Non-epileptic Control Research Articles

The Sodium Channel B4-Subunits are Dysregulated in Temporal Lobe Epilepsy Drug-Resistant Patients.

Temporal lobe epilepsy (TLE) is the most common type of partial epilepsy referred for surgery due to antiepileptic drug (AED) resistance. A common molecular target for many of these drugs is the voltage-gated sodium channel (VGSC). The VGSC consists of four domains of pore-forming α-subunits and two auxiliary β-subunits, several of which have been well studied in epileptic conditions. However, despite the β4-subunits’ role having been reported in some neurological conditions, there is little research investigating its potential significance in epilepsy. Therefore, the purpose of this work was to assess the role of SCN4β in epilepsy by using a combination of molecular and bioinformatics approaches. We first demonstrated that there was a reduction in the relative expression of SCN4B in the drug-resistant TLE patients compared to non-epileptic control specimens, both at the mRNA and protein levels. By analyzing a co-expression network in the neighborhood of SCN4B we then discovered a linkage between the expression of this gene and K+ channels activated by Ca2+, or K+ two-pore domain channels. Our approach also inferred several potential effector functions linked to variation in the expression of SCN4B. These observations support the hypothesis that SCN4B is a key factor in AED-resistant TLE, which could help direct both the drug selection of TLE treatments and the development of future AEDs.

GAT-1 (rs2697153) and GAT-3 (rs2272400) polymorphisms are associated with febrile seizures and temporal lobe epilepsy.

The purpose of this study was to determine a possible association between two GABA transporter (GAT) single-nucleotide polymorphisms (SNPs), rs2697153 G>A in SLC6A1 (GAT-1) and rs2272400 C>T in SLC6A11 (GAT-3), and drug-resistant temporal lobe epilepsy (TLE). DNA was isolated from 138 TLE patients (from the neocortex) and 94 non-epileptic controls (from blood/buccal swaps), and amplified by polymerase chain reaction and subjected to restriction fragment length polymorphism assays. A subgroup of patients with a positive history of febrile seizures (FS+) and traumatic brain injury (TBI+) were investigated in a separate analysis. P values were obtained using the Chi-Square test and Fishers exact test. The GAT-1 SNP was different between patients and controls (p<0.05); the AA genotype was observed in 40% of the cases vs 23% of the controls (p<0.05). Thirty-one patients were FS+ and the GAT-3 CT genotype was observed significantly more frequently in the FS+ group (14%) than in the FS- group (1%; p<0.01). Thirteen patients were TBI+, and genotyping for GAT-1 and GAT-3 in these patients did not result in statistical differences between TBI+ and TBI- groups. The findings suggest that TLE is associated with GAT-1 and GAT-3 SNPs. More specifically, GAT-3 c1572T seems to be associated with TLE in patients with FS+. However, the pathophysiological consequences of these SNPs remain to be elucidated.

Cognitive Impairments in Touchscreen-based Visual Discrimination and Reversal Learning in Genetic Absence Epilepsy Rats from Strasbourg

Absence Epilepsy (AE) is associated with recurrent losses of awareness and synchronous bilateral spike-wave discharges (SWDs). While seizures do not generally continue into adulthood, cognitive and behavioral comorbidities persist. One preclinical model used to investigate AE is the Genetic Absence Epilepsy Rats from Strasbourg (GAERS) which consistently have bilateral SWDs and similar behavioral profiles. In this experiment, we characterized discrimination learning and behavioral flexibility in female and male GAERS (n = 7 per sex) and Non-Epileptic Controls (NEC; n = 8 per sex) in a touchscreen-based version of visual discrimination (VD) and reversal learning (RL). We found that, on average, female GAERS required more sessions (12.3) to complete pretraining compared to female and male NEC (8.2 and 7.3, respectively) and male GAERS (9.4). In contrast, there was a sex-specific impairment during VD with male GAERS requiring more sessions on average (12.3) than male and female NEC (both 7.5) and female GAERS (8.3). Additionally, male GAERS completed >30% more selection and correction trials during VD and made >30% more errors.Both female and male GAERS required more sessions on average (9.1 and 10.7, respectively) of RL compared to female and male NEC (6.4 and 6.0 sessions, respectively). Accordingly, GAERS performed ∼30% more selection trials and correction trials compared to NEC, although only male GAERS made significantly more errors (>40%). Deficits in VD and RL were not associated with differences in correct or incorrect response latency, or reward collection latency, suggesting impairments are not due to alterations in locomotor activity or motivation. Together, these data suggest that GAERS have impaired behavioral flexibility and identify some sex-dependent differences. Thus, GAERS may be suitable for assessing the potential benefit of antiepileptic drugs on comorbid behavioral and cognitive deficits.

