RESUMEN
Temporal lobe epilepsy (TLE) development is associated with dysregulation of glutamatergic transmission in the hippocampus; however, detailed molecular mechanisms of pathological changes are still poorly understood. In the present study, we performed the complex analysis of glutamatergic system in the hippocampus of Krushinsky-Molodkina (KM) rats genetically prone to audiogenic seizures (AGS). Daily AGS stimulations (audiogenic kindling) were used to reproduce the dynamics of TLE development. Naïve KM rats were used as a control. After 14 AGS, at the stage of developing TLE, KM rats demonstrated significant upregulation of extracellular signal-regulated kinases (ERK) 1 and 2, cAMP response element-binding protein (CREB), and c-Fos in the hippocampus indicating activation of the hippocampal cells. These changes were accompanied with an increase in glutaminase and vesicular glutamate transporter (VGLUT) 2 suggesting the activation of glutamate production and loading into the synaptic vesicles. After 21 AGS, when TLE was fully-established, alterations were similar but more pronounced, with higher activation of glutaminase, increase in glutamate production, upregulation of VGLUT1 and 2, and Fos-related antigen 1 (Fra-1) along with c-Fos. Analysis of glutamate receptors showed variable changes. Thus, after 14 AGS, simultaneous increase in metabotropic glutamate receptor mGluR1 and decrease in ionotropic N-methyl-D-aspartate (NMDA) receptors could reflect compensatory anti-epileptic mechanism, while further kindling progression induced upregulation of ionotropic receptors, probably, contributing to the hippocampal epileptization. However, we revealed practically no alterations in the expression of synaptic proteins. Altogether, obtained results suggested that overactivation of glutamate production in the hippocampus strongly contributed to TLE development in KM rats.
Asunto(s)
Epilepsia Refleja , Epilepsia del Lóbulo Temporal , Excitación Neurológica , Ratas , Animales , Glutaminasa/metabolismo , Hipocampo/metabolismo , Epilepsia Refleja/metabolismo , Excitación Neurológica/fisiología , Epilepsia del Lóbulo Temporal/metabolismo , Predisposición Genética a la Enfermedad , Ácido Glutámico/metabolismo , Convulsiones/metabolismo , Estimulación AcústicaRESUMEN
To explore the processes of epileptogenesis/ictogenesis, this study determined the age-dependent development of the functional abnormalities in astroglial transmission associated with pannexin1-hemichannel using a genetic rat model of autosomal dominant sleep-related hypermotor epilepsy (ADSHE) named 'S286L-TG'. Pannexin1 expression in the plasma membrane of primary cultured cortical astrocytes and the orbitofrontal cortex (OFC), which is an ADSHE focus region, were determined using capillary immunoblotting. Astroglial D-serine releases induced by artificial high-frequency oscillation (HFO)-evoked stimulation, the removal of extracellular Ca2+, and the P2X7 receptor agonist (BzATP) were determined using ultra-high performance liquid chromatography (UHPLC). The expressions of pannexin1 in the plasma membrane fraction of the OFC in S286L-TG at four weeks old were almost equivalent when compared to the wild type. The pannexin1 expression in the OFC of the wild type non-statistically decreased age-dependently, whereas that in S286L-TG significantly increased age-dependently, resulting in relatively increasing pannexin1 expression from the 7- (at the onset of interictal discharge) and 10-week-old (after the ADSHE seizure onset) S286L-TG compared to the wild type. However, no functional abnormalities of astroglial pannexin1 expression or D-serine release through the pannexin1-hemichannels from the cultured astrocytes of S286L-TG could be detected. Acutely HFO-evoked stimulation, such as physiological ripple burst (200 Hz) and epileptogenic fast ripple burst (500 Hz), frequency-dependently increased both pannexin1 expression in the astroglial plasma membrane and astroglial D-serine release. Neither the selective inhibitors of pannexin1-hemichannel (10PANX) nor connexin43-hemichannel (Gap19) affected astroglial D-serine release during the resting stage, whereas HFO-evoked D-serine release was suppressed by both inhibitors. The inhibitory effect of 10PANX on the ripple burst-evoked D-serine release was more predominant than that of Gap19, whereas fast ripple burst-evoked D-serine release was predominantly suppressed by Gap19 rather than 10PANX. Astroglial D-serine release induced by acute exposure to BzATP was suppressed by 10PANX but not by Gap19. These results suggest that physiological ripple burst during the sleep spindle plays important roles in the organization of some components of cognition in healthy individuals, but conversely, it contributes to the initial development of epileptogenesis/ictogenesis in individuals who have ADSHE vulnerability via activation of the astroglial excitatory transmission associated with pannexin1-hemichannels.
