Myocardial glycophagy flux dysregulation and glycogen accumulation characterize diabetic cardiomyopathy.

Diabetic heart disease morbidity and mortality is escalating. No specific therapeutics exist and mechanistic understanding of diabetic cardiomyopathy etiology is lacking. While lipid accumulation is a recognized cardiomyocyte phenotype of diabetes, less is known about glycolytic fuel handling and storage. Based on in vitro studies, we postulated the operation of an autophagy pathway in the myocardium specific for glycogen homeostasis - glycophagy. Here we visualize occurrence of cardiac glycophagy and show that the diabetic myocardium is characterized by marked glycogen elevation and altered cardiomyocyte glycogen localization. We establish that cardiac glycophagy flux is disturbed in diabetes. Glycophagy may represent a potential therapeutic target for alleviating the myocardial impacts of metabolic disruption in diabetic heart disease.

Transmembrane α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor regulatory protein expression during the development of absence seizures in genetic absence epilepsy rats from Strasbourg.

The transmembrane α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPAR) regulatory proteins (TARPs), γ2 (stargazin), γ3, γ4, γ5, γ7, and γ8, are a family of proteins that regulate AMPAR trafficking, expression, and biophysical properties that could have a role in the development of absence seizures. Here, we evaluated the expression of TARPs and AMPARs across the development of epilepsy in the genetic absence epilepsy rats from Strasbourg (GAERS) model of idiopathic generalized epilepsy (IGE) with absence seizures. Pre-epileptic (7-day-old), early epileptic (6-week-old), and chronically epileptic (16-week-old) GAERS, and age-matched male nonepileptic control rats (NEC) were used. Electroencephalographic (EEG) recordings were acquired from the 6- and 16-week-old animals to quantify seizure expression. Somatosensory cortex (SCx) and whole thalamus were collected from all the animals to evaluate TARP and AMPAR mRNA expression. Analysis of the EEG demonstrated a gradual increase in the number and duration of seizures across GAERS development. mRNA expression of the TARPs γ2, γ3, γ4, γ5, and γ8 in the SCx, and γ4 and γ5 in the thalamus, increased as the seizures started and progressed in the GAERS compared to NEC. There was a temporal association between increased TARP expression and seizures in GAERS, highlighting TARPs as potential targets for developing novel treatments for IGE with absence seizures.

Effects of the T-type calcium channel CaV3.2 R1584P mutation on absence seizure susceptibility in GAERS and NEC congenic rats models.

