Status Epilepticus Animals Research Articles

Investigating the Effects of Perampanel on Autophagy-mediated Regulation of GluA2 and PSD95 in Epilepsy.

Epilepsy is a chronic neurological disorder characterized by recurrent seizures. Despite various treatment approaches, a significant number of patients continue to experience uncontrolled seizures, leading to refractory epilepsy. The emergence of novel anti-epileptic drugs, such as perampanel (PER), has provided promising options for effective epilepsy treatment. However, the specific mechanisms underlying the therapeutic effects of PER remain unclear. This study aimed to investigate the intrinsic molecular regulatory mechanisms involved in the downregulation of GluA2, a key subunit of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors, following epileptic seizures. Primary mouse hippocampal neurons were cultured and subjected to an epilepsy cell model. The expression levels of GluA2 and autophagy-related proteins were assessed using Western blotting and real-time fluorescent quantitative PCR. Immunofluorescence and immunohistochemistry techniques were employed to investigate the nuclear translocation of CREB-regulated transcriptional coactivator 1 (CRTC1). Additionally, status epilepticus animal models were established to further validate the findings. The epilepsy cell model exhibited a significant decrease in GluA2 expression, accompanied by elevated levels of autophagy-related proteins. Immunofluorescence analysis revealed the nuclear translocation of CRTC1, which correlated with the expression of autophagy-related genes. Treatment with an autophagy inhibitor reversed the decreased expression of GluA2 in the epilepsy cell model. Furthermore, the calcium/calmodulin-dependent protein phosphatase inhibitor FK506 and CaN overexpression affected the dephosphorylation and nuclear translocation of CRTC1, consequently influencing GluA2 expression. Animal model results further supported the involvement of these molecular mechanisms in epilepsy. Our findings suggest that the downregulation of GluA2 following epileptic seizures involves the activation of autophagy and the regulation of CRTC1 nuclear translocation. These intrinsic molecular regulatory mechanisms provide potential targets for developing novel therapeutic strategies to alleviate refractory epilepsy and preserve cognitive functions in patients.

Alpha-2a adrenergic receptor activation in genetic absence epilepsy: An absence status model?

The objective of the study was to propose a candidate animal model of absence status epilepticus induced by specific alpha-2a adrenergic receptor (α2AR) activation. We also aim to investigate the responsiveness of this model to classical anti-status or anti-absence medications. An α2AR agonist, dexmedetomidine (DEX), was injected intracerebroventricularly into adult rats with genetic absence epilepsy, and their electroencephalography (EEG) was recorded. The total duration, number, and mean duration of each spike-and-wave discharges (SWDs) were evaluated. The blocks of absence status events were classified as the initial and second sets of absence statuses. Ethosuximide (ETX) was administered as a pretreatment to another group of rats and later injected with 2.5 μg DEX. In addition, ETX, valproic acid (VPA), diazepam (DIAZ), and atipamezole (ATI) were administered after induced status-like events following DEX administration. Power spectral characteristics and coherence analysis were performed on the EEG to assess the absence status events and sleep. The 2.5 μg dose of DEX increased the total SWD duration and induced continuous SWDs up to 26 min. Following the initial absence status event, sleep was induced; then, the second period of absence status-like activities were initiated. ETX pretreatment blocked the occurrence of absence status-like activities. Power spectral density analyses revealed that DEX-induced post-sleep activities had higher power in delta frequency band (1-4 Hz) and attenuated power of 7 Hz harmonics (14 and 21 Hz) than the pre-injection seizure. The mean duration of SWDs were decreased in all the groups, but occasional prolonged activities were seen in ETX or VPA-injected rats but not with DIAZ or ATI. This study presents an absence status epilepticus animal model that is activated by α2AR activation to investigate the pathophysiological role of absence status. Unlike other agents ATI switched off the second set of absence statuses to normal SWDs, without sedation or lethargy, can show it may preferentially block absence status-like activity. This study proposes a rat model for prolonged seizures, resembling absence status epilepticus. Activating the brain's alpha-2a adrenergic receptor with dexmedetomidine induced seizures lasting up to 26 minutes. Ethosuximide pretreatment and post-treatment with valproic acid, diazepam, and atipamezole decreased induced seizures. The findings suggest this model is valuable for studying absence status epilepticus. In addition, atipamezole normalized abnormal seizures without sedation, hinting at its potential for targeted treatment and further research.