Asunto(s)
Conexinas , Epilepsia Refleja , Animales , Ratas , Astrocitos/metabolismo , Conexina 43/metabolismo , Epilepsia Refleja/metabolismo , Corteza Prefrontal/metabolismo , Serina/metabolismo , Sueño , Conexinas/metabolismoRESUMEN
Epilepsy is one of the common neurological diseases that affects not only adults but also infants and children. Because epilepsy has been studied for a long time, there are several pharmacologically effective anticonvulsants, which, however, are not suitable as therapy for all patients. The genesis of epilepsy has been extensively investigated in terms of its occurrence after injury and as a concomitant disease with various brain diseases, such as tumors, ischemic events, etc. However, in the last decades, there are multiple reports that both genetic and epigenetic factors play an important role in epileptogenesis. Therefore, there is a need for further identification of genes and loci that can be associated with higher susceptibility to epileptic seizures. Use of mouse knockout models of epileptogenesis is very informative, but it has its limitations. One of them is due to the fact that complete deletion of a gene is not, in many cases, similar to human epilepsy-associated syndromes. Another approach to generating mouse models of epilepsy is N-Ethyl-N-nitrosourea (ENU)-directed mutagenesis. Recently, using this approach, we generated a novel mouse strain, soc (socrates, formerly s8-3), with epileptiform activity. Using molecular biology methods, calcium neuroimaging, and immunocytochemistry, we were able to characterize the strain. Neurons isolated from soc mutant brains retain the ability to differentiate in vitro and form a network. However, soc mutant neurons are characterized by increased spontaneous excitation activity. They also demonstrate a high degree of Ca2+ activity compared to WT neurons. Additionally, they show increased expression of NMDA receptors, decreased expression of the Ca2+-conducting GluA2 subunit of AMPA receptors, suppressed expression of phosphoinositol 3-kinase, and BK channels of the cytoplasmic membrane involved in protection against epileptogenesis. During embryonic and postnatal development, the expression of several genes encoding ion channels is downregulated in vivo, as well. Our data indicate that soc mutation causes a disruption of the excitation-inhibition balance in the brain, and it can serve as a mouse model of epilepsy.
Asunto(s)
Epilepsia Refleja , Niño , Animales , Humanos , Ratones , Epilepsia Refleja/genética , Epilepsia Refleja/metabolismo , Etilnitrosourea/toxicidad , Canales de Potasio de Gran Conductancia Activados por el Calcio/metabolismo , Anticonvulsivantes/farmacología , Encéfalo/metabolismo , Modelos Animales de EnfermedadRESUMEN
Epileptogenesis can be associated with altered genetic control of the GABAergic system. Here we analyzed Krushinsky-Molodkina (KM) rats genetically prone to audiogenic epilepsy. KM rats express fully formed audiogenic seizures (AGSs) not early, then they reach 3 months. At the age of 1-2 months, KM rats either do not express AGS or demonstrate an incomplete pattern of seizure. Such long-term development of AGS susceptibility makes KM rats an especially convenient model to investigate the mechanisms and dynamics of the development of inherited epilepsy. The analysis of the GABAergic system of the hippocampus of KM rats was done during postnatal development at the 15th, 60th, and 120th postnatal days. Wistar rats of corresponding ages were used as a control. In the hippocampus of KM pups, we observed a decrease in the expression of glutamic acid decarboxylase 67 (GAD67) and parvalbumin (PV), which points to a decrease in the activity of GABAergic neurons. Analysis of the 2-month-old KM rats showed an increase in GAD67 and PV expression while synapsin I and vesicular GABA transporter (VGAT) were decreased. In adult KM rats, the expression of GAD67, PV, and synapsin I was upregulated. Altogether, the obtained data indicate significant alterations in GABAergic transmission in the hippocampus of audiogenic KM rats during the first postnatal months.
Asunto(s)
Epilepsia Refleja , Ratas , Animales , Epilepsia Refleja/genética , Epilepsia Refleja/metabolismo , Ratas Wistar , Sinapsinas/metabolismo , Convulsiones , Hipocampo/metabolismo , Glutamato Descarboxilasa/genética , Glutamato Descarboxilasa/metabolismo , Estimulación AcústicaRESUMEN
Distributions of brain H3 histamine receptors in regions of the prefrontal cortex were studied by assessing regional binding densities for [3 H](R)α-methylhistamine in coronal brain slices of normal rats and rats with genetically determined absence and/or audiogenic epilepsies. The three groups of epileptic rats displayed widespread significant decreases in H3 histamine receptor binding densities. A 20-25% decline was seen in the rostral aspects of the lateral prefrontal cortex, namely the granular, dysgranular, and dorsal agranular insular regions. The reduction was not specific for the epilepsy types. The same was observed in the rostral part of the primary cingulate cortex and the secondary midcingulate cortex. On borders of this core effect, several seizure-type specific declines were seen. Namely, the infralimbic, prelimbic and posterior agranular insular cortices demonstrated absence-epilepsy related reductions in the H3 histamine receptor binding densities. A decrease related to audiogenic seizures was noted in the rostral part of the piriform cortex. The pattern of widespread and seizure-type unspecific decline in H3 histamine receptor binding densities points to a common part of brain loops underlying generalized convulsive and non-convulsive types of epilepsy. It also might hint at putative seizure-related changes in the release of histamine from specific fibers innervating the prefrontal area.