Brain Aminergic Deficiency in Absence Epileptic Rats: Dependency on Seizure Severity and Their Functional Coupling at Rest

Brain aminergic tone was examined in rats with different genetically determined generalized epilepsies and in normal rats. Our previously published database was analyzed to examine if there were functional correlations between brain biogenic amines and their metabolic rates within particular cerebral regions. Rats with genetically determined absence (AbS) and/or audiogenic seizures (AGS) served as models for human generalized non-convulsive and convulsive epilepsies. Tissue concentrations of 5-HT, dopamine (DA) and their main metabolites, as well as tryptophan (TRT), histamine (HA) and noradrenaline (NA) were assayed in different brain regions and analyzed for within-regions correlations in non-stressed conditions. It was found that AbS rats had higher indices of DA-ergic metabolism than non-epileptic controls, but the severity of AbS correlated with a widespread dopaminergic deficiency, as expressed in lowered indices of DA metabolism. AGS exerted no major effects on tissue neurochemistry. Brain DA-ergic and 5HT-ergic metabolic indices (measured as the ratio of the metabolite to the parent transmitter) were closely linked in striatum and pons-medulla of AbS rats. Remarkably, the tissue content of HA in pons-medulla of AbS rats displayed correlations with metabolic ratios of 5HT and DA, which was not seen in normal rats. It is hypothesized that enhanced metabolism of DA and 5HT are at least partial compensatory antiepileptic mechanisms in the brain, and a disturbance of this compensation leads to an aggravation of absence seizures.

GABA bouton subpopulations in the human dentate gyrus are differentially altered in mesial temporal lobe epilepsy.

Medically intractable temporal lobe epilepsy is a devastating disease, for which surgical removal of the seizure onset zone is the only known cure. Multiple studies have found evidence of abnormal dentate gyrus network circuitry in human mesial temporal lobe epilepsy (MTLE). Principal neurons within the dentate gyrus gate entorhinal input into the hippocampus, providing a critical step in information processing. Crucial to that role are GABA-expressing neurons, particularly parvalbumin (PV)-expressing basket cells (PVBCs) and chandelier cells (PVChCs), which provide strong, temporally coordinated inhibitory signals. Alterations in PVBC and PVChC boutons have been described in epilepsy, but the value of these studies has been limited due to methodological hurdles associated with studying human tissue. We developed a multilabel immunofluorescence confocal microscopy and a custom segmentation algorithm to quantitatively assess PVBC and PVChC bouton densities and to infer relative synaptic protein content in the human dentate gyrus. Using en bloc specimens from MTLE subjects with and without hippocampal sclerosis, paired with nonepileptic controls, we demonstrate the utility of this approach for detecting cell-type specific synaptic alterations. Specifically, we found increased density of PVBC boutons, while PVChC boutons decreased significantly in the dentate granule cell layer of subjects with hippocampal sclerosis compared with matched controls. In contrast, bouton densities for either PV-positive cell type did not differ between epileptic subjects without sclerosis and matched controls. These results may explain conflicting findings from previous studies that have reported both preserved and decreased PV bouton densities and establish a new standard for quantitative assessment of interneuron boutons in epilepsy.NEW & NOTEWORTHY A state-of-the-art, multilabel immunofluorescence confocal microscopy and custom segmentation algorithm technique, developed previously for studying synapses in the human prefrontal cortex, was modified to study the hippocampal dentate gyrus in specimens surgically removed from patients with temporal lobe epilepsy. The authors discovered that chandelier and basket cell boutons in the human dentate gyrus are differentially altered in mesial temporal lobe epilepsy.

