Spatiotemporal Correlation of Epileptiform Activity and Gene Expression in vitro.
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ABSTRACT:
SUBMITTER: Schlabitz S
PROVIDER: S-EPMC8042243 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Epilepsy is a chronic brain disorder involving recurring seizures often precipitated by an earlier neuronal insult. The mechanisms that link the transient neuronal insult to the lasting state of epilepsy are unknown. Here we tested the possible role of DNA methylation in mediating long-term induction of epileptiform activity by transient kainic acid exposure using in vitro and in vivo rodent models. We analyzed changes in the gria2 gene, which encodes for the GluA2 subunit of the ionotropic glutamate, alpha-amino-3-hydroxy-5-methyl-4-isoxazole proprionic acid receptor and is well documented to play a role in epilepsy. We show that kainic acid exposure for two hours to mouse hippocampal slices triggers methylation of a 5' regulatory region of the gria2 gene. Increase in methylation persists one week after removal of the drug, with concurrent suppression of gria2 mRNA expression levels. The degree of kainic acid-induced hypermethylation of gria2 5' region varies between individual slices and correlates with the changes in excitability induced by kainic acid. In a rat in vivo model of post kainic acid-induced epilepsy, we show similar hypermethylation of the 5' region of gria2. Inter-individual variations in gria2 methylation, correlate with the frequency and intensity of seizures among epileptic rats. Luciferase reporter assays support a regulatory role for methylation of gria2 5' region. Inhibition of DNA methylation by RG108 blocked kainic acid-induced hypermethylation of gria2 5' region in hippocampal slice cultures and bursting activity. Our results suggest that DNA methylation of such genes as gria2 mediates persistent epileptiform activity and inter-individual differences in the epileptic response to neuronal insult and that pharmacological agents that block DNA methylation inhibit epileptiform activity raising the prospect of DNA methylation inhibitors in epilepsy therapeutics.
| S-EPMC3788713 | biostudies-literature
Project description:PurposeIn vitro brain preparations have been used extensively to study the generation and propagation of epileptiform activity. Transverse and longitudinal slices of the rodent hippocampus have revealed various patterns of propagation. Yet intact connections between the transverse and longitudinal pathways should generate orthogonal (both transverse and longitudinal) propagation of seizures involving the entire hippocampus. This study utilizes the planar unfolded mouse hippocampus preparation to reveal simultaneous orthogonal epileptiform propagation and to test a method of arresting propagation.MethodsThis study utilized an unfolded mouse hippocampus preparation. It was chosen due to its preservation of longitudinal neuronal processes, which are thought to play an important role in epileptiform hyperexcitability. 4-Aminopyridine (4-AP), microelectrodes, and voltage-sensitive dye imaging were employed to investigate tissue excitability.Key findingsIn 50-μm 4-AP, stimulation of the stratum radiatum induced transverse activation of CA3 cells but also induced a longitudinal wave of activity propagating along the CA3 region at a speed of 0.09 m/s. Without stimulation, a wave originated at the temporal CA3 and propagated in a temporal-septal direction could be suppressed with glutamatergic receptor antagonists. Orthogonal propagation traveled longitudinally along the CA3 pathway, secondarily invading the CA1 region at a velocity of 0.22 ± 0.024 m/s. Moreover, a local lesion restricted to the CA3 region could arrest wave propagation.SignificanceThese results reveal a complex two-dimensional epileptiform wave propagation pattern in the hippocampus that is generated by a combination of synaptic transmission and axonal propagation in the CA3 recurrent network. Epileptiform propagation block via a transverse selective CA3 lesion suggests a potential surgical technique for the treatment of temporal lobe epilepsy.
