Genetic Epilepsy Model Derived from Common Inbred Mouse Strains

The recombinant inbred mouse strain, SWXL-4, exhibits tonic-clonic and generalized seizures similar to the commonest epilepsies in humans. In SWXL-4 animals, seizures are observed following routine handling at about 80 days of age and may be induced as early as 55 days by rhythmic gentle tossing. Seizures are accompanied by rapid, bilateral high frequency spike cortical discharges and followed by a quiescent post-ictal phase. Immunohistochemistry of the immediate early gene products c-Fos and c-Jun revealed abnormal activation within cortical and limbic structures. The seizure phenotype of SWXL-4 can be explained and replicated fully by the inheritance of susceptibility alleles from its progenitor strains, SWR/J and C57L/J. Outcrosses of SWXL-4 with most other common inbred strains result in F(1) hybrids that have seizures at least as frequently as SWXL-4 itself. Quantitative trait locus mapping reveals a seizure frequency determinant, Szf1, near the pink-eyed dilution locus on chromosome 7, accounting for up to 32% of the genetic variance in an F(2) intercross between SWXL-4 and the linkage testing strain ABP/Le. These studies demonstrate that common strains of mice such as SWR and C57L contain latent epilepsy susceptibility alleles. Although the inheritance of susceptibility may be complex, these results imply that a number of potentially important and practical, noninvasive models for this disorder can be constructred and studied in crosses between common mouse strains.     

Altered Cortical GABAA Receptor Composition,Physiology, and Endocytosis in a Mouse Model of a Human Genetic Absence Epilepsy Syndrome

Patients with generalized epilepsy exhibit cerebral cortical disinhibition. Likewise, mutations in the inhibitory ligand-gated ion channels, GABA_A receptors (GABA_ARs), cause generalized epilepsy syndromes in humans. Recently, we demonstrated that heterozygous knock-out (Het_α1KO) of the human epilepsy gene, the GABA_AR α1 subunit, produced absence epilepsy in mice. Here, we determined the effects of Het_α1KO on the expression and physiology of GABA_ARs in the mouse cortex. We found that Het_α1KO caused modest reductions in the total and surface expression of the β2 subunit but did not alter β1 or β3 subunit expression, results consistent with a small reduction of GABA_ARs. Cortices partially compensated for Het_α1KO by increasing the fraction of residual α1 subunit on the cell surface and by increasing total and surface expression of α3, but not α2, subunits. Co-immunoprecipitation experiments revealed that Het_α1KO increased the fraction of α1 subunits, and decreased the fraction of α3 subunits, that associated in hybrid α1α3βγ receptors. Patch clamp electrophysiology studies showed that Het_α1KO layer VI cortical neurons exhibited reduced inhibitory postsynaptic current peak amplitudes, prolonged current rise and decay times, and altered responses to benzodiazepine agonists. Finally, application of inhibitors of dynamin-mediated endocytosis revealed that Het_α1KO reduced base-line GABA_AR endocytosis, an effect that probably contributes to the observed changes in GABA_AR expression. These findings demonstrate that Het_α1KO exerts two principle disinhibitory effects on cortical GABA_AR-mediated inhibitory neurotransmission: 1) a modest reduction of GABA_AR number and 2) a partial compensation with GABA_AR isoforms that possess physiological properties different from those of the otherwise predominant α1βγ GABA_ARs.     

A Mesiotemporal Lobe Epilepsy Mouse Model

Among the different forms of epilepsies, mesiotemporal lobe epilepsy (MTLE) is one of the most common and represents the main pharmaco-resistant form of epilepsy. There is therefore an urgent need to better understand this form of epilepsy to develop better anti-epileptic drugs. Many rodent models are mimicking some aspects of the human temporal lobe epilepsy but only few are addressing most of the human mesiotemporal lobe epilepsy. In this article, we describe the main characteristics of a mouse of model of mesial temporal lobe epilepsy. This model is generated by a single injection of kainic acid into the dorsal hippocampus which reproduces most of the morphological and electrophysiological features of human MTLE in a mouse. This model may help to better understand mesial temporal lobe epilepsy and the development of new therapeutic drugs.     

