RESUMEN
Voltage gated K(+) channels (Kv) are a highly diverse group of channels critical in determining neuronal excitability. Deficits of Kv channel subunit expression and function have been implicated in the pathogenesis of epilepsy. In this study, we investigate whether the expression of the specific subunit Kv3.4 is affected during epileptogenesis following pilocarpine-induced status epilepticus. For this purpose, we used immunohistochemistry, Western blotting assays and comparative analysis of gene expression using TaqMan-based probes and delta-delta cycle threshold (ΔΔCT) method of quantitative real-time polymerase chain reaction (qPCR) technique in samples obtained from age-matched control and epileptic rats. A marked down-regulation of Kv3.4 immunoreactivity was detected in the stratum lucidum and hilus of dentate gyrus in areas corresponding to the mossy fiber system of chronically epileptic rats. Correspondingly, a 20% reduction of Kv3.4 protein levels was detected in the hippocampus of chronic epileptic rats. Real-time quantitative PCR analysis of gene expression revealed that a significant 33% reduction of transcripts for Kv3.4 (gene Kcnc4) occurred after 1 month of pilocarpine-induced status epilepticus and persisted during the chronic phase of the model. These data indicate a reduced expression of Kv3.4 channels at protein and transcript levels in the epileptic hippocampus. Down-regulation of Kv3.4 in mossy fibers may contribute to enhanced presynaptic excitability leading to recurrent seizures in the pilocarpine model of temporal lobe epilepsy.
Asunto(s)
Epilepsia/metabolismo , Hipocampo/metabolismo , Canales de Potasio Shaw/metabolismo , Animales , Giro Dentado/metabolismo , Modelos Animales de Enfermedad , Regulación hacia Abajo/efectos de los fármacos , Epilepsia/inducido químicamente , Expresión Génica/efectos de los fármacos , Fibras Musgosas del Hipocampo/metabolismo , Pilocarpina , Ratas , Ratas Sprague-Dawley , Canales de Potasio Shaw/genética , Factores de TiempoRESUMEN
Functional properties of large conductance Ca(2+) activated potassium (BK) channels are determined by complex alternative splicing of the Kcnma1 gene encoding the alpha pore-forming subunit. Inclusion of the STREX exon in a C-terminal splice site is dynamically regulated and confers enhanced Ca(2+) sensitivity and channel inhibition via cAMP-dependent phosphorylation. Here, we describe a real time quantitative PCR (qPCR) approach to investigate relative changes in the expression of STREX and ZERO splice variants using a newly designed set of probes and primers for TaqMan-based qPCR analysis of cDNA from the rat dentate gyrus at different time points following pilocarpine-induced status epilepticus. Reduction in Kcnma1 gene expression is associated with a relative increase of STREX splice variant. Relative expression of STREX variant mRNA was increased at 10 days and at more than 1 month following status epilepticus. The biological consequences of seizure-related changes in alternative splicing of Kcnma1 deserve additional investigation.
Asunto(s)
Empalme Alternativo/genética , Calcio/metabolismo , Epilepsia del Lóbulo Temporal/genética , Subunidades alfa de los Canales de Potasio de Gran Conductancia Activados por Calcio/metabolismo , Animales , Giro Dentado/efectos de los fármacos , Exones , Subunidades alfa de los Canales de Potasio de Gran Conductancia Activados por Calcio/genética , Modelos Animales , Fosforilación , Pilocarpina/farmacología , Reacción en Cadena de la Polimerasa/métodos , ARN Mensajero/biosíntesis , ARN Mensajero/genética , Ratas , Ratas Wistar , Convulsiones/inducido químicamente , Estado Epiléptico/inducido químicamente , Regulación hacia ArribaRESUMEN
Small conductance calcium (Ca(2+)) activated SK channels are critical regulators of neuronal excitability in hippocampus. Accordingly, these channels are thought to play a key role in controlling neuronal activity in acute models of epilepsy. In this study, we investigate the expression and function of SK channels in the pilocarpine model of mesial temporal lobe epilepsy. For this purpose, protein expression was assessed using western blotting assays and gene expression was analyzed using TaqMan-based probes and the quantitative real-time polymerase chain reaction (qPCR) comparative method delta-delta cycle threshold ( big up tri, open big up tri, openCT) in samples extracted from control and epileptic rats. In addition, the effect of SK channel antagonist UCL1684 and agonist NS309 on CA1 evoked population spikes was studied in hippocampal slices. Western blotting analysis showed a significant reduction in the expression of SK1 and SK2 channels at 10days following status epilepticus (SE), but levels recovered at 1month and at more than 2months after SE. In contrast, a significant down-regulation of SK3 channels was detected after 10days of SE. Analysis of gene expression by qPCR revealed a significant reduction of transcripts for SK2 (Kcnn1) and SK3 (Kcnn3) channels as early as 10days following pilocarpine-induced SE and during the chronic phase of the pilocarpine model. Moreover, bath application of UCL1684 (100nM for 15min) induced a significant increase of the population spike amplitude and number of spikes in the hippocampal CA1 area of slices obtained control and chronic epileptic rats. This effect was obliterated by co-administration of UCL1684 with SK channel agonist NS309 (1microM). Application of NS309 failed to modify population spikes in the CA1 area of slices taken from control and epileptic rats. These data indicate an abnormal expression of SK channels and a possible dysfunction of these channels in experimental MTLE.