RATIONALE: Low-voltage-activated or T-type Ca2+ channels play a key role in the generation of seizures in absence epilepsy. We have described a homozygous, gain of function substitution mutation (R1584P) in the CaV3.2 T-type Ca2+ channel gene (Cacna1h) in the Genetic Absence Epilepsy Rats from Strasbourg (GAERS). The non-epileptic control (NEC) rats, derived from the same original Wistar strains as GAERS but selectively in-breed not to express seizures, are null for the R1584P mutation. To study the effects of this mutation in rats who otherwise have a GAERS or NEC genetic background, we bred congenic GAERS-Cacna1hNEC (GAERS null for R1584P mutation) and congenic NEC-Cacna1hGAERS (NEC homozygous for R1584P mutation) and evaluated the seizure and behavioral phenotype of these strains in comparison to the original GAERS and NEC strains. METHODS: To evaluate seizure expression in the congenic strains, EEG electrodes were implanted in NEC, GAERS, GAERS-Cacna1hNEC without the R1584P mutation, and NEC-Cacna1hGAERS with the R1584P mutation rats. In the first study, continuous EEG recordings were acquired from week 4 (when seizures begin to develop in GAERS) to week 14 of age (when GAERS display hundreds of seizures per day). In the second study, the seizure and behavioral phenotype of GAERS and NEC-Cacna1hGAERS strains were evaluated during young age (6 weeks of age) and adulthood (16 weeks of age) of GAERS, NEC, GAERS-Cacna1hNEC and NEC-Cacna1hGAERS. The Open field test (OFT) and sucrose preference test (SPT) were performed to evaluate anxiety-like and depressive-like behavior, respectively. This was followed by EEG recordings at 18 weeks of age to quantify the seizures, and spike-wave discharge (SWD) cycle frequency. At the end of the study, the whole thalamus was collected for T-type calcium channel mRNA expression analysis. RESULTS: GAERS had a significantly shorter latency to first seizures and an increased number of seizures per day compared to GAERS-Cacna1hNEC. On the other hand, the presence of the R1584P mutation in the NEC-Cacna1hGAERS was not enough to generate spontaneous seizures in their seizure-resistant background. 6 and 16-week-old GAERS and GAERS-Cacna1hNEC rats showed anxiety-like behavior in the OFT, in contrast to NEC and NEC-Cacna1hGAERS. Results from the SPT showed that the GAERS developed depressive-like in the SPT compared to GAERS-Cacna1hNEC, NEC, and NEC-Cacna1hGAERS. Analysis of the EEG at 18 weeks of age showed that the GAERS had an increased number of seizures per day, increased total seizure duration and a higher cycle frequency of SWD relative to GAERS-Cacna1hNEC. However, the average seizure duration was not significantly different between strains. Quantitative real-time PCR showed that the T-type Ca2+ channel isoform CaV3.2 channel expression was significantly increased in GAERS compared to NEC, GAERS-Cacna1hNEC and NEC-Cacna1hGAERS. The presence of the R1584P mutation increased the total ratio of CaV3.2 + 25/-25 splice variants in GAERS and NEC-Cacna1hGAERS compared to NEC and GAERS-Cacna1hNEC. DISCUSSION: The data from this study demonstrate that the R1584P mutation in isolation on a seizure-resistant NEC genetic background was insufficient to generate absence seizures, and that a GAERS genetic background can cause seizures even without the mutation. However, the study provides evidence that the R1584P mutation acts as a modulator of seizures development and expression, and depressive-like behavior in the SPT, but not the anxiety phenotype of the GAERS model of absence epilepsy.

Integrative genomics reveals pathogenic mediator of valproate-induced neurodevelopmental disability.

Prenatal exposure to the anti-seizure medication sodium valproate (VPA) is associated with an increased risk of adverse postnatal neurodevelopmental outcomes, including lowered intellectual ability, autism spectrum disorder and attention-deficit hyperactivity disorder. In this study, we aimed to clarify the molecular mechanisms underpinning the neurodevelopmental consequences of gestational VPA exposure using integrative genomics. We assessed the effect of gestational VPA on foetal brain gene expression using a validated rat model of valproate teratogenicity that mimics the human scenario of chronic oral valproate treatment during pregnancy at doses that are therapeutically relevant to the treatment of epilepsy. Two different rat strains were studied-inbred Genetic Absence Epilepsy Rats from Strasbourg, a model of genetic generalized epilepsy, and inbred non-epileptic control rats. Female rats were fed standard chow or VPA mixed in standard chow for 2 weeks prior to conception and then mated with same-strain males. In the VPA-exposed rats maternal oral treatment was continued throughout pregnancy. Foetuses were extracted via C-section on gestational Day 21 (1 day prior to birth) and foetal brains were snap-frozen and genome-wide gene expression data generated. We found that gestational VPA exposure via chronic maternal oral dosing was associated with substantial drug-induced differential gene expression in the pup brains, including dysregulated splicing, and observed that this occurred in the absence of evidence for significant neuronal gain or loss. The functional consequences of VPA-induced gene expression were explored using pathway analysis and integration with genetic risk data for psychiatric disease and behavioural traits. The set of genes downregulated by VPA in the pup brains were significantly enriched for pathways related to neurodevelopment and synaptic function and significantly enriched for heritability to human intelligence, schizophrenia and bipolar disorder. Our results provide a mechanistic link between chronic foetal VPA exposure and neurodevelopmental disability mediated by VPA-induced transcriptional dysregulation.

Altered cardiac structure and function is related to seizure frequency in a rat model of chronic acquired temporal lobe epilepsy.