Asunto(s)
Epilepsia Generalizada , Epilepsia Refleja , Animales , Encéfalo/metabolismo , Corteza Cerebral , Epilepsia Generalizada/genética , Epilepsia Generalizada/metabolismo , Epilepsia Refleja/metabolismo , Corteza Prefrontal , Ratas , Convulsiones/metabolismoRESUMEN
BACKGROUND: Levetiracetam (LEV, 5-100 mg/kg) has been shown to prevent audiogenic seizures in a dose-dependent manner. This chemical is known to bind to synaptic vesicle protein 2A and inhibit l-type calcium channels, affecting neurotransmitter release. We hypothesize that the drug prevents audiogenic seizures partially by affecting cochlear neural response. METHODS: To test this hypothesis, rats were given 1000, 500, 50, or 0 mg/kg (saline control) LEV-injection. Distortion product otoacoustic emissions (DPOAE), reflecting outer hair cell (OHC) function, and cochlear compound action potentials (CAP), reflecting cochlear neural output, were recorded and compared pre- and post-LEV. RESULTS: 1000 mg/kg LEV-injection did not significantly affect DPOAE. The high dose LEV-injection, however, significantly reduced CAP amplitude resulting threshold shift (TS), prolonged CAP latency, and enhanced CAP forward masking. CAP latency and forward masking were significantly affected at the 500 mg/kg dose, but CAP-TS remained unchanged after LEV-injection. Interestingly, CAP latency wassignificantly prolonged, at least at the low stimulation levels, although the amplitude of CAP remained constant after a clinical dose of LEV-injection (50 mg/kg). DISCUSSION: Since the clinical dose of LEV-injection does not reduce CAP amplitude, the reduction of cochlear neural output is unlikely to be the underlying mechanism of LEV in the treatment of audiogenic seizure. The delayed cochlear neural response may be partially related to the prevention of audiogenic seizure. However, neuropharmacological changes in the central nervous system must play a major role in the treatment of audiogenic seizure, as it does in the treatment of focal epilepsy.
Asunto(s)
Epilepsia Refleja , Piracetam , Animales , Anticonvulsivantes/farmacología , Anticonvulsivantes/uso terapéutico , Epilepsia Refleja/metabolismo , Levetiracetam , Piracetam/metabolismo , Piracetam/farmacología , Ratas , Vesículas SinápticasRESUMEN
The Wistar audiogenic rat (WAR) strain is used as an animal model of epilepsy, which when submitted to acute acoustic stimulus presents tonic-clonic seizures, mainly dependent on brainstem (mesencephalic) structures. However, when WARs are exposed to chronic acoustic stimuli (audiogenic kindling-AK), they usually present tonic-clonic seizures, followed by limbic seizures, after recruitment of forebrain structures such as the cortex, hippocampus and amygdala. Although some studies have reported that hypothalamic-hypophysis function is also altered in WAR through modulating vasopressin (AVP) and oxytocin (OXT) secretion, the role of these neuropeptides in epilepsy still is controversial. We analyzed the impact of AK and consequent activation of mesencephalic neurocircuits and the recruitment of forebrain limbic (LiR) sites on the hypothalamic-neurohypophysial system and expression of Avpr1a and Oxtr in these structures. At the end of the AK protocol, nine out of 18 WARs presented LiR. Increases in both plasma vasopressin and oxytocin levels were observed in WAR when compared to Wistar rats. These results were correlated with an increase in the expressions of heteronuclear (hn) and messenger (m) RNA for Oxt in the paraventricular nucleus (PVN) in WARs submitted to AK that presented LiR. In the paraventricular nucleus, the hnAvp and mAvp expressions increased in WARs with and without LiR, respectively. There were no significant differences in Avp and Oxt expression in supraoptic nuclei (SON). Also, there was a reduction in the Avpr1a expression in the central nucleus of the amygdala and frontal lobe in the WAR strain. In the inferior colliculus, Avpr1a expression was lower in WARs after AK, especially those without LiR. Our results indicate that both AK and LiR in WARs lead to changes in the hypothalamic-neurohypophysial system and its receptors, providing a new molecular basis to better understaind epilepsy.
