstanediol. These two steroids increase extrasynaptic thalamic tonic GABAergic inhibition by selectively targeting neurosteroid-selective subunits of GABAA receptors (GABAARs). Neurosteroids also modulate the expression of GABAAR containing the γ2, α4, and δ subunits. It is hypothesized that differences in subunit expression during pregnancy and ovarian cycle contribute to the opposite effects of progesterone in these two hormonal states.

Seizure expression, behavior, and brain morphology differences in colonies of Genetic Absence Epilepsy Rats from Strasbourg.

OBJECTIVE: Originally derived from a Wistar rat strain, a proportion of which displayed spontaneous absence-type seizures, Genetic Absence Epilepsy Rats from Strasbourg (GAERS) represent the most widely utilized animal model of genetic generalized epilepsy. Here we compare the seizure, behavioral, and brain morphometric characteristics of four main GAERS colonies that are being actively studied internationally: two from Melbourne (MELB and STRAS-MELB), one from Grenoble (GREN), and one from Istanbul (ISTAN). METHODS: Electroencephalography (EEG) recordings, behavioral examinations, and structural magnetic resonance imaging (MRI) studies were conducted on GAERS and Non-Epileptic Control (NEC) rats to assess and compare the following: (1) characteristics of spike-and-wave discharges, (2) anxiety-like and depressive-like behaviors, and (3) MRI brain morphology of regions of interest. RESULTS: Seizure characteristics varied between the colonies, with MELB GAERS exhibiting the least severe epilepsy phenotype with respect to seizure frequency, and GREN GAERS exhibiting four times more seizures than MELB. MELB and STRAS-MELB colonies both displayed consistent anxiety and depressive-like behaviors relative to NEC. MELB and GREN GAERS showed similar changes in brain morphology, including increased whole brain volume and increased somatosensory cortical width. A previously identified mutation in the Cacna1h gene controlling the CaV 3.2 T-type calcium channel (R1584P) was present in all four GAERS colonies, but absent in all NEC rats. SIGNIFICANCE: This study demonstrates differences in epilepsy severity between GAERS colonies that were derived from the same original colony in Strasbourg. This multi-institute study highlights the potential impact of environmental conditions and/or genetic drift on the severity of epileptic and behavioral phenotypes in rodent models of epilepsy.

The effect of amygdala kindling on neuronal firing patterns in the lateral thalamus in the GAERS model of absence epilepsy.

OBJECTIVE: The co-occurrence of absence and mesial temporal lobe epilepsy is rare in both humans and animal models. Consistent with this, rat models of absence epilepsy, including genetic absence epilepsy rats from Strasbourg (GAERS), are resistant to experimental temporal lobe epileptogenesis, in particular by amygdala kindling. Structures within the cortical-thalamocortical system are critically involved in the generation and maintenance of the electrographic spike-and-wave discharges (SWDs) that characterize absence seizures. Using in vivo electrophysiologic recordings, this study investigated the role of thalamocortical circuitry in the generalization of amygdala-kindling induced seizures in the GAERS and the nonepileptic control (NEC) strain of Wistar rats. METHODS: GAERS and NEC rats were implanted with a stimulating electrode in amygdala and stimulated at afterdischarge threshold twice daily to a maximum number of 30 stimulations. Thereafter extracellular single neuron recordings were performed in vivo under neuroleptanesthesia in the thalamocortical network. RESULTS: In NEC rats, amygdala kindling induced convulsive class V seizures and altered characteristics of neuronal activity in the thalamic reticular nucleus (TRN), in particular decreased firing rates and increased burst firing patterns. Less marked changes were seen in other regions examined: the ventroposteromedial nucleus of thalamus (VPM), the CA3 region of the hippocampus, and the deep layers (V/VI) of the cortex. GAERS did not progress beyond class II seizures, with a matched number of kindling stimulations, and the thalamic neuronal firing alterations observed in NEC rats were not seen. SIGNIFICANCE: These data suggest that the TRN plays an important role in kindling resistance in GAERS and is central to the control of secondary generalization of limbic seizures.

Electroencephalographic and behavioral effects of intracerebroventricular or intraperitoneal injections of toxic honey extract in adult Wistar rats and GAERS.

Toxic honey, containing grayanotoxin, is obtained from nectar and polen of rhododendron. Consumed in excess it produces seizures and convulsions. In order to investigate whether the toxic honey extract can be used as a seizure model, we examined the electroencephalographic (EEG) and motor effects of intracerebroventricular (icv) or intraperitoneal (ip) injection of toxic honey extract in Wistar rats or in genetic absence epilepsy rats from Strasbourg (GAERS). Male Wistar rats or GAERS were stereotaxically implanted with bilateral cortical recording electrodes in all ip groups and cannula in the icv groups. Based on the previous study, an extract was obtained from the non-toxic and toxic honey. After the injection of the non-toxic or toxic honey extract, seizure stages and changes in EEG were evaluated from 9 am to noon. The icv administration of toxic honey extract produced stage 4 seizures and bilateral cortical spikes within 30-60 min and these effects disappeared after 120 min in Wistar rats or GAERS. The mean of bilateral cortical spike acitivity in EEG of Wistar rats was 804.2 ± 261.0 s in the 3-h period. After the icv administration of toxic honey extract to GAERS, the mean duration of spike-and-wave discharges (SWDs) in GAERS significantly decreased during the first 60 min and then returned to baseline level. Ip injection of toxic honey extract caused no seizure and no change in EEG in either GAERS or Wistars. These results suggest that the icv administration of toxic honey extract can be used as a seizure model.

Proteomic changes in the cortex membrane fraction of genetic absence epilepsy rats from Strasbourg.

Epilepsy is a serious neurodegenerative disorder with a high incidence and a variety of presentations and causes. Studies on brain from various animal models including chronic models: Genetic Absence Epilepsy Rats from Strasbourg (GAERS) are very useful for understanding the fundamental mechanisms associated with human epilepsy. Individual regions of the brain have different protein composition in different conditions. Therefore, proteomic analyses of the brain compartments are preferred for the development of new therapeutic targets in different pathophysiological conditions like neurodegenerative disorders. In this study, we describe a proteomic profiling of membrane fraction of cortex tissue from epileptic GAERS and non-epileptic Wistar rat brain by two-dimensional gel electrophoresis coupled with matrix-assisted laser desorption/ionization mass spectroscopy. Comparing the optical density of spots between groups, we found that one protein expression was significantly down-regulated (guanine nucleotide-binding protein G(I)/G(S)/G(T) subunit beta-1) and one protein expression was significantly up-regulated (14-3-3 protein epsilon isoform) in GAERS group. Our results indicate that these proteins might have played a significant role in epilepsy and may be considered as valuable therapeutic targets in the absence of epilepsy.