Quantification of chloride channel 2 (CLCN2) gene isoforms in normal versus lesion- and epilepsy-associated brain tissue

The chloride channel 2 (CLCN2) gene codes for a protein organized in N- and C-terminal regions with regulatory functions and a transmembrane region which forms the ring of the pore. Mutations in the gene have previously been described in patients with idiopathic familial epilepsy. In this study we looked for new isoforms of CLCN2 and we estimated expression levels by real time PCR in brain tissue containing epileptic foci. Samples used in this study were first analyzed and selected to exclude mutations in the coding region of the gene. Four isoforms (skipping exons 3, 16, 22 and 6/7) were identified and quantified by Real Time PCR and compared with total expression of the gene. Expression of the region common to all CLCN2 isoforms was 50% less in epilepsy-associated brain tissue than in controls. The ratio of the various isoforms was slightly greater in epileptic than control tissue. The greatest difference was recorded in the temporal lobe for the isoform with skipped exon 22. Analysis of these isoforms in brain tissue containing epileptic foci suggests that CLCN2 could be implicated in epilepsy, even in the absence of mutations.     

Increased plasma glutamic acid in a genetic model of epilepsy

A significant increase in the plasma levels of glutamic acid and a significant decrease in aspartic acid and taurine in epileptic patients and their first degree relatives was reported more than a decade ago and an underlying genetic basis for these amino acid changes was suggested. The main objective of the present study was to determine the plasma levels of glutamic acid, aspartic acid and taurine in El mice which are an inbred epileptic mutant mouse strain. The results show a significant increase in plasma glutamic acid but no changes in aspartic acid or taurine in the epileptic mice as compared to controls. The data provide the first evidence of a significant increase in plasma glutamic acid in an animal model of hereditary epilepsy and substantiate the hypothesis that a genetic defect underlies the elevated plasma glutamic acid levels in association with epilepsy. The findings are also compatible with neurochemical and neurophysiological evidence implicating glutamic acid in the mechanism of seizures.     

Inhibition of miR-203 Reduces Spontaneous Recurrent Seizures in Mice

Inhibitory synaptic receptors are dysfunctional in epileptic brains, and agents that selectively target these receptors may be effective for the treatment of epilepsy. MicroRNAs interfere with the translation of target genes, including various synaptic proteins. Here, we show that miR-203 regulates glycine receptor-β (Glrb) in epilepsy models. miR-203 is upregulated in the hippocampus of epileptic mice and human epileptic brains and is predicted to target inhibitory synaptic receptors, including Glrb. In vitro transfection, target gene luciferase assays, and analysis of human samples confirmed the direct inhibition of GLRB by miR-203, and AM203, an antagomir targeting miR-203, reversed the effect of miR-203. When intranasal AM203 was administered, AM203 reached the brain and restored hippocampal GLRB levels in epileptic mice. Finally, intranasal AM203 reduced the epileptic seizure frequency of mice. Overall, this study suggests that GLRB expression in the epileptic brain is controlled by miR-203, and intranasal delivery of AM203 showed therapeutic effects in chronic epilepsy mice.     

Alterations in miRNA Levels in the Dentate Gyrus in Epileptic Rats

The aim of this study was to characterize changes in miRNA expression in the epileptic dentate gyrus. Status epilepticus evoked by amygdala stimulation was used to induce epilepsy in rats. The dentate gyri were isolated at 7 d, 14 d, 30 d and 90 d after stimulation (n=5). Sham-operated time-matched controls were prepared for each time point (n=5). The miRNA expression was evaluated using Exiqon microarrays. Additionally, mRNA from the same animals was profiled using Affymetrix microarrays. We detected miRNA expression signatures that differentiate between control and epileptic animals. Significant changes in miRNA expression between stimulated and sham operated animals were observed at 7 and 30 d following stimulation. Moreover, we found that there are ensembles of miRNAs that change expression levels over time. Analysis of the mRNA expression from the same animals revealed that the expression of several mRNAs that are potential targets for miRNA with altered expression level is regulated in the expected direction. The functional characterization of miRNAs and their potential mRNA targets indicate that miRNA can participate in several molecular events that occur in epileptic tissue, including immune response and neuronal plasticity. This is the first report on changes in the expression of miRNA and the potential functional impact of these changes in the dentate gyrus of epileptic animals. Complex changes in the expression of miRNAs suggest an important role for miRNA in the molecular mechanisms of epilepsy.     