Dapsone Protects Against Lithium-Pilocarpine-Induced Status Epilepticus in Rats through Targeting Tumor Necrosis Factor-α and Nitrergic Pathway.

Status epilepticus (SE) results in permanent neuronal brain damage in the central nervous system. One of the complex etiologies underlying SE pathogenesis is neuroinflammation. Dapsone has been recently considered as a potential neuroprotective agent in neuroinflammatory conditions. Therefore, the present study aims to investigate effects of dapsone on lithium-pilocarpine-induced SE in rats and assess whether tumor necrosis factor-alpha (TNF-α) and nitric oxide (NO) pathway participate in this effect. SE was established by injecting lithium chloride (127 mg/kg, intraperitoneally [i.p.]) and pilocarpine (60 mg/kg, i.p.). The animals received pre-treatment dapsone (2, 5, 10, and 20 mg/kg, oral gavage) and post-treatment dapsone (10 mg/kg). Subsequently, seizure score and mortality rate were documented. To assess the underlying signaling pathway, L-Nω-Nitro-L-arginine methyl ester hydrochloride (a non-specific NO synthase [NOS] inhibitor), 7-nitroindazole (a specific neuronal NOS inhibitor), and aminoguanidine (a specific inducible NOS inhibitor) were administered 15 minutes before dapsone (10 mg/kg) pre- or post-treatment. Hippocampal tissue TNF-α and NO concentrations were quantified using the enzyme-linked immunosorbent assay method. Dapsone (10 mg/kg) pre-and post-treatment significantly attenuated the increased seizure score and mortality rate due to lithium-pilocarpine-induced SE. The development of SE in animals was associated with higher TNF-α and NO metabolites levels, which notably decreased in the dapsone-treated rats. Moreover, co-administration of NOS inhibitors with dapsone markedly reversed the anti-epileptic effects of dapsone and caused an escalation in TNF-α level but a significant reduction in NO concentration level. It seems that dapsone may exert an anti-epileptic effect on lithium-pilocarpine-induced SE through TNF-α inhibition and modulation of the nitrergic pathway.

Neuroprotective effect and herbal-drug pharmacokinetic interaction of Gastrodia elata extract on valproic acid

Valproic acid (VPA) is a widely used antiepileptic drug, and the herbal extract of Gastrodia elata exerts an anticonvulsant effect. However, few studies have investigated the pharmacokinetic and pharmacodynamic interactions between G. elata extract and VPA. We hypothesize that G. elata extract increases the VPA levels in the brain and enhances the antiepileptic effects of VPA, and this synergistic effect is mediated by transporters at the bloodbrain barrier (BBB). We performed microdialysis on pilocarpine-induced epileptic model rats in vivo to investigate this hypothesis. The results demonstrated that cotreatment with G. elata extract and VPA ameliorated drug-resistant epilepsy by increasing the VPA levels in the brain. In addition, G. elata extract and VPA exerted synergistic anticonvulsive effects to decrease the seizure severity by protecting neurons in the hippocampus and altering the DOPAC and 5-HT levels. However, these phenomena were partially blocked by the organic anion transporter peptide (OATP) inhibitor cyclosporine A (CsA; 20 mg/kg, i.p.), which demonstrated that the increase in the VPA level in the brain was modulated by the transporter OATP. This study provides a comprehensive strategy for assessing the interaction between traditional medicines and conventional antiepileptic drugs in a status epilepticus animal model.

Histopathological modeling of status epilepticus-induced brain damage based on in vivo diffusion tensor imaging in rats.