Unilateral and Bilateral Cortical Resection: Effects on Spike-Wave Discharges in a Genetic Absence Epilepsy Model

Research Question

Recent discoveries have challenged the traditional view that the thalamus is the primary source driving spike-and-wave discharges (SWDs). At odds, SWDs in genetic absence models have a cortical focal origin in the deep layers of the perioral region of the somatosensory cortex. The present study examines the effect of unilateral and bilateral surgical resection of the assumed focal cortical region on the occurrence of SWDs in anesthetized WAG/Rij rats, a well described and validated genetic absence model.

Methods

Male WAG/Rij rats were used: 9 in the resected and 6 in the control group. EEG recordings were made before and after craniectomy, after unilateral and after bilateral removal of the focal region.

Results

SWDs decreased after unilateral cortical resection, while SWDs were no longer noticed after bilateral resection. This was also the case when the resected areas were restricted to layers I-IV with layers V and VI intact.

Conclusions

These results suggest that SWDs are completely abolished after bilateral removal of the focal region, most likely by interference with an intracortical columnar circuit. The evidence suggests that absence epilepsy is a network type of epilepsy since interference with only the local cortical network abolishes all seizures.     

Disease-Modifying Effects of Neural Regeneration Peptide 2945 in the GAERS Model of Absence Epilepsy

Neurochemical Research - Epilepsy is a common neurological condition characterised by spontaneous recurrent seizures. Current anti-epileptic drugs are only effective and tolerated in ~70% of...     

Cytokines and Absence Seizures in a Genetic Rat Model

We investigated the role of two cytokines, IL-1β and TNF-α, in the development of absence seizures using a genetic model of absence epilepsy in WAG/Rij rats. We administered these cytokines to animals systemically and measured the number of spike-wave discharges (SWDs) in the EEG. We also coadministered IL-1β with the GABA reuptake inhibitor tiagabine and measured the levels of IL-1β and TNF-α in the brain and blood plasma of 2-, 4-, and 6-month-old WAG/Rij rats and animals that served as a non-epileptic control (ACI). We found that IL-1β induced a significant increase in SWDs 2-5 h after administration, while TNF-α enhanced SWDs much later. Both cytokines enhanced passive behavior; body temperature was elevated only after TNF-α. The action of tiagabine was potentiated by earlier IL-1β injection, even when IL-1β was no longer active. Young WAG/Rij rats showed higher levels of TNF-α in blood serum than young ACI rats; the effects in the brain tended to be opposite. The marked differences in timing of the increase in SWDs suggest different time scales for the action of both cytokines tested. It is proposed that the results found after TNF-α are due to the de novo synthesis of IL-1β. TNF-α may possess neuroprotective effects. IL-1β might increase GABA-ergic neurotransmission. The changes in the efficacy of antiepileptic drugs related to changes in the cytokine systems may have some clinical relevance.     

Urokinase-Type Plasminogen Activator Receptor Modulates Epileptogenesis in Mouse Model of Temporal Lobe Epilepsy

Mutation in Plaur gene encoding urokinase-type plasminogen activator receptor (uPAR) results in epilepsy and autistic phenotype in mice. In humans, a single nucleotide polymorphism in PLAUR gene represents a risk for autism spectrum disorders. Importantly, the expression of uPAR is elevated in the brain after various epileptogenic insults like traumatic brain injury and status epilepticus. So far, the consequences of altered uPAR expression on brain networks are poorly known. We tested a hypothesis that uPAR regulates post-injury neuronal reorganization and consequent functional outcome, particularly epileptogenesis. Epileptogenesis was induced by intrahippocampal injection of kainate in adult male wild type (Wt) or uPAR knockout (uPAR?/?) mice, and animals were monitored with continuous (24/7) video-electroencephalogram for 30 days. The severity of status epilepticus did not differ between the genotypes. The spontaneous electrographic seizures which developed were, however, longer and their behavioral manifestations were more severe in uPAR?/? than Wt mice. The more severe epilepsy phenotype in uPAR?/? mice was associated with delayed but augmented inflammatory response and more severe neurodegeneration in the hippocampus. Also, the distribution of newly born cells in the dentate gyrus was more scattered, and the recovery of hippocampal blood vessel length from status epilepticus-induced damage was compromised in uPAR?/? mice as compared to Wt mice. Our data demonstrate that a deficiency in uPAR represents a mechanisms which results in the development of a more severe epilepsy phenotype and progressive brain pathology after status epilepticus. We suggest that uPAR represents a rational target for disease-modifying treatments after epileptogenic brain insults.     