Effects of phenazepam and aliphatic alcohols on epileptic complex created in the rat brain cortex

Effects of phenazepam and aliphatic alcohols (methanol, ethanol, butanol, and propanol) on associated epileptic complex created in the rat brain cortex by applications of strychnine were studied. Phenazepam considerably suppressed the epileptic foci and their complexes in a dose-dependent manner. A comparison of anticonvulsive effects in a series of aliphatic alcohols methanol-ethanol-propanol-butanol showed that all these substances are potent anticonvulsants. Propanol and butanol reduce epileptic seizures most intensively, but, at the same time, they are most toxic. Application of labelled⁴C-ethanol showed that anticonvulsive effects strongly correlate with changes in the ethanol concentration in the blood.Neirofiziologiya/Neurophysiology, Vol. 26, No. 6, pp. 443–448, November–December, 1994.     

Long-lasting alterations in neuronal calcium homeostasis in an in vitro model of stroke-induced epilepsy

Altered calcium homeostatic mechanisms have been implicated in the development of acquired epilepsy in numerous models. Stroke is one of the leading brain injuries that cause acquired epilepsy, yet little is known concerning the molecular mechanisms underlying stroke-induced epileptogenesis. Recently an in vitro model of stroke-induced epilepsy was developed and characterized as a powerful tool to study the pathophysiology of injury and stroke-induced epileptogenesis. Using this glutamate injury-induced epileptogenesis model, we have investigated the role of altered calcium homeostatic mechanisms in the development and maintenance of stroke-induced epilepsy. Epileptic neurons manifested elevated intracellular calcium levels compared to control neurons independent of neuronal activity and seizure discharge for the remainder of the life of the neurons in culture. In addition, epileptic neurons were found to have alterations in the ability to reduce intracellular calcium levels following a calcium load. These long-term epileptic changes in calcium homeostasis were dependent on calcium during the initial glutamate injury. The data demonstrate that significant alterations in calcium homeostatic mechanisms occur in association with stroke-induced epilepsy and suggest that these changes may play a role in both the induction and maintenance of the epileptic phenotype in this model.     

Comparative study of the epileptogenic effect of kainic acid injected into the cerebral cortex, hippocampus and amygdala in adult cats chronically implanted

Intracerebral injection of kainic acid in cerebral cortex, hippocampus or amygdala in cats chronically implanted showed that: 1) Hippocampus and amygdala presented a greater sensitivity than the cerebral cortex, while hippocampus presented a greater sensitivity than the amygdala to the generation of an epileptic focus. 2) Comparison of latency, mean duration of afterdischarges, and the mean time period to obtain the peak intensity of the afterdischarge in the three cited structures, showed that mean latency of the first afterdischarge was significantly shorter in hippocampus and amygdala compared with the cerebral cortex. Moreover the mean time period to reach the peak intensity of the afterdischarge was again shorter in the subcortical structures. 3) The epileptic foci both in hippocampus and amygdala were blocked by CNQX and muscimol. 4) The behavioral changes depended on the intensity of the epileptic process. Tonic-clonic convulsions appeared only when the motor cerebral cortex was involved. Finally, 5) kainic acid injections in hippocampus and amygdala elicited an intense neuronal destruction and gliosis of these structures. We conclude that intracerebral injection of low doses of kainic acid in cats represent a good model to study focal epileptic thresholds in the CNS.     

Effect of diazepam on electrical activity and Na,K-ATP-ase level in a penicillin-induced epileptic focus in the rat cerebral cortex

Application of penicillin solution to the motor cortex in rats evoked the appearance of interictal discharges and epileptic seizures. After administration of diazepam in a dose of 2 mg/kg, Na,K-ATPase activity in the unpurified synaptosomes fraction of the cortex in the zone of the focus was increased by practically 100% compared with the level of activity of the enzyme in the focus without diazepam. Interictal discharges and epileptic seizures underwent different changes following intramuscular injection of diazepam. The frequency and variability of amplitude of the interictal discharges increased after administration of diazepam, whereas epileptic seizures were depressed. This effect was potentiated with an increase in the dose of diazepam. It is suggested that the opposite action of diazepam on epileptic seizures and interictal discharges may be evidence that the mechanisms lying at the basis of the development of these phenomena are different.Institute of General Pathology and Pathological Physiology, Academy of Medical Sciences of the USSR, Moscow. Translated from Neirofiziologiya, Vol. 12, No. 4, pp. 349–357, July–August, 1980.     