Epilepsy and migraine in adolescent and adult subjects in West Africa

Objective The aim of this work was to study migraine exposure in epileptics and non-epileptics in Parakou in 2018. Framework and methods This study was conducted in Parakou in Northen Benin.It was a case - control study of adolescents and adults of both sexes in Parakou. Each cases that were epileptic subjects were matched to three non-epileptic controls by age, sex and place of residence. Thirty epileptics ans ninegthy controls were included. The EPIDATA analysis EPI INFO 7.1 and SPSS2.1 software. Were used for the analysis. The p Results The mean age of the two groups was 32.33 ± 15.28 years [15–64 years]; with a sex ratio at 1.45. 43.33% of epileptics had at least one seizure per month; 56.67% suffered from generalized epilepsy. Frequency of migraine in epileptics was 63.33% and 17.78% in non-epileptics. There was a statistically significant association between migraine and epilepsy (p = .000). The relationship between migraine and epilepsy adjusted to religion, socioeconomic level, stress, temperature change, excessive light, menstrual period shows that epileptics are 6.18 times more likely to have migraine compared to non-epileptic subjects. Conclusion Epileptics are more exposed to migraine and migraine is more common in these subjects compared to non-epileptics.

Early reduced dopaminergic tone mediated by D3 receptor and dopamine transporter in absence epileptogenesis.

In Genetic Absence Epilepsy Rats From Strasbourg (GAERSs), epileptogenesis takes place during brain maturation and correlates with increased mRNA expression of D3 dopamine receptors (D3R). Whether these alterations are the consequence of seizure repetition or contribute to the development of epilepsy remains to be clarified. Here, we addressed the involvement of the dopaminergic system in epilepsy onset in GAERSs. Experiments were performed using rats at different stages of brain maturation from three strains according to their increasing propensity to develop absence seizures: nonepileptic control rats (NECs), Wistar Hannover rats, and GAERSs. Changes in dopaminergic neurotransmission were investigated using different behavioral and neurochemical approaches: autoradiography of D3R and dopamine transporter, single photon emission computed tomographic imaging, acute and chronic drug effects on seizure recordings (dopaminergic agonists and antagonists), quinpirole-induced yawns and dopamine synaptosomal uptake, microdialysis, brain tissue monoamines, and brain-derived neurotrophic factor quantification. Autoradiography revealed an increased expression of D3R in 14-day-old GAERSs, before absence seizure onset, that persists in adulthood, as compared to age-matched NECs. This was confirmed by increased yawns, a marker of D3R activity, and increased seizures when animals were injected with quinpirole at low doses to activate D3R. We also observed a concomitant increase in the expression and activity of the dopamine transporter in GAERSs before seizure onset, consistent with both lowered dopamine basal level and increased phasic responses. Our data show that the dopaminergic system is persistently altered in GAERSs, which may contribute not only to behavioral comorbidities but also as an etiopathogenic factor in the development of epilepsy. The data suggest that an imbalanced dopaminergic tone may contribute to absence epilepsy development and seizure onset, as its reversion by a chronic treatment with a dopamine stabilizer significantly suppressed epileptogenesis. Our data suggest a potential new target for antiepileptic therapies and/or improvement of quality of life of epileptic patients.

