RESUMEN
Hyperpolarization-activated cation channels are involved, among other functions, in learning and memory, control of synaptic transmission and epileptogenesis. The importance of the HCN1 and HCN2 isoforms for brain function has been demonstrated, while the role of HCN4, the third major neuronal HCN subunit, is not known. Here we show that HCN4 is essential for oscillatory activity in the thalamocortical (TC) network. HCN4 is selectively expressed in various thalamic nuclei, excluding the thalamic reticular nucleus. HCN4-deficient TC neurons revealed a massive reduction of Ih and strongly reduced intrinsic burst firing, whereas the current was normal in cortical pyramidal neurons. In addition, evoked bursting in a thalamic slice preparation was strongly reduced in the mutant mice probes. HCN4-deficiency also significantly slowed down thalamic and cortical oscillations during active wakefulness. Taken together, these results establish that thalamic HCN4 channels are essential for the production of rhythmic intrathalamic oscillations and determine regular TC oscillatory activity during alert states.
Asunto(s)
Ondas Encefálicas , Corteza Cerebral/fisiología , Canales Regulados por Nucleótidos Cíclicos Activados por Hiperpolarización/fisiología , Neuronas/fisiología , Tálamo/fisiología , Potenciales de Acción , Animales , Femenino , Masculino , Ratones Endogámicos C57BL , Ratones Noqueados , Modelos Neurológicos , Vías Nerviosas/fisiologíaRESUMEN
The polygenic origin of generalized absence epilepsy results in dysfunction of ion channels that allows the switch from physiological asynchronous to pathophysiological highly synchronous network activity. Evidence from rat and mouse models of absence epilepsy indicates that altered Ca(2+) channel activity contributes to cellular and network alterations that lead to seizure activity. Under physiological circumstances, high voltage-activated (HVA) Ca(2+) channels are important in determining the thalamic firing profile. Here, we investigated a possible contribution of HVA channels to the epileptic phenotype using a rodent genetic model of absence epilepsy. In this study, HVA Ca(2+) currents were recorded from neurons of three different thalamic nuclei that are involved in both sensory signal transmission and rhythmic-synchronized activity during epileptic spike-and-wave discharges (SWD), namely the dorsal part of the lateral geniculate nucleus (dLGN), the ventrobasal thalamic complex (VB) and the reticular thalamic nucleus (NRT) of epileptic Wistar Albino Glaxo rats from Rijswijk (WAG/Rij) and non-epileptic August Copenhagen Irish (ACI) rats. HVA Ca(2+) current densities in dLGN neurons were significantly increased in epileptic rats compared with non-epileptic controls while other thalamic regions revealed no differences between the strains. Application of specific channel blockers revealed that the increased current was carried by L-type Ca(2+) channels. Electrophysiological evidence of increased L-type current correlated with up-regulated mRNA and protein expression of a particular L-type channel, namely Cav1.3, in dLGN of epileptic rats. No significant changes were found for other HVA Ca(2+) channels. Moreover, pharmacological inactivation of L-type Ca(2+) channels results in altered firing profiles of thalamocortical relay (TC) neurons from non-epileptic rather than from epileptic rats. While HVA Ca(2+) channels influence tonic and burst firing in ACI and WAG/Rij differently, it is discussed that increased Cav1.3 expression may indirectly contribute to increased robustness of burst firing and thereby the epileptic phenotype of absence epilepsy.
Asunto(s)
Canales de Calcio/metabolismo , Epilepsia/patología , Potenciales de la Membrana/fisiología , Núcleos Talámicos/metabolismo , Regulación hacia Arriba/fisiología , Agonistas de Receptores Adrenérgicos beta 2/farmacología , Albuterol/análogos & derivados , Albuterol/farmacología , Animales , Animales Recién Nacidos , Fenómenos Biofísicos/efectos de los fármacos , Fenómenos Biofísicos/genética , Fenómenos Biofísicos/fisiología , Bloqueadores de los Canales de Calcio/farmacología , Canales de Calcio/genética , Modelos Animales de Enfermedad , Estimulación Eléctrica , Epilepsia/genética , Epilepsia/fisiopatología , Inmunosupresores/farmacología , Potenciales de la Membrana/efectos de los fármacos , Potenciales de la Membrana/genética , Tasa de Mutación , Proteínas del Tejido Nervioso/metabolismo , Neuronas/efectos de los fármacos , Neuronas/fisiología , Ratas , Ratas Wistar , Xinafoato de Salmeterol , Tacrolimus/análogos & derivados , Tacrolimus/farmacología , Núcleos Talámicos/patología , Regulación hacia Arriba/genéticaRESUMEN
OBJECTIVE: The outbreak of hemolytic-uremic syndrome and diarrhea caused by Shiga toxin-producing Escherichia coli O104:H4 in Germany during May to July 2011 involved severe and characteristic neurologic manifestations with a strong female preponderance. Owing to these observations, we designed a series of experimental studies to evaluate the underlying mechanism of action of this clinical picture. METHODS: A magnetic resonance imaging and electroencephalographic study of patients was performed to evaluate the clinical picture in detail. Thereafter, combinations of different experimental settings, including electrophysiological and histological analyses, as well as calcium imaging in brain slices of rats, were conducted. RESULTS: We report on 7 female patients with neurologic symptoms and signs including bilateral thalamic lesions and encephalopathic changes indicative of a predominant involvement of the thalamus. Experimental studies in rats revealed an enhanced expression of the Shiga toxin receptor globotriaosylceramide on thalamic neurons in female rats as compared to other brain regions in the same rats and to male animals. Incubation of brain slices with Shiga toxin 2 evoked a strong membrane depolarization and intracellular calcium accumulation in neurons, associated with neuronal apoptosis, predominantly in the thalamic area. INTERPRETATION: These findings suggest that the direct cytotoxic effect of Shiga toxin 2 in the thalamus might contribute to the pathophysiology of neuronal complications in hemolytic-uremic syndrome.