OBJECTIVE: This study aimed to prospectively examine cardiac structure and function in the kainic acid-induced post-status epilepticus (post-KA SE) model of chronic acquired temporal lobe epilepsy (TLE), specifically to examine for changes between the pre-epileptic, early epileptogenesis and the chronic epilepsy stages. We also aimed to examine whether any changes related to the seizure frequency in individual animals. METHODS: Four hours of SE was induced in 9 male Wistar rats at 10 weeks of age, with 8 saline treated matched control rats. Echocardiography was performed prior to the induction of SE, two- and 10-weeks post-SE. Two weeks of continuous video-EEG and simultaneous ECG recordings were acquired for two weeks from 11 weeks post-KA SE. The video-EEG recordings were analyzed blindly to quantify the number and severity of spontaneous seizures, and the ECG recordings analyzed for measures of heart rate variability (HRV). PicroSirius red histology was performed to assess cardiac fibrosis, and intracellular Ca2+ levels and cell contractility were measured by microfluorimetry. RESULTS: All 9 post-KA SE rats were demonstrated to have spontaneous recurrent seizures on the two-week video-EEG recording acquired from 11 weeks SE (seizure frequency ranging from 0.3 to 10.6 seizures/day with the seizure durations from 11 to 62 s), and none of the 8 control rats. Left ventricular wall thickness was thinner, left ventricular internal dimension was shorter, and ejection fraction was significantly decreased in chronically epileptic rats, and was negatively correlated to seizure frequency in individual rats. Diastolic dysfunction was evident in chronically epileptic rats by a decrease in mitral valve deceleration time and an increase in E/E` ratio. Measures of HRV were reduced in the chronically epileptic rats, indicating abnormalities of cardiac autonomic function. Cardiac fibrosis was significantly increased in epileptic rats, positively correlated to seizure frequency, and negatively correlated to ejection fraction. The cardiac fibrosis was not a consequence of direct effect of KA toxicity, as it was not seen in the 6/10 rats from separate cohort that received similar doses of KA but did not go into SE. Cardiomyocyte length, width, volume, and rate of cell lengthening and shortening were significantly reduced in epileptic rats. SIGNIFICANCE: The results from this study demonstrate that chronic epilepsy in the post-KA SE rat model of TLE is associated with a progressive deterioration in cardiac structure and function, with a restrictive cardiomyopathy associated with myocardial fibrosis. Positive correlations between seizure frequency and the severity of the cardiac changes were identified. These results provide new insights into the pathophysiology of cardiac disease in chronic epilepsy, and may have relevance for the heterogeneous mechanisms that place these people at risk of sudden unexplained death.

A rat model of valproate teratogenicity from chronic oral treatment during pregnancy.

OBJECTIVE: Sodium valproate (VPA), the most effective antiepileptic drug for patients with genetic generalized epilepsy (GGE), is a potent human teratogen that increases the risk of a range of congenital malformations, including spina bifida. The mechanisms underlying this teratogenicity are not known, but may involve genetic risk factors. This study aimed to develop an animal model of VPA-induced birth defects. METHODS: We used three different rat strains: inbred Genetic Absence Epilepsy Rats From Strasbourg (GAERS), a model of GGE with absence seizures; inbred Non-Epileptic Controls (NEC); and outbred nonepileptic Wistars. Female rats were fed standard chow or VPA (20 g/kg food) mixed in standard chow for 2 weeks prior to conception, and then mated with same-strain males. Treatment continued throughout pregnancy. Fetuses were extracted via C-section on gestational day 21 and examined for birth defects, including external assessment and spinal measurements. RESULTS: VPA-exposed pups showed significant reductions in weight, length, and whole-body development compared with controls of all three strains (P < .0001). Gestational VPA treatment altered intravertebral distances, and resulted in underdeveloped vertebral arches between thoracic region T11 and caudal region C2 in most pups (GAERS, 100%; NEC, 95%; Wistar, 80%), more frequently than in controls (9%, 13%, 19%). SIGNIFICANCE: Gestational VPA treatment results in similar developmental and morphological abnormalities in three rat strains, including one with GGE, indicating that the genetic underpinnings of epilepsy do not contribute markedly to VPA-induced birth defects. This model may be used in future studies to investigate mechanisms involved in the pathogenesis of antiepileptic drug-induced birth defects.