Brain Purines in a Genetic Mouse Model of Lesch-Nyhan Disease

Abstract: Mice carrying a mutation in the gene encoding the purine salvage enzyme hypoxanthine-guanine phosphoribosyltransferase (HPRT) have recently been produced to provide an animal model for Lesch-Nyhan disease. The current-studies were conducted to characterize the consequences of the mutation on the expression of HPRT and to characterize potential changes in brain purine content in these mutants. Our results indicate that the mutant animals have no detectable HPRT-immunoreactive material on western blots and no detectable HPRT enzyme activity in brain tissue homogenates, confirming that they are completely HPRT deficient (HPRT^-). Despite the absence of HPRT-mediated purine salvage, the animals have apparently normal brain purine content. However, de novo purine synthesis, as measured by [¹⁴C]formate incorporation into brain purines, is accelerated four- to fivefold in the mutant animals. This increase in the synthesis of purines may protect the HPRT^- mice from potential depletion of brain purines despite complete impairment of HPRT-mediated purine salvage.     

Anticonvulsant Effect of Asparagus racemosus Willd. in a Mouse Model of Catamenial Epilepsy

Neurochemical Research - Asparagus racemosus Willd. (Family Liliaceae), also known as female reproductive tonic, is traditionally used across the Sub-Himalayan region in Uttarakhand, India for...     

Genetic Analysis of a New Mouse Model for Non-Insulin-Dependent Diabetes

The TallyHo (TH) mouse strain is a newly established model for non-insulin-dependent diabetes mellitus (NIDDM). TH mice show obesity, hyperinsulinemia, hyperlipidemia, and male-limited hyperglycemia. A genetic dissection of the diabetes syndrome has been carried out using male backcross 1 progeny obtained from crosses between (C57BL/6J x TH)F1 and TH mice or (CAST/Ei x TH)F1 and TH mice. A genome-wide scan reveals three quantitative trait loci (QTLs), Tanidd1-3 (TH-associated NIDDM) linked to hyperglycemia. The major QTL (common in both crosses), Tanidd1, maps to chromosome (Chr) 19. Additionally, gene-gene interactions contributing to hyperglycemia have been observed between Tanidd1 and a locus on Chr 18 as well as between Tanidd2 and a locus on Chr 16. The overt hyperglycemia in TH mice is, therefore, likely due to a mutation in a major diabetes susceptibility locus on Chr 19, which interacts with additional genes to lead to an observable phenotype.     

Hunger in the Absence of Caloric Restriction Improves Cognition and Attenuates Alzheimer's Disease Pathology in a Mouse Model

It has been shown that caloric restriction (CR) delays aging and possibly delays the development of Alzheimer''s disease (AD). We conjecture that the mechanism may involve interoceptive cues, rather than reduced energy intake per se. We determined that hunger alone, induced by a ghrelin agonist, reduces AD pathology and improves cognition in the APP-SwDI mouse model of AD. Long-term treatment with a ghrelin agonist was sufficient to improve the performance in the water maze. The treatment also reduced levels of amyloid beta (Aβ) and inflammation (microglial activation) at 6 months of age compared to the control group, similar to the effect of CR. Thus, a hunger-inducing drug attenuates AD pathology, in the absence of CR, and the neuroendocrine aspects of hunger also prevent age-related cognitive decline.     