Asunto(s)
Epilepsia Refleja , Hipotálamo/metabolismo , Excitación Neurológica/fisiología , Sistemas Neurosecretores/metabolismo , Neurohipófisis/metabolismo , Estimulación Acústica , Animales , Modelos Animales de Enfermedad , Epilepsia Refleja/genética , Epilepsia Refleja/metabolismo , Epilepsia Refleja/patología , Epilepsia Refleja/fisiopatología , Regulación de la Expresión Génica , Hipocampo/metabolismo , Hipocampo/patología , Hipocampo/fisiopatología , Hipotálamo/patología , Hipotálamo/fisiopatología , Excitación Neurológica/patología , Masculino , Sistemas Neurosecretores/patología , Sistemas Neurosecretores/fisiopatología , Oxitocina/sangre , Oxitocina/genética , Oxitocina/metabolismo , Neurohipófisis/patología , Neurohipófisis/fisiopatología , Ratas , Ratas Wistar , Convulsiones/genética , Convulsiones/metabolismo , Convulsiones/fisiopatología , Convulsiones/psicología , Vasopresinas/sangre , Vasopresinas/genética , Vasopresinas/metabolismoRESUMEN
The loss-of-function S284L-mutant α4 subunit of the nicotinic acetylcholine receptor (nAChR) is considered to contribute to the pathomechanism of autosomal dominant sleep-related hypermotor epilepsy (ADSHE); however, the age-dependent and sleep-related pathomechanisms of ADSHE remain to be clarified. To explore the age-dependent and sleep-induced pathomechanism of ADSHE, the present study determined the glutamatergic transmission abnormalities associated with α4ß2-nAChR and the astroglial hemichannel in the hyperdirect and corticostriatal pathways of ADSHE model transgenic rats (S286L-TG) bearing the rat S286L-mutant Chrna4 gene corresponding to the human S284L-mutant CHRNA4 gene of ADSHE, using multiprobe microdialysis and capillary immunoblotting analyses. This study could not detect glutamatergic transmission in the corticostriatal pathway from the orbitofrontal cortex (OFC) to the striatum. Before ADSHE onset (four weeks of age), functional abnormalities of glutamatergic transmission compared to the wild-type in the cortical hyperdirect pathway, from OFC to the subthalamic nucleus (STN) in S286L-TG, could not be detected. Conversely, after ADSHE onset (eight weeks of age), glutamatergic transmission in the hyperdirect pathway of S286L-TG was enhanced compared to the wild-type. Notably, enhanced glutamatergic transmission of S286L-TG was revealed by hemichannel activation in the OFC. Expression of connexin43 (Cx43) in the OFC of S286L-TG was upregulated after ADSHE onset but was almost equal to the wild-type prior to ADSHE onset. Differences in the expression of phosphorylated protein kinase B (pAkt) before ADSHE onset between the wild-type and S286L-TG were not observed; however, after ADSHE onset, pAkt was upregulated in S286L-TG. Conversely, the expression of phosphorylated extracellular signal-regulated kinase (pErk) was already upregulated before ADSHE onset compared to the wild-type. Both before and after ADSHE onset, subchronic nicotine administration decreased and did not affect the both expression of Cx43 and pErk of respective wild-type and S286L-TG, whereas the pAkt expression of both the wild-type and S286L-TG was increased by nicotine. Cx43 expression in the plasma membrane of the primary cultured astrocytes of the wild-type was increased by elevation of the extracellular K+ level (higher than 10 mM), and the increase in Cx43 expression in the plasma membrane required pErk functions. These observations indicate that a combination of functional abnormalities, GABAergic disinhibition, and upregulated pErk induced by the loss-of-function S286L-mutant α4ß2-nAChR contribute to the age-dependent and sleep-induced pathomechanism of ADSHE via the upregulation/hyperactivation of the Cx43 hemichannels.
Asunto(s)
Epilepsia Refleja/patología , Genes Dominantes , Convulsiones/complicaciones , Trastornos del Sueño-Vigilia/complicaciones , Factores de Edad , Animales , Conexina 43/metabolismo , Epilepsia Refleja/etiología , Epilepsia Refleja/metabolismo , Ácido Glutámico/metabolismo , Ratas , Ratas Sprague-Dawley , Receptores Nicotínicos/metabolismoRESUMEN
Rodent models of audiogenic seizures, in which seizures are precipitated by an abnormal response of the brain to auditory stimuli, are crucial to investigate the neural bases underlying ictogenesis. Despite significant advances in understanding seizure generation in the inferior colliculus, namely the epileptogenic nucleus, little is known about the contribution of lower auditory stations to the seizure-prone network. Here, we examined the cochlea and cochlear nucleus of the genetic audiogenic seizure hamster from Salamanca (GASH/Sal), a model of reflex epilepsy that exhibits generalized tonic-clonic seizures in response to loud sound. GASH/Sal animals under seizure-free conditions were compared with matched control hamsters in a multi-technical approach that includes auditory brainstem responses (ABR) testing, histology, scanning electron microscopy analysis, immunohistochemistry, quantitative morphometry and gene expression analysis (RT-qPCR). The cochlear histopathology of the GASH/Sal showed preservation of the sensory hair cells, but a significant loss of spiral ganglion neurons and mild atrophy of the stria vascularis. At the electron microscopy level, the reticular lamina exhibited disarray of stereociliary tufts with blebs, loss or elongated stereocilia as well as non-parallel rows of outer hair cells due to protrusions of Deiters' cells. At the molecular level, the abnormal gene expression patterns of prestin, cadherin 23, protocadherin 15, vesicular glutamate transporters 1 (Vglut1) and -2 (Vglut2) indicated that the hair-cell mechanotransduction and cochlear amplification were markedly altered. These were manifestations of a cochlear neuropathy that correlated to ABR waveform I alterations and elevated auditory thresholds. In the cochlear nucleus, the distribution of VGLUT2-immunolabeled puncta was differently affected in each subdivision, showing significant increases in magnocellular regions of the ventral cochlear nucleus and drastic reductions in the granule cell domain. This modified inputs lead to disruption of Vglut1 and Vglut2 gene expression in the cochlear nucleus. In sum, our study provides insight into the morphological and molecular traits associated with audiogenic seizure susceptibility in the GASH/Sal, suggesting an upward spread of abnormal glutamatergic transmission throughout the primary acoustic pathway to the epileptogenic region.