Acquired cardiac channelopathies in epilepsy: Evidence, mechanisms, and clinical significance.

There is growing evidence that cardiac dysfunction in patients with chronic epilepsy could play a pathogenic role in sudden unexpected death in epilepsy (SUDEP). Recent animal studies have revealed that epilepsy secondarily alters the expression of cardiac ion channels alongside abnormal cardiac electrophysiology and remodeling. These molecular findings represent novel evidence for an acquired cardiac channelopathy in epilepsy, distinct from inherited ion channels mutations associated with cardiocerebral phenotypes. Specifically, seizure activity has been shown to alter the messenger RNA (mRNA) and protein expression of voltage-gated sodium channels (Nav 1.1, Nav 1.5), voltage-gated potassium channels (Kv 4.2, Kv 4.3), sodium-calcium exchangers (NCX1), and nonspecific cation-conducting channels (HCN2, HCN4). The pathophysiology may involve autonomic dysfunction and structural cardiac disease, as both are independently associated with epilepsy and ion channel dysregulation. Indeed, in vivo and in vitro studies of cardiac pathology reveal a complex network of signaling pathways and transcription factors regulating ion channel expression in the setting of sympathetic overactivity, cardiac failure, and hypertrophy. Other mechanisms such as circulating inflammatory mediators or exogenous effects of antiepileptic medications lack evidence. Moreover, an acquired cardiac channelopathy may underlie the electrophysiologic cardiac abnormalities seen in chronic epilepsy, potentially contributing to the increased risk of malignant arrhythmias and sudden death. Therefore, further investigation is necessary to establish whether cardiac ion channel dysregulation similarly occurs in patients with epilepsy, and to characterize any pathogenic relationship with SUDEP.

Gene therapy mediated seizure suppression in Genetic Generalised Epilepsy: Neuropeptide Y overexpression in a rat model.

Neuropeptide Y (NPY) is an important 36 amino acid peptide that is abundantly expressed in the mammalian CNS and is known to be an endogenous modulator of seizure activity, including in rat models of Genetic Generalised Epilepsy (GGE) with absence seizures. Studies have shown that viral-mediated "gene therapy" with overexpression of NPY in the hippocampus can suppress seizures in acquired epilepsy animal models. This study investigated whether NPY gene delivery to the thalamus or somatosensory cortex, using recombinant adeno-associated viral vector (rAAV), could produce sustained seizure suppression in the GAERS model of GGE with absence seizures. Three cohorts of GAERS were injected bilaterally into the thalamus (short term n = 14 and long term n = 8) or the somatosensory cortex (n = 26) with rAAV-NPY or rAAV-empty. EEG recordings were acquired weekly post-treatment and seizure expression was quantified. Anxiety levels were tested using elevated plus maze and open field test. NPY and NPY receptor mRNA and protein expression were evaluated using quantitative PCR, immunohistochemistry and immunofluorescence. Viral overexpression of human NPY in the thalamus and somatosensory cortex in GAERS significantly reduced the time spent in seizure activity and number of seizures, whereas seizure duration was only reduced after thalamic NPY overexpression. Human and rat NPY and rat Y2 receptor mRNA expression was significantly increased in the somatosensory cortex. NPY overexpression in the thalamus was observed in rAAV-NPY treated rats compared to controls in the long term cohort. No effect was observed on anxiety behaviour. We conclude that virally-mediated human NPY overexpression in the thalamus or somatosensory cortex produces sustained anti-epileptic effects in GAERS. NPY gene therapy may represent a novel approach for the treatment of patients with genetic generalised epilepsies.

Delayed myelination and neurodevelopment in male seizure-prone versus seizure-resistant rats.