| S-EPMC3169769 | biostudies-literature
Project description:Temporal lobe epilepsy (TLE) is the most common form of focal epilepsy with about 30% of patients developing pharmacoresistance. These patients continue to suffer from seizures despite polytherapy with antiepileptic drugs (AEDs) and have an increased risk for premature death, thus requiring further efforts for the development of new antiepileptic therapies. The molecule dimethylethanolamine (DMEA) has been tested as a potential treatment in various neurological diseases, albeit the functional mechanism of action was never fully understood. In this study, we investigated the effects of DMEA on neuronal activity in single-cell recordings of primary neuronal cultures. DMEA decreased the frequency of spontaneous synaptic events in a concentration-dependent manner with no apparent effect on resting membrane potential (RMP) or action potential (AP) threshold. We further tested whether DMEA can exert antiepileptic effects in human brain tissue ex vivo. We analyzed the effect of DMEA on epileptiform activity in the CA1 region of the resected hippocampus of TLE patients in vitro by recording extracellular field potentials in the pyramidal cell layer. Epileptiform burst activity in resected hippocampal tissue from TLE patients remained stable over several hours and was pharmacologically suppressed by lacosamide, demonstrating the applicability of our platform to test antiepileptic efficacy. Similar to lacosamide, DMEA also suppressed epileptiform activity in the majority of samples, albeit with variable interindividual effects. In conclusion, DMEA might present a new approach for treatment in pharmacoresistant TLE and further studies will be required to identify its exact mechanism of action and the involved molecular targets.
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Project description:The optogenetic approach to gain control over neuronal excitability both in vitro and in vivo has emerged as a fascinating scientific tool to explore neuronal networks, but it also opens possibilities for developing novel treatment strategies for neurologic conditions. We have explored whether such an optogenetic approach using the light-driven halorhodopsin chloride pump from Natronomonas pharaonis (NpHR), modified for mammalian CNS expression to hyperpolarize central neurons, may inhibit excessive hyperexcitability and epileptiform activity. We show that a lentiviral vector containing the NpHR gene under the calcium/calmodulin-dependent protein kinase IIalpha promoter transduces principal cells of the hippocampus and cortex and hyperpolarizes these cells, preventing generation of action potentials and epileptiform activity during optical stimulation. This study proves a principle, that selective hyperpolarization of principal cortical neurons by NpHR is sufficient to curtail paroxysmal activity in transduced neurons and can inhibit stimulation train-induced bursting in hippocampal organotypic slice cultures, which represents a model tissue of pharmacoresistant epilepsy. This study demonstrates that the optogenetic approach may prove useful for controlling epileptiform activity and opens a future perspective to develop it into a strategy to treat epilepsy.
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Project description:Otoconia are minute biocrystals composed of glycoproteins, proteoglycans, and CaCO3 , and are indispensable for sensory processing in the utricle and saccule. Otoconia abnormalities and degeneration can cause or facilitate crystal dislocation to the ampulla, leading to vertigo and imbalance in humans. In order to better understand the molecular mechanism controlling otoconia formation and maintenance, we have examined the spatial and temporal expression differences of otoconial genes in the mouse inner ear at developmental, mature and aging stages using whole transcriptome sequencing (RNA-Seq) and quantitative RT-PCR. We show that the expression levels of most otoconial genes are much higher in the utricle and saccule compared with other inner ear tissues before postnatal stages in C57Bl/6J mice, and the expression of a few of these genes is restricted to the embryonic utricle and saccule. After the early postnatal stages, expression of all otoconial genes in the utricle and saccule is drastically reduced, while a few genes gain expression dominance in the aging ampulla, indicating a potential for ectopic debris formation in the latter tissue at old ages. The data suggest that the expression of otoconial genes is tightly regulated spatially and temporally during developmental stages and can become unregulated at aging stages. Birth Defects Research (Part A) 106:613-625, 2016. © 2016 Wiley Periodicals, Inc.