Asunto(s)
Regulación de la Expresión Génica/efectos de los fármacos , Potenciales de la Membrana/efectos de los fármacos , Agonistas Muscarínicos/efectos adversos , Pilocarpina/efectos adversos , Canales de Potasio de Pequeña Conductancia Activados por el Calcio/fisiología , Estado Epiléptico , Factores de Edad , Alcanos/farmacología , Análisis de Varianza , Animales , Modelos Animales de Enfermedad , Interacciones Farmacológicas , Hipocampo/patología , Técnicas In Vitro , Indoles/farmacología , Masculino , Potenciales de la Membrana/fisiología , Neuronas/efectos de los fármacos , Neuronas/fisiología , Oximas/farmacología , Compuestos de Quinolinio/farmacología , Ratas , Ratas Sprague-Dawley , Canales de Potasio de Pequeña Conductancia Activados por el Calcio/antagonistas & inhibidores , Canales de Potasio de Pequeña Conductancia Activados por el Calcio/efectos de los fármacos , Estado Epiléptico/inducido químicamente , Estado Epiléptico/patología , Estado Epiléptico/fisiopatología , Factores de TiempoRESUMEN
Group II metabotropic (mGlu II) receptor subtypes mGlu2 and mGlu3 are important modulators of synaptic plasticity and glutamate release in the brain. Accordingly, several pharmacological ligands have been designed to target these receptors for the treatment of neurological disorders characterized by anomalous glutamate regulation including epilepsy. In this study, we examine whether the expression level and function of mGlu2 and mGlu3 are altered in experimental epilepsy by using immunohistochemistry, Western blot analysis, RT-PCR and extracellular recordings. A down-regulation of mGlu2/3 protein expression at the mossy fiber pathway was associated with a significant reduction in mGlu2/3 protein expression in the hippocampus and cortex of chronically epileptic rats. Moreover, a reduction in mGlu2 and mGlu3 transcripts levels was noticed as early as 24 h after pilocarpine-induced status epilepticus (SE) and persisted during subsequent "latent" and chronic periods. In addition, a significant impairment of mGlu II-mediated depression of field excitatory postsynaptic potentials at mossy fiber-CA3 synapses was detected in chronically epileptic rats. Application of mGlu II agonists (2S,2'R,3'R)-2-(2',3'-dicarboxycyclopropyl)glycine (DCG-IV) induced a significant reduction of the fEPSP amplitude in control rats, but not in chronic epileptic rats. These data indicate a long-lasting impairment of mGlu2/3 expression that may contribute to abnormal presynaptic plasticity, exaggerate glutamate release and hyperexcitability in temporal lobe epilepsy.
Asunto(s)
Encéfalo/metabolismo , Epilepsia/metabolismo , Plasticidad Neuronal/fisiología , Receptores de Glutamato Metabotrópico/metabolismo , Animales , Anticonvulsivantes/farmacología , Western Blotting , Encéfalo/efectos de los fármacos , Enfermedad Crónica , Convulsivantes/toxicidad , Ciclopropanos/farmacología , Epilepsia/inducido químicamente , Potenciales Postsinápticos Excitadores/fisiología , Glicina/análogos & derivados , Glicina/farmacología , Inmunohistoquímica , Técnicas de Placa-Clamp , Pilocarpina/toxicidad , ARN Mensajero/análisis , Ratas , Ratas Wistar , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
Epileptogenesis in mesial temporal lobe epilepsy is determined by several factors including abnormalities in the expression and function of ion channels. Here, we report a long-lasting deficit in gene expression of Kcnma1 coding for the large-conductance calcium-activated potassium (BK, MaxiK) channel alpha-subunits after pilocarpine-induced status epilepticus. By using comparative real-time PCR, Taqman gene expression assays, and the delta-delta comparative threshold method we detected a significant reduction in Kcnma1 expression in microdissected dentate gyrus at different intervals after status epilepticus (24 h, 10 days, 1 month, and more than 2 months). BK channels are key regulators of neuronal excitability and transmitter release. Hence, defective Kcnma1 expression may play a critical role in the pathogenesis of mesial temporal lobe epilepsy.