OBJECTIVE: Aberrant myelination and developmental delay have been reported in epilepsy. However, it is unclear whether these are linked to intrinsic mechanisms that support a predisposition toward seizures and the development of epilepsy. Thus, we compared rates of myelination and neurodevelopment in male rats selectively bred for enhanced susceptibility to kindling epileptogenesis (FAST) with male rats bred for resistance (SLOW). METHODS: Myelin-specific gene expression was compared in the brainstem, cerebellum, and cerebral hemisphere of FAST and SLOW rats on postnatal days (PNDs) 5, 11, 17, 23, and 90 to determine strain-specific myelination rates. Myelin protein levels were also compared at PNDs 5 and 23 in the brainstem. Relative rates of neurodevelopment were evaluated between PNDs 5 and 21 using physical growth landmarks and neuromotor tests including righting reflex, cliff avoidance, negative geotaxis, and locomotor activity. RESULTS: Myelin-specific mRNA expression was significantly down-regulated in FAST rats on PNDs 5 and 11 in all 3 brain structures, indicating relatively delayed myelination. Likewise, corresponding protein levels were significantly lower in FAST brainstem on PND 5. Developmental delay was evident in the FAST strain such that only 9% of FAST pups, compared to 81% of SLOW, had open eyes by PND 13, locomotor activity was significantly reduced between PNDs 12 and 16, and neuromotor task acquisition was delayed between PNDs 5 and 10. SIGNIFICANCE: Relative delays in myelination and neurodevelopment co-occurred in the seizure-prone FAST strain in the absence of seizures. These findings suggest these symptoms are not seizure-induced and may be mechanistically linked to an underlying pathophysiology supporting a predisposition toward developing epilepsy.

CaV 3.2 drives sustained burst-firing, which is critical for absence seizure propagation in reticular thalamic neurons.

OBJECTIVE: Genetic alterations have been identified in the CACNA1H gene, encoding the CaV 3.2 T-type calcium channel in patients with absence epilepsy, yet the precise mechanisms relating to seizure propagation and spike-wave-discharge (SWD) pacemaking remain unknown. Neurons of the thalamic reticular nucleus (TRN) express high levels of CaV 3.2 calcium channels, and we investigated whether a gain-of-function mutation in the Cacna1h gene in Genetic Absence Epilepsy Rats from Strasbourg (GAERS) contributes to seizure propagation and pacemaking in the TRN. METHODS: Pathophysiological contributions of CaV 3.2 calcium channels to burst firing and absence seizures were assessed in vitro using acute brain slice electrophysiology and quantitative real-time polymerase chain reaction (PCR) and in vivo using free-moving electrocorticography recordings. RESULTS: TRN neurons from GAERS display sustained oscillatory burst-firing that is both age- and frequency-dependent, occurring only in the frequencies overlapping with GAERS SWDs and correlating with the expression of a CaV 3.2 mutation-sensitive splice variant. In vivo knock-down of CaV 3.2 using direct thalamic injection of lipid nanoparticles containing CaV 3.2 dicer small interfering (Dsi) RNA normalized TRN burst-firing, and in free-moving GAERS significantly shortened seizures. SIGNIFICANCE: This supports a role for TRN CaV 3.2 T-type channels in propagating thalamocortical network seizures and setting the pacemaking frequency of SWDs.

Differences in white matter structure between seizure prone (FAST) and seizure resistant (SLOW) rat strains.