Asunto(s)
Umbral Auditivo , Conducta Animal , Cóclea/fisiopatología , Epilepsia Refleja/fisiopatología , Epilepsia Tónico-Clónica/fisiopatología , Audición , Animales , Cóclea/metabolismo , Cóclea/ultraestructura , Cricetinae , Modelos Animales de Enfermedad , Epilepsia Refleja/genética , Epilepsia Refleja/metabolismo , Epilepsia Refleja/psicología , Epilepsia Tónico-Clónica/genética , Epilepsia Tónico-Clónica/metabolismo , Epilepsia Tónico-Clónica/psicología , Ácido Glutámico/metabolismo , Masculino , Ruido , Proteína 1 de Transporte Vesicular de Glutamato/genética , Proteína 1 de Transporte Vesicular de Glutamato/metabolismo , Proteína 2 de Transporte Vesicular de Glutamato/genética , Proteína 2 de Transporte Vesicular de Glutamato/metabolismoRESUMEN
The present study analysed the effects of audiogenic kindling on the functional state of the vasopressinergic system of Krushinsky-Molodkina (KM) rats. KM rats represent a genetic model of audiogenic reflex epilepsy. Multiple audiogenic seizures in KM rats lead to the involvement of the limbic structures and neocortex in the epileptic network. The phenomenon of epileptic activity that overspreads from the brain stem to the forebrain is called audiogenic kindling and represents a model of limbic epilepsy. In the present study, audiogenic kindling was induced by 25 repetitive audiogenic seizures (AGS) with 1 AGS per day. A proportion of KM rats did not express AGS to sound stimuli, and these rats were characterised as the AGS-resistant group. The data demonstrated that audiogenic kindling did not change activity of extracellular signal-regulated kinase 1/2 or cAMP response element-binding protein, although it led to an increase in vasopressin (VP) expression in the supraoptic nucleus (SON) and in the magnocellular division of the paraventricular nucleus (PVN). Additionally, we observed a decrease in GABAergic innervation of the hypothalamic neuroendocrine neurones after audiogenic kindling, whereas glutamatergic innervation of the SON and PVN was not altered. By contrast, analysis of AGS-resistant KM rats did not reveal any changes in the activity of the VP-ergic system, confirming that the activation of VP expression was caused by repetitive AGS expression, rather than by repetitive acoustic stress. Thus, we suggest that overspread of epileptiform activity in the brain is the main factor that affects VP expression in the hypothalamic magnocellular neurones.
Asunto(s)
Epilepsia Refleja/metabolismo , Hipotálamo/metabolismo , Excitación Neurológica/fisiología , Vasopresinas/metabolismo , Estimulación Acústica , Animales , Conducta Animal/fisiología , Epilepsia Refleja/genética , Neuronas GABAérgicas/metabolismo , Ácido Glutámico/metabolismo , Ratas , Transducción de Señal/fisiologíaRESUMEN
Focal epileptic seizures can in some patients be managed by inhibiting γ-aminobutyric acid (GABA) uptake via the GABA transporter 1 (GAT1) using tiagabine (Gabitril®). Synergistic anti-seizure effects achieved by inhibition of both GAT1 and the betaine/GABA transporter (BGT1) by tiagabine and EF1502, compared to tiagabine alone, suggest BGT1 as a target in epilepsy. Yet, selective BGT1 inhibitors are needed for validation of this hypothesis. In that search, a series of BGT1 inhibitors typified by (1R,2S)-2-((4,4-bis(3-methylthiophen-2-yl)but-3-en-yl)(methyl)amino)cyclohexanecarboxylic acid (SBV2-114) was developed. A thorough pharmacological characterization of SBV2-114 using a cell-based [3H]GABA uptake assay at heterologously expressed BGT1, revealed an elusive biphasic inhibition profile with two IC50 values (4.7 and 556 µM). The biphasic profile was common for this structural class of compounds, including EF1502, and was confirmed in the MDCK II cell line endogenously expressing BGT1. The possibility of two binding sites for SBV2-114 at BGT1 was assessed by computational docking studies and examined by mutational studies. These investigations confirmed that the conserved residue Q299 in BGT1 is involved in, but not solely responsible for the biphasic inhibition profile of SBV2-114. Animal studies revealed anti-seizure effects of SBV2-114 in two mouse models, supporting a function of BGT1 in epilepsy. However, as SBV2-114 is apparent to be rather non-selective for BGT1, the translational relevance of this observation is unknown. Nevertheless, SBV2-114 constitutes a valuable tool compound to study the molecular mechanism of an emerging biphasic profile of BGT1-mediated GABA transport and the putative involvement of two binding sites for this class of compounds.