Identification of Genetic Modifiers of TDP-43 Neurotoxicity in Drosophila

Cytosolic aggregation of the nuclear RNA-binding protein TDP-43 is a histopathologic signature of degenerating neurons in amyotrophic lateral sclerosis (ALS), and mutations in the TARDBP gene encoding TDP-43 cause dominantly inherited forms of this condition. To understand the relationship between TDP-43 misregulation and neurotoxicity, we and others have used Drosophila as a model system, in which overexpression of either wild-type TDP-43 or its ALS-associated mutants in neurons is sufficient to induce neurotoxicity, paralysis, and early death. Using microarrays, we have examined gene expression patterns that accompany TDP-43-induced neurotoxicity in the fly system. Constitutive expression of TDP-43 in the Drosophila compound eye elicited widespread gene expression changes, with strong upregulation of cell cycle regulatory genes and genes functioning in the Notch intercellular communication pathway. Inducible expression of TDP-43 specifically in neurons elicited significant expression differences in a more restricted set of genes. Genes that were upregulated in both paradigms included SpindleB and the Notch target Hey, which appeared to be a direct TDP-43 target. Mutations that diminished activity of Notch or disrupted the function of downstream Notch target genes extended the lifespan of TDP-43 transgenic flies, suggesting that Notch activation was deleterious in this model. Finally, we showed that mutation of the nucleoporin Nup50 increased the lifespan of TDP-43 transgenic flies, suggesting that nuclear events contribute to TDP-43-dependent neurotoxicity. The combined findings identified pathways whose deregulation might contribute to TDP-43-induced neurotoxicity in Drosophila.     

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

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

Decreased Bone Formation Explains Osteoporosis in a Genetic Mouse Model of Hemochromatosiss

Osteoporosis may complicate iron overload diseases such as genetic hemochromatosis. However, molecular mechanisms involved in the iron-related osteoporosis remains poorly understood. Recent in vitro studies support a role of osteoblast impairment in iron-related osteoporosis. Our aim was to analyse the impact of excess iron in Hfe^-/- mice on osteoblast activity and on bone microarchitecture. We studied the bone formation rate, a dynamic parameter reflecting osteoblast activity, and the bone phenotype of Hfe^−/− male mice, a mouse model of human hemochromatosis, by using histomorphometry. Hfe^−/− animals were sacrificed at 6 months and compared to controls. We found that bone contains excess iron associated with increased hepatic iron concentration in Hfe^−/− mice. We have shown that animals with iron overload have decreased bone formation rate, suggesting a direct impact of iron excess on active osteoblasts number. For bone mass parameters, we showed that iron deposition was associated with bone loss by producing microarchitectural impairment with a decreased tendency in bone trabecular volume and trabecular number. A disorganization of trabecular network was found with marrow spaces increased, which was confirmed by enhanced trabecular separation and star volume of marrow spaces. These microarchitectural changes led to a loss of connectivity and complexity in the trabecular network, which was confirmed by decreased interconnectivity index and increased Minkowski’s fractal dimension. Our results suggest for the first time in a genetic hemochromatosis mouse model, that iron overload decreases bone formation and leads to alterations in bone mass and microarchitecture. These observations support a negative effect of iron on osteoblast recruitment and/or function, which may contribute to iron-related osteoporosis.     

Complexity of Multi-Channel Electroencephalogram Signal Analysis in Childhood Absence Epilepsy