Non-invasive magnetic resonance imaging (MRI) methods have proved useful in the diagnosis and prognosis of neurodegenerative diseases. However, the interpretation of imaging outcomes in terms of tissue pathology is still challenging. This study goes beyond the current interpretation of in vivo diffusion tensor imaging (DTI) by constructing multivariate models of quantitative tissue microstructure in status epilepticus (SE)-induced brain damage. We performed in vivo DTI and histology in rats at 79 days after SE and control animals. The analyses focused on the corpus callosum, hippocampal subfield CA3b, and layers V and VI of the parietal cortex. Comparison between control and SE rats indicated that a combination of microstructural tissue changes occurring after SE, such as cellularity, organization of myelinated axons, and/or morphology of astrocytes, affect DTI parameters. Subsequently, we constructed a multivariate regression model for explaining and predicting histological parameters based on DTI. The model revealed that DTI predicted well the organization of myelinated axons (cross-validated R = 0.876) and astrocyte processes (cross-validated R = 0.909) and possessed a predictive value for cell density (CD) (cross-validated R = 0.489). However, the morphology of astrocytes (cross-validated R > 0.05) was not well predicted. The inclusion of parameters from CA3b was necessary for modeling histopathology. Moreover, the multivariate DTI model explained better histological parameters than any univariate model. In conclusion, we demonstrate that combining several analytical and statistical tools can help interpret imaging outcomes to microstructural tissue changes, opening new avenues to improve the non-invasive diagnosis and prognosis of brain tissue damage.

Theta and gamma hippocampal–neocortical oscillations during the episodic-like memory test: Impairment in epileptogenic rats

Cortical oscillations in different frequency bands have been shown to be intimately involved in exploration of environment and cognition. Here, the local field potentials in the hippocampus, the medial prefrontal cortex (mPFC), and the medial entorhinal cortex (mEC) were recorded simultaneously in rats during the execution of the episodic-like memory task. The power of theta (~4–10 Hz), slow gamma (~25–50 Hz), and fast gamma oscillations (~55–100 Hz) was analyzed in all structures examined. Particular attention was paid to the theta coherence between three mentioned structures. The modulation of the power of gamma rhythms by the phase of theta cycle during the execution of the episodic-like memory test by rats was also closely studied. Healthy rats and rats one month after kainate-induced status epilepticus (SE) were examined. Paroxysmal activity in the hippocampus (high amplitude interictal spikes), excessive excitability of animals, and the death of hippocampal and dentate granular cells in rats with kainate-evoked SE were observed, which indicated the development of seizure focus in the hippocampus (epileptogenesis). One month after SE, the rats exhibited a specific impairment of episodic memory for the what-where-when triad: unlike healthy rats, epileptogenic SE animals did not identify the objects during the test. This impairment was associated with the changes in the characteristics of theta and gamma rhythms and specific violation of theta coherence and theta/gamma coupling in these structures in comparison with the healthy animals. We believe that these disturbances in the cortical areas play a role in episodic memory dysfunction in kainate-treated animals. These findings can shed light on the mechanisms of cognitive deficit during epileptogenesis.

Beneficial effect of moringa stenopetala (bak.f) cuf. on lithium–pilocarpine-induced temporal lobe epilepsy in experimental animals

Background: Moringa stenopetala is traditionally used for the treatment of epilepsy in southern Ethiopia. Additionally, the plant material is used for the treatment of various ailments such as pain, diabetes, hypertension, and hyperlipemia. Aim of the Study: The aim of the study was to investigate the effect of M. stenopetala crude extract on the development of lithium–pilocarpine-induced temporal lobe epilepsy model in rats. Materials and Methods: Male Sprague-Dawley rats weighing 200–225 g were randomly divided into nine groups. Groups I, V, and VI served as normal control, positive control, and negative control, respectively. While Groups II–IV were treated with three different doses (400, 600, and 800 mg/kg) levels of crude extract in pre- and postinduction of status epilepticus (SE). On the other hand, Groups VII–IX were treated with the same three dose levels but after induction of SE only. All the rats, except those in normal control, were subjected to lithium (3 meq/kg) and pilocarpine (35 mg/kg) for the induction of SE. In addition, the elevated plusmaze and infrared actimeter models were used to evaluate anxiolytic and antidepressant efficacy in SE animals. Results: The rats treated with the crude extract of M. stenopetala significantly increased the latency period to the first motor seizure (58.1 min) and SE (80.4 min) compared to the control group; those developed seizure and SE at 13.3 and 35.4 min, respectively. The chronic spontaneous seizure was less severity just below spontaneous recurrent seizure at Pinel and Rovner scale 6 in rats treated with extract pre- and postinduction. The crude extract also showed significant dose-dependent anxiolytic activity (P < 0.001) and improved locomotor activity (P < 0.001). The histopathological studies of hippocampus formation revealed that the extract prevented damages compared to control animals. Conclusion: The current findings suggest that a crude extract of M. stenopetala has anticonvulsant activity in a lithium–pilocarpine-induced epilepsy model M. stenopetala also reduced depression and anxiety associated with epilepsy that indicates that it is having antidepressant and anxiolytic activity.