Coding and non-coding transcriptome of mesial temporal lobe epilepsy: Critical role of small non-coding RNAs

Our understanding of mesial temporal lobe epilepsy (MTLE), one of the most common form of drug-resistant epilepsy in humans, is derived mainly from clinical, imaging, and physiological data from humans and animal models. High-throughput gene expression studies of human MTLE have the potential to uncover molecular changes underlying disease pathogenesis along with novel therapeutic targets. Using RNA- and small RNA-sequencing in parrallel, we explored differentially expressed genes in the hippocampus and cortex of MTLE patients who had undergone surgical resection and non-epileptic controls. We identified differentially expressed genes in the hippocampus of MTLE patients and differentially expressed small RNAs across both the cortex and hippocampus. We found significant enrichment for astrocytic and microglial genes among up-regulated genes, and down regulation of neuron specific genes in the hippocampus of MTLE patients. The transcriptome profile of the small RNAs reflected disease state more robustly than mRNAs, even across brain regions which show very little pathology. While mRNAs segregated predominately by brain region for MTLE and controls, small RNAs segregated by disease state. In particular, our data suggest that specific miRNAs (e.g., let-7b-3p and let-7c-3p) may be key regulators of multiple pathways related to MTLE pathology. Further, we report a strong association of other small RNA species with MTLE pathology. As such we have uncovered novel elements that may contribute to the establishment and progression of MTLE pathogenesis and that could be leveraged as therapeutic targets.

Multiunit cluster firing patterns of piriform cortex and mediodorsal thalamus in absence epilepsy

ObjectiveThe objective of the study were to investigate patterns of multiunit cluster firing in the piriform cortex (PC) and mediodorsal thalamus (MDT) in a rat model of genetic generalized epilepsy (GGE) with absence seizures and to assess whether these regions contribute to the initiation or spread of generalized epileptiform discharges. MethodsMultiunit clusters and their corresponding local field potentials (LFPs) were recorded from microelectrode arrays implanted in the PC and MDT in urethane anesthetized Genetic Absence Epilepsy Rats from Strasbourg (GAERS) and nonepileptic control (NEC) rats. Peristimulus time histograms (PSTHs) and cross-correlograms were used to observe transient changes in both the rate of firing and synchrony over time. The phase locking of multiunit clusters to LFP signals (spike-LFP phase locking) was calculated for frequency bands associated with olfactory communication between the two brain regions. ResultsThere were significant increases in both rate of firing and synchronous activity at the onset of generalized epileptiform discharges in both PC and MDT. Prior to and following these increases in synchronous activity, there were periods of suppression. Significant increases in spike-LFP phase locking were observed within the PC prior to the onset of epileptiform discharges across all spectral bands. There were also significant increases in spike-LFP phase locking within the theta band of the MDT prior to onset. Between the two brain regions, there was a significant decrease in spike-LFP phase locking −0.5 s prior to onset in the theta band which coincided with a significant elevation in spike-LFP phase locking in the gamma band. ConclusionsBoth the PC and MDT are engaged in the absence epilepsy network. Early spike-LFP phase locking between these two brain regions suggests potential involvement in the initiation of seizure activity.

Cav3.2 T‐type calcium channel mutation influences kindling‐induced thalamic neuronal firing patterns in Genetic Absence Epilepsy Rats From Strasbourg

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. 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. 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). 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.

Delayed cortical maturation at the centrotemporal brain regions in patients with benign childhood epilepsy with centrotemporal spikes (BCECTS)

Benign childhood epilepsy with centrotemporal spikes (BCECTS) is an epilepsy syndrome commonly found in child and adolescent. Although the prognosis is mostly favorable as long as the seizure is well controlled. However, they are often suffering from the cognitive and behavioral problems which might be the consequences of the initial insults. It is still not clear whether the initial epileptiform discharges has long term impact on the resting-state brain activities at later ages. This study investigated the resting-state brain activities in BCECTS patients with clinical seizure remission stage (n = 16; 11 males) and compared with the non-epileptic, age-matched control subjects.Quantitative electroencephalography (qEEG) revealed a significantly higher absolute power of the theta and alpha waves in BCECTS patients with clinical seizure remission as compared with the non-epileptic control subjects. Interestingly, the differences were observed mainly over the centrotemporal electrodes which are the common sites of the initial epileptiform discharges. The differences were more significant in patients with bilateral epileptiform discharges than those with the unilateral epileptic activities. Typically, the brain wave power continuously decreases with increasing ages. Therefore, higher absolute powers of the brain waves indicate more delayed in cortical maturation compared with the non-epileptic control group. These findings indicated that BCECTS patients have delay cortical maturation at the centrotemporal brain regions even at the clinical seizure remission phase.