Absence epilepsy is an important epileptic syndrome in children. Multiscale entropy (MSE), an entropy-based method to measure dynamic complexity at multiple temporal scales, is helpful to disclose the information of brain connectivity. This study investigated the complexity of electroencephalogram (EEG) signals using MSE in children with absence epilepsy. In this research, EEG signals from 19 channels of the entire brain in 21 children aged 5-12 years with absence epilepsy were analyzed. The EEG signals of pre-ictal (before seizure) and ictal states (during seizure) were analyzed by sample entropy (SamEn) and MSE methods. Variations of complexity index (CI), which was calculated from MSE, from the pre-ictal to the ictal states were also analyzed. The entropy values in the pre-ictal state were significantly higher than those in the ictal state. The MSE revealed more differences in analysis compared to the SamEn. The occurrence of absence seizures decreased the CI in all channels. Changes in CI were also significantly greater in the frontal and central parts of the brain, indicating fronto-central cortical involvement of “cortico-thalamo-cortical network” in the occurrence of generalized spike and wave discharges during absence seizures. Moreover, higher sampling frequency was more sensitive in detecting functional changes in the ictal state. There was significantly higher correlation in ictal states in the same patient in different seizures but there were great differences in CI among different patients, indicating that CI changes were consistent in different absence seizures in the same patient but not from patient to patient. This implies that the brain stays in a homogeneous activation state during the absence seizures. In conclusion, MSE analysis is better than SamEn analysis to analyze complexity of EEG, and CI can be used to investigate the functional brain changes during absence seizures.     

Genetic Modifiers of Duchenne Muscular Dystrophy and Dilated Cardiomyopathy

Objective

Dilated cardiomyopathy (DCM) is a major complication and leading cause of death in Duchenne muscular dystrophy (DMD). DCM onset is variable, suggesting modifier effects of genetic or environmental factors. We aimed to determine if polymorphisms previously associated with age at loss of independent ambulation (LoA) in DMD (rs28357094 in the SPP1 promoter, rs10880 and the VTTT/IAAM haplotype in LTBP4) also modify DCM onset.

Methods

A multicentric cohort of 178 DMD patients was genotyped by TaqMan assays. We performed a time-to-event analysis of DCM onset, with age as time variable, and finding of left ventricular ejection fraction < 50% and/or end diastolic volume > 70 mL/m² as event (confirmed by a previous normal exam < 12 months prior); DCM-free patients were censored at the age of last echocardiographic follow-up.

Results

Patients were followed up to an average age of 15.9 ± 6.7 years. Seventy-one/178 patients developed DCM, and median age at onset was 20.0 years. Glucocorticoid corticosteroid treatment (n = 88 untreated; n = 75 treated; n = 15 unknown) did not have a significant independent effect on DCM onset. Cardiological medications were not administered before DCM onset in this population. We observed trends towards a protective effect of the dominant G allele at SPP1 rs28357094 and recessive T allele at LTBP4 rs10880, which was statistically significant in steroid-treated patients for LTBP4 rs10880 (< 50% T/T patients developing DCM during follow-up [n = 13]; median DCM onset 17.6 years for C/C-C/T, log-rank p = 0.027).

Conclusions

We report a putative protective effect of DMD genetic modifiers on the development of cardiac complications, that might aid in risk stratification if confirmed in independent cohorts.     

A circuit of mossy cells controls the efficacy of memory retrieval by Gria2I inhibition of Gria2

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Up-regulation of d-serine Might Induce GABAergic Neuronal Degeneration in the Cerebral Cortex and Hippocampus in the Mouse Pilocarpine Model of Epilepsy

Epilepsy is a serious neurological disorder with neuronal loss and spontaneous recurrent seizures, but the neurochemical basis remains largely unclear. We hypothesize that d-serine, a newly identified endogenous co-agonist of N-methyl-d-aspartate (NMDA) receptor, may trigger excitotoxicity and neuronal damage in epileptogenesis. By using a mouse pilocarpine model, immunohistochemistry, Fluoro-Jade staining and double-labeling, the present study revealed up-regulation of d-serine expression in a proportion (41%) of neurons in the cerebral cortex and hippocampus. The d-serine-positive neurons occurred at 4 h, reached peak levels at 12–24 h, and gradually went down at 3–14 days. Moreover, most of d-serine-positive neurons were GABAergic (98%), underwent degenerating death (93%), and were accompanied enhancing phosphorylation of NMDA receptor subunit 1. This study has provided new evidence that up-regulation of d-serine production might induce GABAergic neuronal degeneration through excitotoxic mechanism in the pilocarpine model and may be involved in early pathogenesis and recurrent seizure of chronic epilepsy. Ms. L. Wang is on leave from Department of Neurology, Kunming General Hospital of Chengdu Military Region, China.