Asunto(s)
Giro Dentado/metabolismo , Epilepsia del Lóbulo Temporal/genética , Perfilación de la Expresión Génica , Subunidades alfa de los Canales de Potasio de Gran Conductancia Activados por Calcio/genética , Análisis de Varianza , Animales , Modelos Animales de Enfermedad , Regulación hacia Abajo/efectos de los fármacos , Expresión Génica/efectos de los fármacos , Inyecciones Intraperitoneales , Inyecciones Subcutáneas , Agonistas Muscarínicos/administración & dosificación , Agonistas Muscarínicos/toxicidad , Pilocarpina/administración & dosificación , Pilocarpina/toxicidad , ARN Mensajero/genética , ARN Mensajero/metabolismo , Ratas , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Convulsiones/inducido químicamente , Convulsiones/genética , Estado Epiléptico/inducido químicamente , Estado Epiléptico/genética , Factores de TiempoRESUMEN
Group II metabotropic glutamate (mGlu II) receptors subtype 2 and 3 (mGlu2 and mGlu3) are subtle regulators of neuronal excitability and synaptic plasticity in the hippocampus. In recent years, researchers have investigated the potential neuroprotective and anticonvulsant effects of compounds acting on mGlu II receptors. However, abnormal expression and function of mGlu2 and mGlu3 have been reported in temporal lobe epilepsy, a phenomena that may limit the therapeutic effectiveness of these potentially new antiepileptic drugs. Here, we investigated seizure-induced changes in mGlu2 and mGlu3 mRNA following pilocarpine-inducted status epilepticus (SE) and subsequent epileptogenesis. Relative changes in gene expression were assessed by comparative analysis of quantitative real-time PCR (qrtPCR) by the delta-delta CT method. Pilocarpine-treated and control rats were sacrificed at different periods (24 h, 10 days, one month and more than two months) following SE. Total RNA was isolated from microdissected dentate gyrus and processed for RT-PCR and qrtPCR using glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as an endogenous control gene. Analysis of relative quantification (RQ) ratios of mGlu2 and mGlu3 mRNA expression revealed a significant down-regulation of both targets at 24 h after SE. Gene expression partially recovered at 10 days following SE reaching control levels at one month after SE. Two month after SE, mGlu2 mRNA expression was significantly down-regulated to approximately 41% of control expression whereas mGlu3 mRNA was comparable to control levels. Our data indicate that mGlu2 and mGlu3 expression is dynamically down-regulated or selectively enhanced during critical periods of epileptogenesis. Seizure-induced differential dysregulation of mGlu2 and mGlu3 receptors may affect the availability of these molecular targets for therapeutic compounds in epilepsy.
Asunto(s)
Regulación de la Expresión Génica/efectos de los fármacos , Pilocarpina , Receptores de Glutamato Metabotrópico/metabolismo , Estado Epiléptico/inducido químicamente , Estado Epiléptico/metabolismo , Análisis de Varianza , Animales , Modelos Animales de Enfermedad , Pilocarpina/farmacología , ARN Mensajero/metabolismo , Ratas , Ratas Wistar , Receptores de Glutamato Metabotrópico/genética , Factores de TiempoRESUMEN
In the hippocampus, BK channels are preferentially localized in presynaptic glutamatergic terminals including mossy fibers where they are thought to play an important role regulating excessive glutamate release during hyperactive states. Large conductance calcium-activated potassium channels (BK, MaxiK, Slo) have recently been implicated in the pathogenesis of genetic epilepsy. However, the role of BK channels in acquired mesial temporal lobe epilepsy (MTLE) remains unknown. Here we used immunohistochemistry, laser scanning confocal microscopy (LSCM), Western immunoblotting and RT-PCR to investigate the expression pattern of the alpha-pore-forming subunit of BK channels in the hippocampus and cortex of chronically epileptic rats obtained by the pilocarpine model of MTLE. All epileptic rats experiencing recurrent spontaneous seizures exhibited a significant down-regulation of BK channel immunostaining in the mossy fibers at the hilus and stratum lucidum of the CA3 area. Quantitative analysis of immunofluorescence signals by LSCM revealed a significant 47% reduction in BK channel immunofluorescent signals in epileptic rats when compared to age-matched non-epileptic control rats. These data correlate with a similar reduction in BK channel protein levels and transcripts in the cortex and hippocampus. Our data indicate a seizure-related down-regulation of BK channels in chronically epileptic rats. Further functional assays are necessary to determine whether altered BK channel expression is an acquired channelopathy or a compensatory mechanism affecting the network excitability in MTLE. Moreover, seizure-mediated BK down-regulation may disturb neuronal excitability and presynaptic control at glutamatergic terminals triggering exaggerated glutamate release and seizures.