Genomic DNA methylation distinguishes subtypes of human focal cortical dysplasia.

ObjectivesFocal cortical dysplasia (FCD) is a major cause of drug‐resistant focal epilepsy in children, and the clinicopathological classification remains a challenging issue in daily practice. With the recent progress in DNA methylation–based classification of human brain tumors we examined whether genomic DNA methylation and gene expression analysis can be used to also distinguish human FCD subtypes.Methods DNA methylomes and transcriptomes were generated from massive parallel sequencing in 15 surgical FCD specimens, matched with 5 epilepsy and 6 nonepilepsy controls.ResultsDifferential hierarchical cluster analysis of DNA methylation distinguished major FCD subtypes (ie, Ia, IIa, and IIb) from patients with temporal lobe epilepsy patients and nonepileptic controls. Targeted panel sequencing identified a novel likely pathogenic variant in DEPDC5 in a patient with FCD type IIa. However, no enrichment of differential DNA methylation or gene expression was observed in mechanistic target of rapamycin (mTOR) pathway–related genes.SignificanceOur studies extend the evidence for disease‐specific methylation signatures toward focal epilepsies in favor of an integrated clinicopathologic and molecular classification system of FCD subtypes incorporating genomic methylation.

Erythropoietin Increases GABAA Currents in Human Cortex from TLE Patients

Erythropoietin (EPO) is a hematopoietic growth factor that has an important role in the erythropoiesis. EPO and its receptor (EPO-R) are expressed all over in the mammalian brain. Furthermore, it has been reported that EPO may exert neuroprotective effect in animal models of brain disorders as ischemia and epilepsy. Here, we investigate whether EPO could modulate the GABA-evoked currents (IGABA) in both human epileptic and non-epileptic control brain tissues.Therefore, we transplanted in Xenopus oocytes cell membranes obtained from autoptic and surgical brain tissues (cortex) of seven temporal lope epilepsy (TLE) patients and of five control patients. Two microelectrodes voltage-clamp technique has been used to record IGABA. Moreover, qRT-PCR assay was performed in the same human tissues to quantify the relative gene expression levels of EPO/EPO-R. To further confirm experiments in oocytes, we performed additional experiments using patch-clamp recording in slices obtained from rat cerebellum.We show that exposure to EPO significantly increased the amplitude of the IGABA in all the patients analyzed. No differences in the expression of EPO and EPO-R in both TLE and control patients have been found. Notably, the increase of IGABA has been recorded also in rat cerebellar slices. Our findings show a new modulatory action of EPO on GABAA receptors (GABAA-Rs). This effect could be relevant to balance the GABAergic dysfunction in human TLE. This article is part of a Special Issue entitled: Honoring Ricardo Miledi - outstanding neuroscientist of XX-XXI centuries.

The T-type calcium channel blocker Z944 reduces conditioned fear in Genetic Absence Epilepsy Rats from Strasbourg and the non-epileptic control strain.

Genetic Absence Epilepsy Rats from Strasbourg (GAERS) are a rodent model of childhood absence epilepsy (CAE) that display a gain-of-function mutation in the gene encoding the Cav3.2 T-type calcium channel. GAERS demonstrate heightened learning and delayed extinction of fear conditioning. Our objective in the present study was to examine the effects of the pan-T-type calcium channel blocker Z944 on the acquisition, consolidation and extinction of conditioned fear in GAERS and the non-epileptic control (NEC) strain. Z944 (10mg/kg; ip) was administered 15min prior to either acquisition, extinction day 1 (24hr later), acquisition and extinction day 1, or during the consolidation (post-acquisition) of tone-cued fear conditioning. Extinction was examined 24 and 48hr after conditioning. In drug naïve GAERS, increased freezing during the acquisition and extinction phases of fear conditioning was found. Short-term effects of Z944 on performance were observed as Z944 increased freezing during testing on the day it was administered. Z944 administered prior to the acquisition phase had a long-term effect on extinction. Specifically, both GAERS and NECs showed a decrease in freezing during extinction relative to drug naïve GAERS and NEC rats respectively. Regardless of strain or treatment, female rats showed reduced extinction of fear relative to male rats. These results demonstrate that T-type calcium channels contribute to the neural systems that mediate the learning and memory of conditioned fear. Overall, these findings suggest that T-type calcium channel blockers show promise in the treatment of learning impairments observed in disorders such as CAE.