| S-EPMC5914167 | biostudies-literature
Project description:Transient receptor potential vanilloid 1 (TRPV1) is a cation-permeable ion channel found in the peripheral and central nervous systems. The membrane surface expression of TRPV1 is known to occur in neuronal cell bodies and sensory neuron axons. TRPV1 receptors are also expressed in the hippocampus, the main epileptogenic region in the brain. Although, previous studies implicate TRPV1 channels in the generation of epilepsy, suppression of ongoing seizures by TRPV1 antagonists has not yet been attempted. Here, we evaluate the role of TRPV1 channels in the modulation of epileptiform activity as well as the anti-convulsant properties of capsazepine (CZP), an established TRPV1 competitive antagonist, using in vitro and in vivo models. To this end, we used 4-aminopyridine (4-AP) to trigger seizure-like activity. We found that CZP suppressed 4-AP induced epileptiform activity in vitro (10-100μM) and in vivo (50mg/kg s.c.). In contrast, capsaicin enhanced 4-AP induced epileptiform activity in vitro (1-100μM) and triggered bursting activity in vivo (100μM dialysis perfusion), which was abolished by the TRPV1 antagonist CZP. To further investigate the mechanisms of TRPV1 modulation, we studied the effect of capsaicin and CZP on evoked potentials. Capsaicin (1-100μM) and CZP (10-100μM) increased and decreased, respectively, the amplitude of extracellular field evoked potentials in a concentration-dependent manner. Additional in vitro studies showed that the effect of the TRPV1 blocker on evoked potentials was similar whether the response was orthodromic or antidromic, suggesting that the effect involves interference with membrane depolarization on cell bodies and axons. The fact that CZP could act directly on axons was confirmed by decreased amplitude of the compound action potential and by an increased delay of both the antidromic potentials and the axonal response. Histological studies using transgenic mice also show that, in addition to the known neural expression, TRPV1 channels are widely expressed in alvear oligodendrocytes in the hippocampus. Taken together, these results indicate that activation of TRPV1 channels leads to enhanced excitability, while their inhibition can effectively suppress ongoing electrographic seizures. These results support a role for TRPV1 channels in the suppression of convulsive activity, indicating that antagonism of TRPV1 channels particularly in axons may possibly be a novel target for effective acute suppression of seizures.
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Project description:Abstract Presurgical evaluation of mesial temporal and neocortical focal pharmacoresistant epilepsy patients using intracranial EEG recordings has led to the generation of extensive data on interictal epileptiform discharges, located within or remotely from seizure onset zones. In this study, we used this data to investigate how interictal epileptiform discharges are modulated and how their spatial distribution changes during wake and sleep and analysed the relationship between these discharge events and seizure onset zones. Preoperative evaluation data from 11 adult patients with focal pharmacoresistant epilepsy were extracted from the Epilepsiae database. Interictal epileptiform discharges were automatically detected during wakefulness and over several hours of continuous seizure-free sleep (total duration of EEG recordings:106.7 h; mean per patient: 9.7 h), and analysed across four brain areas (mesial temporal, lateral neocortical, basal cortical and the temporal pole). Sleep stages were classified manually from scalp EEG. Discharge events were characterized according to their rate and morphology (amplitude, sharpness and duration). Eight patients had a seizure onset zone over mesial areas and three patients over lateral neocortical areas. Overall, discharge rates varied across brain areas during wakefulness and sleep [wake/sleep stages × brain areas interaction; Wald χ2(df = 6) = 31.1, P < 0.0001]. N2–N3 non-rapid eye movement sleep increased interictal epileptiform discharges in mesial areas compared with wakefulness and rapid eye movement sleep (P < 0.0001), and to other areas (P < 0.0001 for all comparisons). This mesial pattern was observed both within and outside of seizure onset zones. During wakefulness, the rate of interictal epileptiform discharges was significantly higher than during N2–N3 non-rapid eye movement sleep (P = 0.04), and rapid eye movement sleep (P = 0.01) in lateral neocortical areas (referred to as lateral neocortical pattern), a finding that was more pronounced in seizures onset zones (P = 0.004). The morphological characteristics of the discharge events were modulated during wakefulness and sleep stages across brain areas. The effect of seizure onset zones on discharge morphology was conditioned by brain area and was particularly marked in temporal pole areas. Our analysis of discharge patterns in relation to cerebral localization, vigilance state and the anatomical affiliation of seizure onset zones revealed the global and local aspects of the complex relationship between interictal discharges, sleep and seizure onset zones. This novel approach may lead to a better understanding of cognitive decline and responses to therapy, as well as to adaptation of surgical interventions for epileptic patients. Fouad et al. report that seizure-free wakefulness and sleep stages, as well as seizure onset zones, modulate interictal discharge rates and morphology in focal epilepsy. The mesial discharges were facilitated by deep sleep, while the neocortical discharges were enhanced by wakefulness. Seizure onset zones influenced discharge rates and morphology. Graphical Abstract Graphical Abstract
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Project description:This retrospective case-controlled study was performed to evaluate whether Epileptiform Activity, suspected clinical seizures, and/or 2HELPS2B/S after nontraumatic Intraparenchymal Hemorrhage or Subarachnoid Hemorrhage can predict Epilepsy. Hundred and thirty-two patients were included-29 (Epilepsy), 103 (Control Group). After matching, the average effect for all three risk factors was significant as follows: (1) Epileptiform Activity (p = 0.012, odds ratio 3.14), (2) suspected seizures (p = 0.021, odds ratio 3.78), and (3) 2HELPS2B/S score (p < 0.001, odds ratio 4.94). This study shows that Epileptiform Activity, suspected seizures, and particularly, the 2HELPS2B/S score in the acute phase are risk factors for the development of epilepsy after nontraumatic intraparenchymal and subarachnoid hemorrhage.