Asunto(s)
Anticonvulsivantes/uso terapéutico , Proteínas Transportadoras de GABA en la Membrana Plasmática/metabolismo , Convulsiones/tratamiento farmacológico , Convulsiones/metabolismo , Estimulación Acústica/efectos adversos , Animales , Anticonvulsivantes/farmacología , Células CHO , Cricetulus , Epilepsia Refleja/tratamiento farmacológico , Epilepsia Refleja/metabolismo , Proteínas Transportadoras de GABA en la Membrana Plasmática/química , Células HEK293 , Humanos , Masculino , Ratones , Ratones Transgénicos , Unión Proteica/fisiología , Estructura Secundaria de Proteína , Convulsiones/etiología , Resultado del TratamientoRESUMEN
The tremor mutant phenotype results from an autosomal recessive spontaneous mutation arisen in a Swiss-Webster mouse colony. The mutant mice displayed normal development until three weeks of age when they began to present motor impairment comprised by whole body tremor, ataxia, and decreased exploratory behavior. These features increased in severity with aging suggesting a neurodegenerative profile. In parallel, they showed audiogenic generalized clonic seizures. Results from genetic mapping identified the mutation tremor on chromosome 14, in an interval of 5 cM between D14Mit37 (33.21â¯cM) and D14Mit115 (38.21â¯cM), making Early Growth Response 3 (Egr3) the main candidate gene. Comparing with wild type (WT) mice, the tremor mice showed higher hippocampal gene expression of Egr3 and Gabra1 and increased concentrations of noradrenalin (NOR; pâ¯=â¯.0012), serotonin (5HT; pâ¯=â¯.0083), 5-hydroxyindoleacetic acid (5-HIAA; pâ¯=â¯.0032), γ-amino butyric acid (GABA; pâ¯=â¯.0123), glutamate (pâ¯=â¯.0217) and aspartate (pâ¯=â¯.0124). In opposition, the content of glycine (pâ¯=â¯.0168) and the vanillylmandelic acid (VMA)/NOR ratio (pâ¯=â¯.032) were decreased. Regarding to dopaminergic system, neither dopamine (DA) and 3,4-dihydroxyphenylacetic acid (DOPAC) contents nor the turnover rate of DA showed statistically significant differences between WT and mutant mice. Data demonstrated that audiogenic seizures of tremor mice are associated with progressive motor impairment as well as to hippocampal alterations of the Egr3 and Gabra1 gene expression and amino acid and monoamine content. In addition, the tremor mice could be useful for study of neurotransmission pathways as modulators of epilepsy and the pathogenesis of epilepsies occurring with generalized clonic seizures.
Asunto(s)
Estimulación Acústica/efectos adversos , Epilepsia Refleja/genética , Epilepsia Refleja/metabolismo , Mutación/genética , Temblor/genética , Temblor/metabolismo , Animales , Modelos Animales de Enfermedad , Dopamina/metabolismo , Femenino , Ácido Glutámico/metabolismo , Hipocampo/química , Hipocampo/metabolismo , Masculino , Ratones , Ratones Transgénicos , Norepinefrina/metabolismo , Convulsiones/genética , Convulsiones/metabolismo , Serotonina/metabolismoRESUMEN
Here, we investigated the participation of pro and anti-inflammatory cytokines in the spread of repeated audiogenic seizures from brainstem auditory structures to limbic areas, including the hippocampus. We used Wistar Audiogenic Rats (WARs) and Wistars submitted to the audiogenic kindling protocol with a loud broad-band noise. We measured pro and anti-inflammatory cytokines and nitrate levels in the hippocampus of stimulated animals. Our results show that all WARs developed audiogenic seizures that evolved to limbic seizures whereas seizure-resistant controls did not present any seizures. However, regardless of seizure severity, we did not observe differences in the pro inflammatory cytokines IL-1ß, IL-6, TNF-α and IFN-α or in the anti-inflammatory IL-10 in the hippocampi of audiogenic and resistant animals. We also did not find any differences in nitrate content. Our data indicate that the spread of seizures during the audiogenic kindling is not dependent on hippocampal release of cytokines or oxidative stress, but the severity of brainstem seizures will be higher in animals with higher levels of cytokines and the oxidative stress marker, nitrate.