Advances on genetic rat models of epilepsy

Considering the suitability of laboratory rats in epilepsy research, we and other groups have been developing genetic models of epilepsy in this species. After epileptic rats or seizure-susceptible rats were sporadically found in outbred stocks, the epileptic traits were usually genetically-fixed by selective breeding. So far, the absence seizure models GAERS and WAG/Rij, audiogenic seizure models GEPR-3 and GEPR-9, generalized tonic-clonic seizure models IER, NER and WER, and Canavan-disease related epileptic models TRM and SER have been established. Dissection of the genetic bases including causative genes in these epileptic rat models would be a significant step toward understanding epileptogenesis. N-ethyl-N-nitrosourea (ENU) mutagenesis provides a systematic approach which allowed us to develop two novel epileptic rat models: heat-induced seizure susceptible (Hiss) rats with an Scn1a missense mutation and autosomal dominant lateral temporal epilepsy (ADLTE) model rats with an Lgi1 missense mutation. In addition, we have established episodic ataxia type 1 (EA1) model rats with a Kcna1 missense mutation derived from the ENU-induced rat mutant stock, and identified a Cacna1a missense mutation in a N-Methyl-N-nitrosourea (MNU)-induced mutant rat strain GRY, resulting in the discovery of episodic ataxia type 2 (EA2) model rats. Thus, epileptic rat models have been established on the two paths: ‘phenotype to gene’ and ‘gene to phenotype’. In the near future, development of novel epileptic rat models will be extensively promoted by the use of sophisticated genome editing technologies.     

Gene expression changes induced by valproate in the process of rat hippocampal neural stem cells differentiation

Valproate (VPA), an effective clinical approved anti‐epileptic drug and mood stabilizer, has been believed to induce neuronal differentiation at the expense of inhibiting astrocytic and oligodendrocytic differentiation. Nevertheless, the involving mechanisms of it remain unclear yet. In the present study, we explored the global gene expression changes of fetus rat hippocampal neural stem cells following VPA treatment by high‐throughput microarray. We obtained 874 significantly upregulated genes and 258 obviously downregulated genes (fold change > 2 and P < 0.05). Then, we performed gene ontology and pathway analyses of these differentially expressed genes and chose several genes associated with nervous system according to gene ontology analysis to conduct expression analysis to validate the reliability of the array results as well as reveal possible mechanisms of VPA. To get a better comprehension of the differentially regulated genes by VPA, we conducted protein–protein association analysis of these genes, which offered a source for further studies. In addition, we made the overlap between the VPA‐downregulated genes and the predicted target genes of VPA‐upregulated microRNAs (miRNAs), which were previously demonstrated. These overlapped genes may provide a source to find functional VPA/miRNA/mRNA axes during neuronal differentiation. This study first constructed a comprehensive potential downstream gene map of VPA in the process of neuronal differentiation.     

Structure and bioelectricity of single neurons of Helix pomatia in the intact nervous tissue during epileptic activity: Simultaneous evaluations by confocal microscopy and intracellular recordings of membrane potential changes

• 1.1. Neuronal shape during epileptic activity was studied with confocal laser scanning microscopy and intracellular recording of identified neuronal individuals in the buccal ganglia of Helixpomatia. Simultaneous observations of single Lucifer Yellow stained fibers and epileptic activity of the same neuron were done.
• 2.2. During epileptic activity, the development of several types of morphological changes were observed: local and extended swellings, constrictions, stretching of neuronal processes and release of intracellular material.
• 3.3. Morphological alterations did not only occur with epileptic activity but could also appear due to extended laser exposure of fluorescent neuronal processes.
• 4.4. Phototoxicity is discussed as a limiting factor for a valid interpretation of the morphological changes observed by confocal microscopy.