Genome-wide microRNA profiling in brain and blood samples in a mouse model of epileptogenesis

ObjectivesThis study profiled circulating and hippocampal microRNAs (miRNAs) to identify alterations associated with the risk of epileptogenesis in a mouse temporal lobe epilepsy model. MethodsNext-generation sequencing was performed to examine the changes in miRNA expression 24 h after pilocarpine-induced status epilepticus (SE) in C57BL/6NCrl mice using both blood and hippocampus samples. Differentially expressed miRNAs were identified from SE animals and matched controls that failed to develop SE after receiving equal doses of pilocarpine (NS animals). Blood and brain miRNA profiles were then compared to identify circulating miRNA alterations reflecting the changes in the brain. ResultsWe identified 3 miRNAs that were significantly up-regulated and 4 miRNAs that were significantly down-regulated in the blood of SE animals compared with NS animals. When hippocampal miRNAs of SE animals and NS animals were compared, 5 miRNAs were up-regulated and 4 were down-regulated. Of these, miR-434-3p and miR-133a-3p were observed to have greatest changes in both blood and brain of SE animals. SignificanceThis study extends current knowledge of changes in miRNAs associated with epileptogenesis by profiling miRNAs in SE and NS animals in an experimental temporal lobe epilepsy model. The study was designed to allow non-specific changes due to the activation of muscarinic cholinergic receptors in peripheral organs by pilocarpine to be ruled out. Significantly altered circulating miRNAs that reflect changes in the brain during epileptogenesis after SE have the potential to be developed as prognostic biomarkers for epileptogenesis.

Cardiac dysregulation following intrahippocampal kainate-induced status epilepticus

Status epilepticus (SE) is a prevalent disorder associated with significant morbidity, including the development of epilepsy and mortality. Cardiac arrhythmias (i.e. inappropriate sinus tachycardia and bradycardia, asystole, and atrioventricular blocks) are observed in patients following SE. We characterized ictal (during a seizure) and interictal (between seizure) cardiac arrhythmogenesis following SE using continuous electrocardiography and video electroencephalography (vEEG) recordings throughout a 14-day monitoring period in an intrahippocampal chemoconvulsant mouse model that develops epilepsy. We quantified heart rhythm abnormalities and examined whether the frequency of cardiac events correlated with epileptiform activity, circadian (light/dark) cycle, the presence of seizures, and survival during this period of early epileptogenesis (the development of epilepsy) following SE. Shortly following SE, mice developed an increased interictal heart rate and heart rhythm abnormalities (i.e. sinus pause and sinus arrhythmias) when compared to control mice. Heart rhythm abnormalities were more frequent during the light cycle and were not correlated with increased epileptiform activity or seizure frequency. Finally, SE animals had early mortality, and a death event captured during vEEG recording demonstrated severe bradycardia prior to death. These cardiac changes occurred within 14 days after SE and may represent an early risk factor for sudden death following SE.

Interaction of GABAA and GABAB antagonists after status epilepticus in immature rats

Among neurotransmitter systems affected by status epilepticus (SE) in adult rats are both GABAergic systems. To analyze possible changes of GABAA and GABAB systems in developing rats lithium-pilocarpine SE was induced at postnatal day 12 (P12). Seizures were elicited by a GABAA antagonist pentylenetetrazol (PTZ) 3, 6, 9, and 13 days after SE (i.e., in P15, P18, P21, and P25 rats), and their possible potentiation by a GABAB receptor antagonist CGP46381 was studied. Pilocarpine was replaced by saline in control animals (lithium-paraldehyde [LiPAR]).Pentylenetetrazol in a dose of 50 mg/kg s.c. elicited generalized seizures in nearly all 15-day-old naive rats and in 40% of 18-day-old ones but not in older animals. After SE, PTZ no longer elicited seizures in these two younger groups, i.e., sensitivity of GABAA system was diminished. The GABAB antagonist exhibited proconvulsant effect in P15 and P18 SE as well as LiPAR rats returning the incidence of PTZ-induced seizures to values of control animals. A decrease in the incidence of minimal clonic seizures was seen in P21 LiPAR animals; these seizures in the oldest group were not affected. Change of the effect from proconvulsant to anticonvulsant (or at least to no action) took place before postnatal day 21.Both SE and LiPAR animals exhibited similar changes but their intensity differed, effects in LiPAR controls were usually more expressed than in SE rats.