513: Obstetric outcomes for women receiving newer generation antiepileptic drugs: retrospective cohort study using claims database

Women with epilepsy are at increased risk for adverse pregnancy outcomes. Since the 1990s, over 11 new antiepileptic drugs (AEDs) have been introduced. We aimed to describe the use of newer AEDs in pregnancy and differences in rates of obstetric outcomes. From the Truven Health MarketScan® Commercial Claims and Encounters Database (years 2011-14), we identified women who had a delivery admission, outpatient pharmacy dispenses for newer generation AEDs (Table) and an indication of epilepsy based on diagnosis codes during the 12 months prior to delivery. Obstetric outcomes included preterm birth, cesarean delivery, postpartum hemorrhage, placental abruption, hypertensive disorders of pregnancy, preterm labor, and severe maternal morbidity (SMM). We described the prevalence of the use of newer AEDs and rates of obstetric outcomes and absolute risk difference compared to women without a diagnosis of epilepsy (Table). In the cohort analyzed (n=1,028,125), the prevalence of epilepsy was 0.44% (n=4,528). Overall, 55% (n=2,508) received any of these AEDs during pregnancy, the most common of which were lamotrigine (49.8%) and levetiracetam (42.5%). Compared to the non-epileptic control group, women on AEDs showed significant increases in the absolute risk of cesarean delivery (5.0%), preeclampsia (0.7%), severe preeclampsia/eclampsia (0.9%) and SMM (1.6%). AEDs with the highest increases in absolute risk were topiramate (6.7%) for cesarean delivery, lamotrigine for pre-eclampsia (1.1%), and lacosamide for severe preeclampsia/eclampsia (8.4%) and SMM (9.3%). Of the 2,508 women, 414 (16.5%) received more than one AED during pregnancy. Compared to women receiving a single AED, those who received multiple AEDs had higher absolute risks of all outcomes except postpartum hemorrhage (Table). Compared to non-epileptic women, women on newer generation AEDs demonstrated an increased absolute risk for cesarean delivery, preeclampsia, severe preeclampsia/eclampsia and severe maternal morbidity. Women receiving multiple medications demonstrated the highest increase in absolute risk for adverse outcomes, similar to findings in older AEDs, possibly also reflecting a more complicated and difficult disease state.

The T-type calcium channel antagonist, Z944, alters social behavior in Genetic Absence Epilepsy Rats from Strasbourg

Abnormalities in social behavior are a co-morbid symptom of idiopathic generalized epilepsies such as childhood absence epilepsy. The Genetic Absence Epilepsy Rats from Strasbourg (GAERS) model is a spontaneously occurring absence epilepsy phenotype closely correlated to that of human absence epilepsies. Similar to the human conditions, GAERS display social abnormalities. Previous studies have only demonstrated social abnormalities in female GAERS, whereas social problems are observed in male and female patients. Seizures in GAERS result in part due to a gain-of-function missense mutation in the Cav3.2 T-type calcium channel gene. This study examined the effects of the pan-T-type calcium channel antagonist, Z944, on social interaction behaviors in male and female GAERS using an open field social interaction test. A second objective of this study was to examine the effects of Z944 on anxiety-like behavior in an open field locomotion test and elevated plus maze. Results showed a decrease in social activity in GAERS relative to non-epileptic control (NEC) rats. Acute, systemic Z944 (5 mg/kg; i.p.) consistently reduced introductory and aggressive behaviors in both GAERS and NECs whereas strain effects were observed for over-and-under crawl behaviors. In the open field locomotion test and elevated plus maze, Z944 increased anxiety-like behaviors in GAERS, whereas, Z944 produced inconsistent effects on anxiety-like behaviors in NECs. The results of this study suggest that the regulation of T-type calcium channel activity may be a useful strategy for the development of new therapeutic approaches for the treatment of social and affective abnormalities observed in absence epilepsy disorders.