     

The epilepsy-linked Lgi1 protein assembles into presynaptic Kv1 channels and inhibits inactivation by Kvbeta1

The voltage-gated potassium (Kv) channel subunit Kv1.1 is a major constituent of presynaptic A-type channels that modulate synaptic transmission in CNS neurons. Here, we show that Kv1.1-containing channels are complexed with Lgi1, the functionally unassigned product of the leucine-rich glioma inactivated gene 1 (LGI1), which is causative for an autosomal dominant form of lateral temporal lobe epilepsy (ADLTE). In the hippocampal formation, both Kv1.1 and Lgi1 are coassembled with Kv1.4 and Kvbeta1 in axonal terminals. In A-type channels composed of these subunits, Lgi1 selectively prevents N-type inactivation mediated by the Kvbeta1 subunit. In contrast, defective Lgi1 molecules identified in ADLTE patients fail to exert this effect resulting in channels with rapid inactivation kinetics. The results establish Lgi1 as a novel subunit of Kv1.1-associated protein complexes and suggest that changes in inactivation gating of presynaptic A-type channels may promote epileptic activity.     

脑电超慢涨落图技术在癫痫研究中的应用

目的：观察脑内多种神经递质对癫痫发作的影响。方法：以癫痫患者和ＳＤ大鼠为实验对象,用脑功能检测的最新脑电超电涨落图分析仪（ｅｎｃｅｐｈａｌｏｆｌｕｃｔｕｏｇｒａｍ ｔｅｃｈｎｏｌｏｇｙ,ＥＴ）长时程采集脑电信号,提取在脑电中载有脑神经递质调节系统的震荡信息（即Ｓ谱线）,分析癫痫发作时的脑神经递质的变化。结果：患儿癫痫发作时,Ｓ谱线中Ｓ２（谷氨酸）增高;Ｓ１（γ－氨基丁酸）降低,造成Ｓ１＜Ｓ２。Ｓ５     

Cellular prion protein: implications in seizures and epilepsy

1. Cellular prion (PrP^c) is a plasma membrane protein involved with copper uptake, protection against oxidative stress, cell adhesion, differentiation, signaling, and survival in the central nervous system2. Deletion of PrP^c gene (Prnp) in mice enhances sensitivity to seizures in vivo and neuronal excitability in vitro which can be related to: (i) disrupted Ca⁺²-activated K⁺ currents, with loss of I _HAP conductance in hippocampus; (ii) abnormal GABA-A inhibition in the hippocampus; (iii) mossy fiber reorganization in the hippocampus; (iv) changes in ectonucleotidases in both hippocampus and neocortex; and (v) higher levels of neocortical and subcortical oxidative stress. Moreover, postnatal Prnp knockout mice showed a significant reduction of after hyperpolarization potentials in hippocampal CA1 cells.3. Taken together, these findings suggest that loss of PrP^c function contributes to the hyperexcitable and synchronized activities underlying epileptic seizures generated in neocortex and hippocampus. Hence, the role of PrP^c on human symptomatic, cryptogenic or idiopathic epileptic syndromes deserves further investigation.     

Clinical whole exome sequencing revealed de novo heterozygous stop-gain and missense variants in the STXBP1 gene associated with epilepsy in Saudi families

Intellectual disability and developmental encephalopathies are mostly linked with infant epilepsy. Epileptic encephalopathy is a term that is used to define association between developmental delay and epilepsy. Mutations in the STXBP1 (Syntaxin-binding protein 1) gene have been previously reported in association with multiple severe early epileptic encephalopathies along with many neurodevelopmental disorders. Among the disorders produced due to any mutations in the STXBP1 gene is developmental and epileptic encephalopathy 4 (OMIM: 612164), is an autosomal dominant neurologic disorder categorized by the onset of tonic seizures in early infancy (usually in the first months of life). In this article, we report two Saudi families one with de novo heterozygous stop-gain mutation c.364C > T and a novel missense c. 305C > A p.Ala102Glu in exon 5 of the STXBP1 gene (OMIM: 602926) lead to development of epileptic encephalopathy 4. The variants identified in the current study broadened the genetic spectrum of STXBP1 gene related with diseases, which will help to add in the literature and benefit to the studies addressing this disease in the future.     