Alterations in white matter integrity have been well documented in chronic epilepsy and during epileptogenesis. However, the relationship between white matter integrity and a predisposition towards epileptogenesis has been understudied. The FAST rat strain exhibit heightened susceptibility towards kindling epileptogenesis whereas SLOW rats are highly resistant. FAST rats also display behavioral phenotypes reminiscent of those observed in neurodevelopmental disorders that commonly comorbid with epilepsy. In this study, we aim to identify differences in white matter integrity that may contribute to a predisposition towards epileptogenesis and its associated comorbidities in 6month old FAST (n=10) and SLOW (n=10) male rats. Open field and water consumption tests were conducted to confirm the behavioral phenotype difference between FAST and SLOW rats followed by ex-vivo diffusion-weighted magnetic resonance imaging to identify differences in white matter integrity. Diffusion tensor imaging scalar values namely fractional anisotropy, mean diffusivity, axial diffusivity and radial diffusivity were compared in the anterior commissure, corpus callosum, external capsule, internal capsule, fimbria and optic tract. Electron microscopy was used to evaluate microstructural alterations in myelinated axons. Behavioral phenotyping confirmed higher activity levels (distance moved on days 2-4, p<0.001; number of rearings on days 2 and 4, p<0.05 at both days) and polydipsia (p<0.001) in FAST rats. Comparative analysis of diffusion tensor imaging scalars found a significant decrease in fractional anisotropy in the corpus callosum (p<0.05) of FAST versus SLOW rats. Using electron microscopy, alterations in myelinated axons including increased axon diameter (p<0.001) and reduced g-ratio (p<0.001) in the midline of the corpus callosum in 6month old FAST (n=3) versus SLOW (n=4) male rats. These findings suggest that differences in white matter integrity between FAST and SLOW rats could be a contributing factor to the differential seizure susceptibility and behavioral phenotypes observed in these strains.

Interleukin-1ß has trophic effects in microglia and its release is mediated by P2X7R pore.

BACKGROUND: Enhanced expression of the purinergic P2X7 receptor (P2X7R) occurs in several neuroinflammatory conditions where increased microglial activation is a co-existing feature. P2X7 receptors can function either as a cation channel or, upon continued stimulation, a large pore. P2X7R-over-expression alone is sufficient to drive microglial activation and proliferation in a process that is P2X7R pore dependent, although the biological signaling pathway through which this occurs remains unclear. Once activated, microglia are known to release a number of bioactive substances that include the proinflammatory cytokine interleukin-1ß (IL-1ß). Previous studies have linked P2X7R stimulation to the processing and release of IL-1ß, but whether the channel or pore state of P2X7R is predominant in driving IL-1ß release is unknown and is a major aim of this study. In addition, we will determine whether IL-1ß has trophic effects on surrounding microglia. METHODS: Electron microscopy and immunohistochemistry were used to delineate the sub-cellular localization of P2X7R and IL-1ß in primary hippocampal rat cultures. FM1-43 fluorescent dye and confocal microscopy were used to quantify vesicular exocytosis from microglia expressing the pore-forming P2X7R versus a non-pore-forming point mutant, P2X7RG345Y. IL-1ß in culture was quantified with an enzyme-linked immunosorbent assay (ELISA). IL-1ß intracellular processing was blocked with inhibition of caspase 1 (with a synthetic peptide antagonist), and its extracellular form was neutralized with an IL-1ß neutralizing antibody. Microglial activation and proliferation was quantified immunohistochemically with confocal microscopy. RESULTS: P2X7R and IL-1ß were co-localized in lysosomes. Vesicular exocytosis was higher in microglia expressing the pore-forming P2X7R compared to those expressing the non-pore-forming mutant. There was increased IL-1ß in cultures expressing the pore-forming P2X7R, and this proinflammatory cytokine was found to mediate the trophic effects of P2X7R pore in microglia. Inhibition of IL-1ß production and function resulted in a significant decrease in P2X7R-mediated microglial activation and proliferation. CONCLUSIONS: IL-1ß is a mediator of microglial activation and proliferation, and its release/production is P2X7R pore dependent. Blockade of P2X7R pore could serve as a therapeutic target in alleviating the degree of inflammation seen in neurodegenerative and neoplastic conditions.

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.

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.

HCN channelopathy and cardiac electrophysiologic dysfunction in genetic and acquired rat epilepsy models.