Asunto(s)
Estimulación Acústica/efectos adversos , Epilepsia Refleja/metabolismo , Hipocampo/metabolismo , Mediadores de Inflamación/metabolismo , Excitación Neurológica/metabolismo , Animales , Epilepsia Refleja/etiología , Femenino , Ratas , Ratas Wistar , Convulsiones/etiología , Convulsiones/metabolismoRESUMEN
Krushinsky-Molodkina (KM) rats genetically prone to audiogenic seizure are characterized by age-dependent expression of audiogenic seizures (AGS). It is known that the critical period of enhanced seizure susceptibility in rodents occurs at 2nd-3rd weeks of postnatal development. However, KM rats do not express AGS at this time-point, but start to demonstrate a stable AGS only after the age of 3â¯months. We hypothesized that this delay in AGS susceptibility in KM rats is genetically determined and may depend on some alterations in the development of the hippocampal glutamatergic system during the early postnatal period. We analyzed the expression and activity of seizure-related proteins, such as vesicular glutamate transporter 2 (VGLUT2), extracellular signal-regulated kinases 1 and 2 (ERK1/2), synapsin I, and NR2B subunit of the N-methyl-d-aspartate (NMDA) receptor (NR2B) in the hippocampus of KM rats during postnatal development. A significantly higher activity of ERK1/2 in KM rats was observed at 14th, 30th, and 60th days of postnatal development (P14, P30, P60) in comparison with control Wistar rats of the corresponding ages, while in adult (P120) KM rats it was at the same level with Wistar rats. Despite the increased activity of ERK1/2 at P14 and P30, the phosphorylation of synapsin I at Ser62/67 was significantly lower in the hippocampus of KM rats than in Wistar rats of the same ages; however, at P60 and P120, the phosphorylation of synapsin I was enhanced. Our data also revealed the increase of VGLUT2 and NR2B expression at P14, which dramatically decreased at the later stages. Our data indicate that a genetically determined increase in ERK1/2 kinase activity during postnatal ontogenesis in KM rats may be associated with the disturbances in synthesis and activity of the proteins, which are responsible for glutamatergic transmission in the KM rat hippocampus during the seizure susceptibility development.
Asunto(s)
Epilepsia Refleja/metabolismo , Epilepsia Refleja/fisiopatología , Hipocampo/metabolismo , Sistema de Señalización de MAP Quinasas/fisiología , Proteína 2 de Transporte Vesicular de Glutamato/metabolismo , Animales , Modelos Animales de Enfermedad , Femenino , Masculino , Ratas , Ratas WistarRESUMEN
The expression of glial cell line-derived neurothrophic factor (GDNF) transcript forms pre-(α)pro-gdnf, pre-(ß)pro-gdnf, and their common region m-gdnf in the pons as well as the inferior (IC) and superior colliculi in Krushinsky-Molodkina (KM) rats and in the strain "0" was analyzed by quantitative real-time polymerase chain reaction (PCR) in the control (unstimulated KM and "0" rats) and 1.5, 4.5, and 8â¯h after auditory stimulation. Such stimulation induced audiogenic seizures (AS) in KM rats. Audiogenic seizure was not observed in "0" rats, which was obtained by selection for the absence of AS in a population of F2 hybrids between KM and Wistar rats not predisposed to AS. A significant drop in the level of all transcripts was observed 1.5â¯h after auditory stimulation in both KM and "0" rats. In most cases, the average expression of α and ß isoforms and m-region 4.5â¯h after stimulation was greater than those after 1.5 and 8â¯h. At the same time, the expression of pre-(ß)pro-gdnf in the IC of KM rats 4.5â¯h after the stimulation was significantly lower than after 1.5 or 8â¯h. This work presents the first demonstration of different time courses of expression of the α and ß GDNF isoforms during physiological processes in genotype-specific pathology.
Asunto(s)
Estimulación Acústica/métodos , Encéfalo/metabolismo , Epilepsia Refleja/metabolismo , Factor Neurotrófico Derivado de la Línea Celular Glial/biosíntesis , ARN Mensajero/biosíntesis , Convulsiones/metabolismo , Animales , Encéfalo/patología , Epilepsia Refleja/genética , Epilepsia Refleja/patología , Expresión Génica , Factor Neurotrófico Derivado de la Línea Celular Glial/genética , Masculino , Neuroglía/metabolismo , Neuroglía/patología , Isoformas de Proteínas/biosíntesis , Isoformas de Proteínas/genética , ARN Mensajero/genética , Ratas , Ratas Wistar , Convulsiones/genética , Convulsiones/patología , Factores de TiempoRESUMEN
Photosensitive epilepsy is the most common subset of reflex epilepsy in humans. It is characterized by an abnormal electroencephalogram trait known as photoparoxysmal response (PPR) corresponding to ictal symptomology in response to intermittent photic stimulation. Increased exposure to photic stimulation through technological and societal advance has had a significant impact on the incidence and interest in photosensitive epilepsy. Paralleled scientific developments have deepened our understanding of the neurophysiology underlying photosensitivity, especially in the context of deep genomic sequencing and neuroimaging. Furthermore, given the relatively predictable nature of photosensitive epilepsy, there is substantial interest in translating advancements in this field to epilepsy as a whole. Herein, we comprehensively review the epidemiology, clinical features and novel insights into the mechanism behind photosensitive epilepsy with a focus on the clinical implications of new advancements.