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Project description:Epilepsy is a common neurological disease, manifested in unprovoked recurrent seizures. Epileptogenesis may develop due to genetic or pharmacological origins or following injury, but it remains unclear how the unaffected brain escapes this susceptibility to seizures. Here, we report that dynamic changes in forebrain microRNA (miR)-211 in the mouse brain shift the threshold for spontaneous and pharmacologically induced seizures alongside changes in the cholinergic pathway genes, implicating this miR in the avoidance of seizures. We identified miR-211 as a putative attenuator of cholinergic-mediated seizures by intersecting forebrain miR profiles that were Argonaute precipitated, synaptic vesicle target enriched, or differentially expressed under pilocarpine-induced seizures, and validated TGFBR2 and the nicotinic antiinflammatory acetylcholine receptor nAChRa7 as murine and human miR-211 targets, respectively. To explore the link between miR-211 and epilepsy, we engineered dTg-211 mice with doxycycline-suppressible forebrain overexpression of miR-211. These mice reacted to doxycycline exposure by spontaneous electrocorticography-documented nonconvulsive seizures, accompanied by forebrain accumulation of the convulsive seizures mediating miR-134. RNA sequencing demonstrated in doxycycline-treated dTg-211 cortices overrepresentation of synaptic activity, Ca2+ transmembrane transport, TGFBR2 signaling, and cholinergic synapse pathways. Additionally, a cholinergic dysregulated mouse model overexpressing a miR refractory acetylcholinesterase-R splice variant showed a parallel propensity for convulsions, miR-211 decreases, and miR-134 elevation. Our findings demonstrate that in mice, dynamic miR-211 decreases induce hypersynchronization and nonconvulsive and convulsive seizures, accompanied by expression changes in cholinergic and TGFBR2 pathways as well as in miR-134. Realizing the importance of miR-211 dynamics opens new venues for translational diagnosis of and interference with epilepsy.
| S-EPMC5488936 | biostudies-literature
Project description:Carbon dioxide (CO2) is one of the most commonly used euthanasia agents for mice, yet it is highly aversive and nociceptive. Inert gases are a possible alternative, however there are qualitative reports of seizures resulting from exposure. Here we evaluate epileptiform activity caused by inert gases (N2, He, Ar and Xe) and CO2 in mice chronically instrumented for EEG/EMG undergoing single-gas euthanasia. We found that N2, He and Ar caused epileptiform activity in all animals, CO2 in half of animals and no epileptiform activity produced by Xe. Atmospheric O2 concentrations at epileptiform activity onset were significantly higher for CO2 than for all other gases and occurred soon after loss of motion, whereas N2 and Ar epileptiform activity occurred at cessation of neocortical activity. Helium caused the longest epileptiform activity and these commenced significantly before isoelectric EEG. We did not detect any epileptiform activity during active behaviour. Taken together, these results demonstrate that whilst epileptiform activity from inert gases and particularly Ar and N2 are more prevalent than for CO2, their occurrence at the onset of an isoelectric EEG is unlikely to impact on the welfare of the animal. Epileptiform activity from these gases should not preclude them from further investigation as euthanasia agents. The genesis of epileptiform activity from CO2 is unlikely to result from hypoxia as with the inert gases. Helium caused epileptiform activity before cessation of neocortical activity and for a longer duration and is therefore less suitable as an alternative to CO2.
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