Astrocytic Atrophy Following Status Epilepticus Parallels Reduced Ca2+ Activity and Impaired Synaptic Plasticity in the Rat Hippocampus.

Epilepsy is a group of neurological disorders commonly associated with the neuronal malfunction leading to generation of seizures. Recent reports point to a possible contribution of astrocytes into this pathology. We used the lithium-pilocarpine model of status epilepticus (SE) in rats to monitor changes in astrocytes. Experiments were performed in acute hippocampal slices 2–4 weeks after SE induction. Nissl staining revealed significant neurodegeneration in the pyramidal cell layers of hippocampal CA1, CA3 areas, and the hilus, but not in the granular cell layer of the dentate gyrus. A significant increase in the density of astrocytes stained with an astrocyte-specific marker, sulforhodamine 101, was observed in CA1 stratum (str.) radiatum. Astrocytes in this area were also whole-cell loaded with a morphological tracer, Alexa Fluor 594, for two-photon excitation imaging. Sholl analyses showed no changes in the size of the astrocytic domain or in the number of primary astrocytic branches, but a significant reduction in the number of distal branches that are resolved with diffraction-limited light microscopy (and are thought to contain Ca2+ stores, such as mitochondria and endoplasmic reticulum). The atrophy of astrocytic branches correlated with the reduced size, but not overall frequency of Ca2+ events. The volume tissue fraction of nanoscopic (beyond the diffraction limit) astrocytic leaflets showed no difference between control and SE animals. The results of spatial entropy-complexity spectrum analysis were also consistent with changes in ratio of astrocytic branches vs. leaflets. In addition, we observed uncoupling of astrocytes through the gap-junctions, which was suggested as a mechanism for reduced K+ buffering. However, no significant difference in time-course of synaptically induced K+ currents in patch-clamped astrocytes argued against possible alterations in K+ clearance by astrocytes. The magnitude of long-term-potentiation (LTP) was reduced after SE. Exogenous D-serine, a co-agonist of NMDA receptors, has rescued the initial phase of LTP. This suggests that the reduced Ca2+-dependent release of D-serine by astrocytes impairs initiation of synaptic plasticity. However, it does not explain the failure of LTP maintenance which may be responsible for cognitive decline associated with epilepsy.

The role of AMPA receptors and their antagonists in status epilepticus.

Status epilepticus (SE) is typically defined as a prolonged self-sustaining seizure or repeated seizures showing an incomplete recovery between them. SE represents a medical emergency often associated with significant disability, morbidity, and mortality. Despite the clinical impact, the mechanisms underlying the transition from self-limited seizures to protracted, medically refractory seizures are not completely understood. About 40% of patients in established SE are refractory to antiepileptic drugs (first-line treatment); therefore, there is a need for more efficacious drugs. In this review, we focused on the current knowledge about the involvement of alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors during SE, the preclinical efficacy of its antagonists, and the currently published clinical studies involving drugs with this mechanism of action. We carried out an extensive literature search to recognize experimental and clinical articles both on AMPA receptors and AMPA antagonists and SE. Recently, the role of AMPA receptors during and after SE has become clearer, and it is now widely accepted that early changes occur in the initial stages and probably contribute both to the maintenance of SE and to its refractoriness to treatment. The therapeutic potential of AMPA receptor inhibition has been sustained by studies in which AMPA receptor antagonists have been shown to terminate seizures in several SE animal models. To date, promising, but limited, data in humans support the use of AMPA receptor antagonists in patients with SE. AMPA receptor antagonists could become a new therapeutic option in patients with established SE when the first trials with second-line agents have failed or probably even better after failure of benzodiazepines as a second-line option.

Early Gabapentin Treatment during the Latency Period Increases Convulsive Threshold, Reduces Microglial Activation and Macrophage Infiltration in the Lithium-Pilocarpine Model of Epilepsy.