Regulatory Architecture of the Neuronal Cacng2/Tarp\u03b32 Gene Promoter: Multiple Repressive Domains, a Polymorphic Regulatory Short Tandem Repeat, and Bidirectional Organization with Co-regulated lncRNAs

CACNG2 (TARPγ2, Stargazin) is a multi-functional regulator of excitatory neurotransmission and has been implicated in the pathological processes of several brain diseases. Cacng2 function is dependent upon expression level, but currently, little is known about the molecular mechanisms that control expression of this gene. To address this deficit and investigate disease-related gene variants, we have cloned and characterized the rat Cacng2 promoter and have defined three major features: (i) multiple repressive domains that include an array of RE-1 silencing transcription factor (REST) elements, and a calcium regulatory element-binding factor (CaRF) element, (ii) a (poly-GA) short tandem repeat (STR), and (iii) bidirectional organization with expressed lncRNAs. Functional activity of the promoter was demonstrated in transfected neuronal cell lines (HT22 and PC12), but although selective removal of REST and CaRF domains was shown to enhance promoter-driven transcription, the enhanced Cacng2 promoter constructs were still about fivefold weaker than a comparable rat Synapsin-1 promoter sequence. Direct evidence of REST activity at the Cacng2 promoter was obtained through co-transfection with an established dominant-negative REST (DNR) construct. Investigation of the GA-repeat STR revealed polymorphism across both animal strains and species, and size variation was also observed in absence epilepsy disease model cohorts (Genetic Absence Epilepsy Rats, Strasbourg [GAERS] and non-epileptic control [NEC] rats). These data provide evidence of a genotype (STR)-phenotype correlation that may be unique with respect to proximal gene regulatory sequence in the demonstrated absence of other promoter, or 3′ UTR variants in GAERS rats. However, although transcriptional regulatory activity of the STR was demonstrated in further transfection studies, we did not find a GAERS vs. NEC difference, indicating that this specific STR length variation may only be relevant in the context of other (Cacna1h and Kcnk9) gene variants in this disease model. Additional studies revealed further (bidirectional) complexity at the Cacng2 promoter, and we identified novel, co-regulated, antisense rat lncRNAs that are paired with Cacng2 mRNA. These studies have provided novel insights into the organization of a synaptic protein gene promoter, describing multiple repressive and modulatory domains that can mediate diverse regulatory inputs.

Serum NT-pro CNP levels in epileptic seizure, psychogenic non-epileptic seizure, and healthy subjects.

Epileptic seizure is the result of uncontrollable neural excitation in the brain. The C-type natriuretic peptide is a member of natriuretic peptide hormone family and is synthesized by brain and blood vessels in CNS. NT-pro CNP is an amino-terminal fragment of C-type natriuretic peptide and is more stable compared to its predecessor. In this study, we aimed to evaluate the role of NT-pro CNP in psychogenic non-epileptic seizures, epileptic seizures, and normal subjects. Thirty-three patients with epilepsy and 43 patients with psychogenic non-epileptic seizures were enrolled in this study. The control group consisted of 28 healthy subjects. Post-ictal serum levels of NT-pro CNP were acquired from all participants. Statistically significant differences between patient groups and controls regarding serum levels of NT-pro CNP were sought. NT-pro CNP levels were significantly lower in the epilepsy group than the psychogenic non-epileptic seizure group and control group with no significant difference between the psychogenic non-epileptic seizure and control group (p < 0.05). Post-ictal serum NT-pro CNP levels were lower in epileptic seizures compared to psychogenic non-epileptic seizures as well as healthy controls. We think that such a difference is associated with C-type natriuretic peptide-related neural mechanisms such as altered microcirculation, increased brain-blood barrier permeability, and synaptic stabilization.