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Epilepsia Refleja/metabolismo , Epilepsia Refleja/terapia , Electroencefalografía/métodos , Epilepsias Mioclónicas/terapia , Humanos , Neuroimagen/métodos , Estimulación Luminosa/efectos adversos , Trastornos por Fotosensibilidad/terapiaRESUMEN
The Wistar audiogenic rat (WAR) is an animal model of tonic-clonic epileptic seizures, developed after genetic selection by sister × brother inbreeding of Wistar rats susceptible to sound stimuli. Although metabolic changes have been described in this strain, nothing is known about its mitochondrial metabolism. Here, we addressed mitochondrial aspects of oxidative phosphorylation, oxidative stress, biogenesis, and dynamics in liver, skeletal muscle, and heart of male WARs and correlating them with physiological aspects of body metabolism. The results showed higher mitochondrial content, respiration rates in phosphorylation and noncoupled states, and H2O2 production in WARs. Liver presented higher content of peroxisome proliferator-activated receptor-γ coactivator 1α (PGC1α) and mammalian target of rapamycin, proteins related to mitochondrial biogenesis. In agreement, isolated liver mitochondria from WARs showed higher respiration rates in phosphorylation state and ADP-to-O ratio, as well as higher content of proteins related to electron transport chain ATP synthase, TCA cycle, and mitochondrial fusion and fission compared with their Wistar counterparts. Mitochondria with higher area and perimeter and more variable shapes were found in liver and soleus from WARs in addition to lower reduced-to-oxidized glutathione ratio. In vivo, WARs demonstrated lower body mass and energy expenditure but higher food and water intake and amino acid oxidation. When exposed to a running test, WARs reached higher speed and resisted for a longer time and distance than their Wistar controls. In conclusion, the WAR strain has mitochondrial changes in liver, skeletal muscle, and heart that improve its mitochondrial capacity of ATP production, making it an excellent rat model to study PGC1α overexpression and mitochondrial function in different physiological conditions or facing pathological challenges.
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Epilepsia Refleja/metabolismo , Adenosina Trifosfato/biosíntesis , Animales , Peso Corporal , Metabolismo Energético , Peróxido de Hidrógeno/metabolismo , Hígado/metabolismo , Masculino , Mitocondrias Hepáticas/metabolismo , Músculo Esquelético/metabolismo , Miocardio/metabolismo , Fosforilación Oxidativa , Estrés Oxidativo , Coactivador 1-alfa del Receptor Activado por Proliferadores de Peroxisomas gamma/biosíntesis , Coactivador 1-alfa del Receptor Activado por Proliferadores de Peroxisomas gamma/metabolismo , Ratas , Ratas Wistar , CarreraRESUMEN
Seizure is a clinical manifestation of a hyperexcitable neuronal network, in which, the electrical balance underlying the normal neuronal activity is altered pathologically-excitation (Glutamatergic activity) predominates over inhibition (GABAergic activity). Arresting of seizure activity is carried out by restoration of neurotransmitter balance. This process has a direct relation with ion channel permeability in cell and ion transmembrane movement. Low frequency EMS may have a neurostimulating and neuromodulating effect that is based on electromagnetic induction of electric field in the brain. Under the conditions of certain amplitude, frequency and relaxation time low-frequency electromagnetic field (EMF) induces depolarization of separate neurons, and changes the total cortical excitability in case of repeatedly carried out procedures. It was shown that the exposure of acoustic range EMS in GEPRs treated with GABA-A or GABA-B receptors antagonists decreased behavior seizure activity in response to audiogenic stimuli. Injection of Glutamate receptor agonist on background EMS causes seizure activity, but seizure manifestations have less degree compared to non-stimulated rats. Thus, in response to electromagnetic stimulation, the reduction or complete cramping of seizures can be explained by a change in the activity of the neurotransmitter systems.
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Epilepsia Refleja/terapia , Glutamatos/metabolismo , Magnetoterapia , Ácido gamma-Aminobutírico/metabolismo , Animales , Campos Electromagnéticos , Epilepsia Refleja/genética , Epilepsia Refleja/metabolismo , Agonistas de Aminoácidos Excitadores/farmacología , Antagonistas de Receptores de GABA-A/farmacología , Antagonistas de Receptores de GABA-B/farmacología , Masculino , RatasRESUMEN
Using immunoblotting, we showed that in rats of audiogenic epilepsy (AE) prone strain (Krushinsky- Molodkina, KM) the superior colliculus tissue (SC) contains significantly less quantity of glial neurotrophic factor (GDNF), beta-tubulin and actin in comparison to the same brain region in "0" rats, nonprone to AE. This fact led to the suggestion that the histological structure of the SC in KM rats could differ significantly from that of the "0" strain. Using neuromorphologÑ technique, we demonstrated that the total number of SC cells, as well as the number of neurons were significantly less in KM rats than in the "0" strain rats. Particularly strong differences were found in the deep layers of SC, the area of terminals from IC. Further studies of the midbrain structures, will help to identify the novel aspects of neural networks, involved in the genesis of AE in rats of KM strain.
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Epilepsia Refleja/patología , Colículos Superiores/patología , Animales , Susceptibilidad a Enfermedades , Epilepsia Refleja/metabolismo , Factores de Crecimiento Nervioso/metabolismo , Ratas , Colículos Superiores/metabolismoRESUMEN
The activity of ERK1/2 kinases in the quadrigemina inferior colliculus of Krushinsky-Molodkina rats of different age, which are characterized by an increased seizure readiness compared to Wistar rats, was analyzed. An increased (probably genetically determined) activity of these enzymes during the development of epileptiform activity in ontogeny was found, which may be the cause of abnormalities in the neurotransmitter system functioning.