OBJECTIVE: Evidence from animal and human studies indicates that epilepsy can affect cardiac function, although the molecular basis of this remains poorly understood. Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels generate pacemaker activity and modulate cellular excitability in the brain and heart, with altered expression and function associated with epilepsy and cardiomyopathies. Whether HCN expression is altered in the heart in association with epilepsy has not been investigated previously. We studied cardiac electrophysiologic properties and HCN channel subunit expression in rat models of genetic generalized epilepsy (Genetic Absence Epilepsy Rats from Strasbourg, GAERS) and acquired temporal lobe epilepsy (post-status epilepticus SE). We hypothesized that the development of epilepsy is associated with altered cardiac electrophysiologic function and altered cardiac HCN channel expression. METHODS: Electrocardiography studies were recorded in vivo in rats and in vitro in isolated hearts. Cardiac HCN channel messenger RNA (mRNA) and protein expression were measured using quantitative PCR and Western blotting respectively. RESULTS: Cardiac electrophysiology was significantly altered in adult GAERS, with slower heart rate, shorter QRS duration, longer QTc interval, and greater standard deviation of RR intervals compared to control rats. In the post-SE model, we observed similar interictal changes in several of these parameters, and we also observed consistent and striking bradycardia associated with the onset of ictal activity. Molecular analysis demonstrated significant reductions in cardiac HCN2 mRNA and protein expression in both models, providing a molecular correlate of these electrophysiologic abnormalities. SIGNIFICANCE: These results demonstrate that ion channelopathies and cardiac dysfunction can develop as a secondary consequence of chronic epilepsy, which may have relevance for the pathophysiology of cardiac dysfunction in patients with epilepsy.

Low threshold T-type calcium channels as targets for novel epilepsy treatments.

Low voltage-activated T-type calcium channels were originally cloned in the 1990s and much research has since focused on identifying the physiological roles of these channels in health and disease states. T-type calcium channels are expressed widely throughout the brain and peripheral tissues, and thus have been proposed as therapeutic targets for a variety of diseases such as epilepsy, insomnia, pain, cancer and hypertension. This review discusses the literature concerning the role of T-type calcium channels in physiological and pathological processes related to epilepsy. T-type calcium channels have been implicated in pathology of both the genetic and acquired epilepsies and several anti-epileptic drugs (AEDs) in clinical use are known to suppress seizures via inhibition of T-type calcium channels. Despite the fact that more than 15 new AEDs have become clinically available over the past 20 years at least 30% of epilepsy patients still fail to achieve seizure control, and many patients experience unwanted side effects. Furthermore there are no treatments that prevent the development of epilepsy or mitigate the epileptic state once established. Therefore there is an urgent need for the development of new AEDs that are effective in patients with drug resistant epilepsy, are anti-epileptogenic and are better tolerated. We also review the mechanisms of action of the current AEDs with known effects on T-type calcium channels and discuss novel compounds that are being investigated as new treatments for epilepsy.

Are alterations in transmitter receptor and ion channel expression responsible for epilepsies?

Neuronal voltage-gated ion channels and ligand-gated synaptic receptors play a critical role in maintaining the delicate balance between neuronal excitation and inhibition within neuronal networks in the brain. Changes in expression of voltage-gated ion channels, in particular sodium, hyperpolarization-activated cyclic nucleotide-gated (HCN) and calcium channels, and ligand-gated synaptic receptors, in particular GABA and glutamate receptors, have been reported in many types of both genetic and acquired epilepsies, in animal models and in humans. In this chapter we review these and discuss the potential pathogenic role they may play in the epilepsies.

Ethosuximide reduces epileptogenesis and behavioral comorbidity in the GAERS model of genetic generalized epilepsy.

PURPOSE: Ethosuximide (ESX) is a drug of choice for the symptomatic treatment of absence seizures. Chronic treatment with ESX has been reported to have disease-modifying antiepileptogenic activity in the WAG/Rij rat model of genetic generalized epilepsy (GGE) with absence seizures. Here we examined whether chronic treatment with ESX (1) possesses antiepileptogenic effects in the genetic absence epilepsy rats from Strasbourg (GAERS) model of GGE, (2) is associated with a mitigation of behavioral comorbidities, and (3) influences gene expression in the somatosensory cortex region where seizures are thought to originate. METHODS: GAERS and nonepileptic control (NEC) rats were chronically treated with ESX (in drinking water) or control (tap water) from 3 to 22 weeks of age. Subsequently, all animals received tap water only for another 12 weeks to assess enduring effects of treatment. Seizure frequency and anxiety-like behaviors were serially assessed throughout the experimental paradigm. Treatment effects on the expression of key components of the epigenetic molecular machinery, the DNA methyltransferase enzymes, were assessed using quantitative polymerase chain reaction (qPCR). KEY FINDINGS: ESX treatment significantly reduced seizures in GAERS during the treatment phase, and this effect was maintained during the 12-week posttreatment phase (p < 0.05). Furthermore, the anxiety-like behaviors present in GAERS were reduced by ESX treatment (p < 0.05). Molecular analysis revealed that ESX treatment was associated with increased expression of DNA methyltransferase enzyme messenger RNA (mRNA) in cortex. SIGNIFICANCE: Chronic ESX treatment has disease-modifying effects in the GAERS model of GGE, with antiepileptogenic effects against absence seizures and mitigation of behavioral comorbidities. The cellular mechanism for these effects may involve epigenetic modifications.