The lithium-pilocarpine model of epilepsy reproduces several features of temporal lobe epilepsy in humans, including the chronological timeline of an initial latency period followed by the development of spontaneous seizures. Epilepsy therapies in humans are implemented, as a rule, after the onset of the spontaneous seizures. We here studied the potential effect on epileptogenesis of starting an early treatment during the latency period, in order to prevent the development of spontaneous seizures. Adult male Wistar rats were treated with 3 mEq/kg LiCl, and 20 h later 30 mg/kg pilocarpine. Once status epilepticus (SE) was achieved, it was allowed to last for 20 min, and then motor seizures were controlled with the administration of 20 mg/kg diazepam. At 1DPSE (DPSE, days post-status epilepticus), animals started to receive 400 mg/kg/day gabapentin or saline for 4 days. At 5DPSE, we observed that SE induced an early profuse microglial and astroglial reactivity, increased synaptogenic trombospondin-1 expression and reduced AQP4 expression in astroglial ending feet. Blood brain barrier (BBB) integrity seemed to be compromised, as infiltrating NG2+ macrophages and facilitated access to the CNS was observed by transplanting eGFP+ blood cells and bone marrow-derived progenitors in the SE animals. The early 4-day gabapentin treatment successfully reduced microglial cell reactivity and blood-borne cell infiltration, without significantly altering the mRNA of proinflammatory cytokines IL-1β and TNFα immediately after the treatment. After 21DSPE, another group of animals that developed SE and received 4 days of gabapentin treatment, were re-exposed to subconvulsive accumulative doses of pilocarpine (10 mg/kg/30 min) and were followed by recording the Racine scale reached. Early 4-day gabapentin treatment reduced the Racine scale reached by the animals, reduced animal mortality, and reduced the number of animals that achieved SE (34% vs. 72%). We conclude that early gabapentin treatment following SE, during the latency period, is able to reduce neuroinflammation and produces a persistent effect that limits seizures and increases convulsive threshold, probably by restricting microglial reactivity and spurious synaptogenesis.

VEGF treatment during status epilepticus attenuates long-term seizure-associated alterations in astrocyte morphology.

Vascular endothelial growth factor (VEGF) treatment during pilocarpine-induced status epilepticus (SE) causes sustained preservation of behavioral function in rats in the absence of enduring neuroprotection (Nicoletti et al., 2010), suggesting the possibility that other cells or mechanisms could be involved in the beneficial effects of VEGF during SE. Astrocytes have been reported to contribute to epileptiform discharges in the hippocampus (Tian et al., 2005; Kang et al., 1998) and to express VEGF receptors (Krum & Rosenstein, 2002). We report here that VEGF treatment significantly alters multiple astrocyte parameters. This study investigated astrocyte morphology one month after SE in animals treated with VEGF or inactivated VEGF control protein during SE. Individual GFAP-immunostained astrocytes from CA1 and dentate gyrus hilus were traced and morphologically quantified, and both somatic and process structures were analyzed. VEGF treatment during SE significantly prevented post-SE increases in number of branch intersections, process length, and node count. Furthermore, analysis of distance to nearest neighboring astrocytes revealed that VEGF treatment significantly increased inter-astrocyte distance. Overall, VEGF treatment during SE did not significantly alter the shape of the astrocytes, but did prevent SE-induced changes in branching complexity, size, and spatial patterning. Because astrocyte morphology may be related to astrocyte function, it is possible that VEGF's enduring effects on astrocyte morphology may impact the functioning of the post-seizure hippocampus.

Which component of treatment is important for changes of cortical epileptic afterdischarges after status epilepticus in immature rats?

Role of lithium chloride and paraldehyde in acute changes after lithium-pilocarpine status epilepticus (SE) induced at postnatal day 12 was studied in 15-day-old rats. In addition to SE group four other groups were formed: naïve animals without any injection, lithium chloride group, paraldehyde group and lithium-paraldehyde group. Cortical epileptic afterdischarges (CxADs) induced by increasing intensities of stimulation current were used as a measure of excitability. SE animals did not exhibit any change in duration of CxADs with increasing stimulation intensity in contrast to naïve control with a progressive prolongation of CxAD. LiCl group was similar to SE rats whereas paraldehyde and lithium-paraldehyde groups exhibited some progress in duration of ADs. Lithium chloride participates in short-term changes of CxADs after SE. Paraldehyde and combination of lithium and paraldehyde are similar to naïve controls.