Deficient post-ictal cardiorespiratory compensatory mechanisms mediated by the periaqueductal gray may lead to death in a mouse model of SUDEP

Post-ictal cardiorespiratory failure is implicated as a major cause of sudden unexpected death in epilepsy (SUDEP) in patients. The DBA/1 mouse model of SUDEP is abnormally susceptible to fatal seizure-induced cardiorespiratory failure (S-CRF) induced by convulsant drug, hyperthermia, electroshock, and acoustic stimulation. Clinical and pre-clinical studies have implicated periaqueductal gray (PAG) abnormalities in SUDEP. Recent functional neuroimaging studies observed that S-CRF resulted in selective changes in PAG neuronal activity in DBA/1 mice. The PAG plays a critical compensatory role for respiratory distress caused by numerous physiological challenges in non-epileptic individuals. These observations suggest that abnormalities in PAG-mediated cardiorespiratory modulation may contribute to S-CRF in DBA/1 mice. To evaluate this, electrical stimulation (20 Hz, 20–100 μA, 10 s) was presented in the PAG of anesthetized DBA/1 and C57BL/6 (non-epileptic) control mice, and post-stimulus changes in respiration [inter-breath interval (IBI)] and heart rate variability (HRV) were examined. The post-stimulus period was considered analogous to the post-ictal period when S-CRF occurred in previous DBA/1 mouse studies. PAG stimulation caused significant intensity-related decreases in IBI in both mouse strains. However, this effect was significantly reduced in DBA/1 vis-a-vis C57BL/6 mice. These changes began immediately following cessation of stimulation and remained significant for 10 s. This time period is critical for initiating resuscitation to successfully prevent seizure-induced death in previous DBA/1 mouse experiments. Significant post-stimulus increases in HRV were also seen at ≥60 μA in the PAG in C57BL/6 mice, which were absent in DBA/1 mice. These data along with previous neuroimaging findings suggest that compensatory cardiorespiratory modulation mediated by PAG is deficient, which may be important to the susceptibility of DBA/1 mice to S-CRF. These observations suggest that correcting this deficit pharmacologically or by electrical stimulation may help to prevent S-CRF. These findings further support the potential importance of PAG abnormalities to human SUDEP.

Abnormal Hippocampal Melatoninergic System: A Potential Link between Absence Epilepsy and Depression-Like Behavior in WAG/Rij Rats?

Absence epilepsy and depression are comorbid disorders, but the molecular link between the two disorders is unknown. Here, we examined the role of the melatoninergic system in the pathophysiology of spike and wave discharges (SWDs) and depression-like behaviour in the Wistar Albino Glaxo from Rijswijk (WAG/Rij) rat model of absence epilepsy. In WAG/Rij rats, SWD incidence was higher during the dark period of the light-dark cycle, in agreement with previous findings. However, neither pinealectomy nor melatonin administration had any effect on SWD incidence, suggesting that the melatoninergic system was not involved in the pathophysiology of absence-like seizures. Endogenous melatonin levels were lower in the hippocampus of WAG/Rij rats as compared to non-epileptic control rats, and this was associated with higher levels of melatonin receptors in the hippocampus, but not in the thalamus. In line with the reduced melatonin levels, cell density was lower in the hippocampus of WAG/Rij rats and was further reduced by pinealectomy. As expected, WAG/Rij rats showed an increased depression-like behaviour in the sucrose preference and forced swim tests, as compared to non-epileptic controls. Pinealectomy abolished the difference between the two strains of rats by enhancing depression-like behaviour in non-epileptic controls. Melatonin replacement displayed a significant antidepressant-like effect in both WAG/Rij and control rats. These findings suggest that a defect of hippocampal melatoninergic system may be one of the mechanisms underlying the depression-like phenotype in WAG/Rij rats and that activation of melatonin receptors might represent a valuable strategy in the treatment of depression associated with absence epilepsy.