Regulators of synaptic transmission: roles in the pathogenesis and treatment of epilepsy.

Synaptic transmission is the communication between a presynaptic and a postsynaptic neuron, and the subsequent processing of the signal. These processes are complex and highly regulated, reflecting their importance in normal brain functioning and homeostasis. Sustaining synaptic transmission depends on the continuing cycle of synaptic vesicle formation, release, and endocytosis, which requires proteins such as dynamin, syndapin, synapsin, and synaptic vesicle protein 2A. Synaptic transmission is regulated by diverse mechanisms, including presynaptic modulators of synaptic vesicle formation and release, postsynaptic receptors and signaling, and modulators of neurotransmission. Neurotransmitters released presynaptically can bind to their postsynaptic receptors, the inhibitory γ-aminobutyric acid (GABA)ergic receptors or the excitatory glutamate receptors. Once released, glutamate activates a variety of postsynaptic receptors including α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA), N-methyl-D-aspartate (NMDA), kainate, and metabotropic receptors. The activation of the receptors triggers downstream signaling cascades generating a vast array of effects, which can be modulated by a numerous auxiliary regulatory subunits. Moreover, different neuropeptides such as neuropeptide Y, brain-derived neurotrophic factor (BDNF), somatostatin, ghrelin, and galanin, act as regulators of diverse synaptic functions and along with the classic neurotransmitters. Abnormalities in the regulation of synaptic transmission play a critical role in the pathogenesis of numerous brain diseases, including epilepsy. This review focuses on the different mechanisms involved in the regulation of synaptic transmission, which may play a role in the pathogenesis of epilepsy: the presynaptic modulators of synaptic vesicle formation and release, postsynaptic receptors, and modulators of neurotransmission, including the mechanism by which drugs can modulate the frequency and severity of epileptic seizures.

The P2X7 receptor drives microglial activation and proliferation: a trophic role for P2X7R pore.

Microglial activation is an integral part of neuroinflammation associated with many neurodegenerative conditions. Interestingly, a number of neurodegenerative conditions exhibit enhanced P2X(7) receptor (P2X(7)R) expression in the neuroinflammatory foci where activated microglia are a coexisting feature. Whether P2X(7)R overexpression is driving microglial activation or, conversely, P2X(7)R overexpression is a consequence of microglial activation is not known. We report that overexpression alone of a purinergic P2X(7)R, in the absence of pathological insults, is sufficient to drive the activation and proliferation of microglia in rat primary hippocampal cultures. The trophic responses observed in microglia were found to be P2X(7)R specific as the P2X(7)R antagonist, oxidized ATP (oxATP), was effective in markedly attenuating microgliosis. oxATP treatment of primary hippocampal cultures expressing exogenous P2X(7)Rs resulted in a significant decrease in the number of activated microglia. P2X(7)R is unusual in exhibiting two conductance states, a cation channel and a plasma membrane pore, and there are no pharmacological agents capable of cleanly discriminating between these two states. We used a point mutant of P2X(7)R (P2X7RG345Y) with intact channel function but ablated pore-forming capacity to establish that the trophic effects of increased P2X(7)R expression are exclusively mediated by the pore conductance. Collectively, and contrary to previous reports describing P2X(7)R as a "death receptor," we provide evidence for a novel trophic role for P2X(7)R pore in microglia.