癫痫发作敏感大鼠前深梨状皮层T区神经病理观察

目的和方法：采用颞叶癫痫红藻氨酸（kainic acid,KA)模型,制备癫痫发作敏感大鼠,并分别以硫瑾染色和GFAP（神经胶质原纤维酸性蛋白,glial fibrilary acidic protein)免疫组化方法检测前深梨状皮层T区（area tempestas,AT)内神经元损伤及星形胶质细胞增生情况,并与经蝎毒（scorpion venom,SV)处理后癫痫发作敏感性明显降低的大鼠进行比较。结果：与对照组比较,癫痫敏感动物前深梨状皮层T区锥体细胞数目明显减少,GFAP免疫反应阳性星形胶质细胞数目明显增加,染色强度明显增强,（P＜0．05）以剂量为100mg/kg/日的蝎毒给予动物连续灌胃三周,可明显降低其癫痫发作敏感性（P＜0．05）,而脑内梨状皮层T区锥体细胞脱失减轻,GPAP免疫反应活性未见明显增强。结论：推测梨状皮层T区硬化（神经元脱失,星形胶质细胞增生肥大）很可能是癫痫发作敏感性长期存在的重要原因。     

Dynamic Imaging of Coherent Sources Reveals Different Network Connectivity Underlying the Generation and Perpetuation of Epileptic Seizures

The concept of focal epilepsies includes a seizure origin in brain regions with hyper synchronous activity (epileptogenic zone and seizure onset zone) and a complex epileptic network of different brain areas involved in the generation, propagation, and modulation of seizures. The purpose of this work was to study functional and effective connectivity between regions involved in networks of epileptic seizures. The beginning and middle part of focal seizures from ictal surface EEG data were analyzed using dynamic imaging of coherent sources (DICS), an inverse solution in the frequency domain which describes neuronal networks and coherences of oscillatory brain activities. The information flow (effective connectivity) between coherent sources was investigated using the renormalized partial directed coherence (RPDC) method. In 8/11 patients, the first and second source of epileptic activity as found by DICS were concordant with the operative resection site; these patients became seizure free after epilepsy surgery. In the remaining 3 patients, the results of DICS / RPDC calculations and the resection site were discordant; these patients had a poorer post-operative outcome. The first sources as found by DICS were located predominantly in cortical structures; subsequent sources included some subcortical structures: thalamus, Nucl. Subthalamicus and cerebellum. DICS seems to be a powerful tool to define the seizure onset zone and the epileptic networks involved. Seizure generation seems to be related to the propagation of epileptic activity from the primary source in the seizure onset zone, and maintenance of seizures is attributed to the perpetuation of epileptic activity between nodes in the epileptic network. Despite of these promising results, this proof of principle study needs further confirmation prior to the use of the described methods in the clinical praxis.     

Oxidative Stress Induced NMDA Receptor Alteration Leads to Spatial Memory Deficits in Temporal Lobe Epilepsy: Ameliorative Effects of Withania somnifera and Withanolide A

In the present study we investigate the effect of Withania somnifera (WS) root extract and Withanolide A (WA) in restoring spatial memory deficit by inhibiting oxidative stress induced alteration in glutamergic neurotransmission. We demonstrate significant cellular loss in hippocampus of epileptic rats, visualized through decreased TOPRO stained neurons. Impaired spatial memory was observed in epileptic rats after Radial arm maze test. Treatment with WS and WA has resulted in increased number of TOPRO stained neurons. Enhanced performance of epileptic rats treated with WS and WA was observed in Radial arm maze test. The antioxidant activity of WS and WA was studied using superoxide dismutase (SOD) and Catalase (CAT) assays in the hippocampus of experimental rats. The SOD activity and CAT activity decreased significantly in epileptic group, treatment with WS and WA significantly reversed the enzymatic activities to near control. Real time gene expression studies of SOD and GPx showed significant up-regulation in epileptic group compared to control. Treatment with WS and WA showed significant reversal to near control. Lipid peroxidation quantified using TBARS assay, significantly increased in epileptic rats. Treatment with WS and WA showed significant reversal to near control. NMDA receptor expression decreased in epileptic rats. The treatment with WS and WA resulted in physiological expression of NMDA receptors. This data suggests that oxidative stress effects membrane constitution resulting in decreased NMDA receptor density leading to impaired spatial memory. Treatment with WS and WA has ameliorated spatial memory deficits by enhancing antioxidant system and restoring altered NMDA receptor density.