Functional properties of granule cells with hilar basal dendrites in the epileptic dentate gyrus.

The maturation of adult-born granule cells and their functional integration into the network is thought to play a key role in the proper functioning of the dentate gyrus. In temporal lobe epilepsy, adult-born granule cells in the dentate gyrus develop abnormally and possess a hilar basal dendrite (HBD). Although morphological studies have shown that these HBDs have synapses, little is known about the functional properties of these HBDs or the intrinsic and network properties of the granule cells that possess these aberrant dendrites. We performed patch-clamp recordings of granule cells within the granule cell layer "normotopic" from sham-control and status epilepticus (SE) animals. Normotopic granule cells from SE animals possessed an HBD (SE+ HBD+ cells) or not (SE+ HBD- cells). Apical and basal dendrites were stimulated using multiphoton uncaging of glutamate. Two-photon Ca2+ imaging was used to measure Ca2+ transients associated with back-propagating action potentials (bAPs). Near-synchronous synaptic input integrated linearly in apical dendrites from sham-control animals and was not significantly different in apical dendrites of SE+ HBD- cells. The majority of HBDs integrated input linearly, similar to apical dendrites. However, 2 of 11 HBDs were capable of supralinear integration mediated by a dendritic spike. Furthermore, the bAP-evoked Ca2+ transients were relatively well maintained along HBDs, compared with apical dendrites. This further suggests an enhanced electrogenesis in HBDs. In addition, the output of granule cells from epileptic tissue was enhanced, with both SE+ HBD- and SE+ HBD+ cells displaying increased high-frequency (>100 Hz) burst-firing. Finally, both SE+ HBD- and SE+ HBD+ cells received recurrent excitatory input that was capable of generating APs, especially in the absence of feedback inhibition. Taken together, these data suggest that the enhanced excitability of HBDs combined with the altered intrinsic and network properties of granule cells collude to promote excitability and synchrony in the epileptic dentate gyrus.

Early cardiac electrographic and molecular remodeling in a model of status epilepticus and acquired epilepsy.

A myriad of acute and chronic cardiac alterations are associated with status epilepticus (SE) including increased sympathetic tone, rhythm and ventricular repolarization disturbances. Despite these observations, the molecular processes underlying SE-associated myocardial remodeling remain to be identified. Here we determined early SE-associated myocardial electrical and molecular alterations using a model of SE and acquired epilepsy. We performed electrocardiography (ECG) assessments in rats beginning at 2 weeks following kainate-induced SE, and calculated short-term variability (STV) of the corrected QT intervals (QTc) as a marker of ventricular stability. Using western blotting, we quantified myocardial β1-adrenergic receptors (β1-AR) and ventricular gap junction protein connexin 43 (Cx43) levels as makers of increased sympathetic tone. We determined the activation status of three kinases associated with sympathetic stimulation and their downstream ion channel targets: extracellular signal-regulated kinase (ERK), protein kinase A (PKA), Ca2+ /calmodulin-dependent protein kinase II (CamKII), hyperpolarization-activated cyclic nucleotide-gated channel subunit 2 (HCN2), and voltage-gated potassium channels 4.2 (Kv4.2 ). We investigated whether SE was associated with altered Ca2+ homeostasis by determining select Ca2+ -handling protein levels using western blotting. Compared with the sham group, SE animals exhibited higher heart rate, longer QTc interval, and higher STV beginning at 2 weeks following SE. Concurrently, the myocardium of SE rats showed lower β1-AR and higher Cx43 protein levels, higher levels of phosphorylated ERK, PKA, and CamKII along with decreased HCN2 and Kv4.2 channel levels. In addition, the SE rats had altered proteins levels of Ca2+ -handling proteins, with decreased Na+ /Ca2+ exchanger-1 and increased calreticulin. SE triggers early molecular alterations in the myocardium consistent with increased sympathetic tone and altered Ca2+ homeostasis. These changes, coupled with early and persistent ECG abnormalities, suggest that the observed molecular alterations may contribute to SE-associated cardiac remodeling. Additional mechanistic studies are needed to determine potential causal roles.

Does status epilepticus induced at early postnatal period change excitability after cortical epileptic afterdischarges?

Possible changes of cortical excitability after status epilepticus (SE) elicited in 12-day-old rats were studied by means of paired cortical afterdischarges (ADs). Consequences of lithium-pilocarpine status were studied in animals with implanted electrodes 3, 6, 9, 13, and 26 days after SE. Paired low-frequency stimulation with a 1-min interval was repeated after 10 min, and duration of ADs was measured. Control rats received saline instead of pilocarpine; other treatments were the same as in SE group. Postictal refractoriness (i.e., the testing response significantly shorter than the conditioning one) appeared at the age of 18 days in lithium-paraldehyde controls, whereas SE animals exhibited this phenomenon since postnatal day 21. The only significant difference between SE and lithium-paraldehyde controls was found in the second conditioning AD in the oldest group studied-it was longer in 38-day-old SE animals. Our results demonstrated moderate signs of higher excitability of SE rats in comparison with control ones long before appearance of spontaneous seizures.

CRTC1 nuclear localization in the hippocampus of the pilocarpine-induced status epilepticus model of temporal lobe epilepsy

cAMP response-element binding protein (CREB)-dependent genes are differentially expressed in brains of temporal lobe epilepsy (TLE) patients and also in animal models of TLE. Previous studies have demonstrated the importance of CREB regulated transcription in TLE. However, the role of the key regulator of CREB activity, CREB-regulated transcription coactivator 1 (CRTC1), has not been explored in epilepsy. In the present study the pilocarpine-induced status epilepticus (SE) model of TLE was used to study the regulation of CRTC1 during and following SE. Nuclear translocation of CRTC1 is critical for its transcriptional activity, and dephosphorylation at serine 151 residue via calcineurin phosphatase regulates cytoplasmic to nuclear transit of CRTC1. Here, we examined the localization and phosphorylation (Ser151) of CRTC1 in SE-induced rat hippocampus at two different time points after SE onset.One hour after SE onset, we found that CRTC1 translocates to the nucleus of CA1 neurons but not CA3 or dentate granule neurons. We further found that this CRTC1 nuclear localization is independent of Ser151 dephosphorylation since we did not detect any difference in dephosphorylation of Ser151 between control and SE animals at this time point.In contrast, 48h after SE CRTC1 shows increased nuclear localization in the dentate gyrus (DG) of the SE-induced rats. At 48h after SE, FK506 treatment blocked CRTC1 nuclear localization and dephosphorylation of Ser151.Our results provide evidence that CREB cofactor CRTC1 translocates into the nucleus of a distinct subset of hippocampal neurons during and following SE and this translocalization is regulated by calcineurin at a later time point following SE. Nuclear CRTC1 can bind to CREB possibly altering transcription during epileptogenesis.

Benzodiazepines induce sequelae in immature mice with inflammation-induced status epilepticus.

Since benzodiazepines (BZPs) became clinically available for the treatment of status epilepticus (SE) in children, the incidence of neurological sequelae has increased. However, the cause-effect relationship is poorly understood. In this paper, we examined the effect of BZPs on an inflammation-induced SE (iSE) animal model. Inflammation was induced by injecting poly(I:C) (pIC 10 mg/kg, postnatal day 12-14), seizure was induced by injecting pilocarpine hydrochloride (PILO 200 mg/kg, postnatal day 15) into C57BL/6J mice, and the pIC+PILO mice were used as the iSE model (miSE). The GABA-A receptor agonist midazolam (MDL 0.5 mg/kg) was used to inhibit seizures. Sequelae were evaluated by performing behavior and immunohistochemical analyses in the chronic phase. The exploratory activity of mice in the miSE plus MDL group increased significantly, indicating that hyperactivity was newly induced by MDL in miSE mice. The contextual fear memory of the miSE mice was also significantly increased and that of miSE treated with MDL returned to the normal level. The parvalbumin-positive GABA neurons were decreased in number by pIC+PILO which was rescued by MDL. Apoptosis marker ssDNA-positive cells were increased by pIC+PILO which could not be rescued by MDL. Therefore, we propose that BZP-dependent therapy for SE needs to be rethought from the perspective of using other treatment approaches.