Asunto(s)
Infecciones por Escherichia coli/complicaciones , Enfermedades del Sistema Nervioso/etiología , Enfermedades del Sistema Nervioso/patología , Toxina Shiga II/toxicidad , Tálamo/patología , Adulto , Anciano , Animales , Animales Recién Nacidos , Apoptosis/efectos de los fármacos , Astrocitos/efectos de los fármacos , Astrocitos/metabolismo , Calcio/metabolismo , Electroencefalografía , Infecciones por Escherichia coli/líquido cefalorraquídeo , Femenino , Regulación de la Expresión Génica/efectos de los fármacos , Humanos , Técnicas In Vitro , Imagen por Resonancia Magnética , Potenciales de la Membrana/efectos de los fármacos , Enfermedades del Sistema Nervioso/líquido cefalorraquídeo , Neuronas/efectos de los fármacos , Técnicas de Placa-Clamp , ARN Mensajero/metabolismo , Ratas , Ratas Long-Evans , Caracteres Sexuales , Tálamo/fisiopatología , Trihexosilceramidas/metabolismo , Adulto JovenRESUMEN
Mutations in genes coding for Ca(2+) channels were found in patients with childhood absence epilepsy (CAE) indicating a contribution of Ca(2+)-dependent mechanisms to the generation of spike-wave discharges (SWD) in humans. Since the involvement of Ca(2+) signals remains unclear, the aim of the present study was to elucidate the function of a Ca(2+)-dependent K(+) channel (BKCa) under physiological conditions and in the pathophysiological state of CAE. The activation of BKCa channels is dependent on both voltage and intracellular Ca(2+) concentrations. Moreover, these channels exhibit an outstandingly high level of regulatory heterogeneity that builds the basis for the influence of BKCa channels on different aspects of neuronal activity. Here, we analyse the contribution of BKCa channels to firing of thalamocortical relay neurons, and we test the hypothesis that BKCa channel activity affects the phenotype of a genetic rat model of CAE. We found that the activation of the ß2-adrenergic receptor/protein kinase A pathway resulted in BKCa channel inhibition. Furthermore, BKCa channels affect the number of action potentials fired in a burst and produced spike frequency adaptation during tonic activity. The latter result was confirmed by a computer modelling approach. We demonstrate that the ß2-adrenergic inhibition of BKCa channels prevents spike frequency adaptation and, thus, might significantly support the tonic firing mode of thalamocortical relay neurons. In addition, we show that BKCa channel functioning differs in epileptic WAG/Rij and thereby likely contributes to highly synchronised, epileptic network activity.
Asunto(s)
Potenciales de Acción , Interneuronas/fisiología , Canales de Potasio de Gran Conductancia Activados por el Calcio/metabolismo , Tálamo/metabolismo , Adaptación Fisiológica , Animales , Calcio/metabolismo , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Modelos Animales de Enfermedad , Epilepsia Tipo Ausencia/genética , Epilepsia Tipo Ausencia/metabolismo , Interneuronas/metabolismo , Modelos Neurológicos , Ratas , Ratas Endogámicas , Receptores Adrenérgicos beta 2/metabolismo , Tálamo/citología , Tálamo/fisiopatologíaRESUMEN
Modulation of the standing outward current (I (SO)) by muscarinic acetylcholine (ACh) receptor (MAChR) stimulation is fundamental for the state-dependent change in activity mode of thalamocortical relay (TC) neurons. Here, we probe the contribution of MAChR subtypes, G proteins, phospholipase C (PLC), and two pore domain K(+) (K(2P)) channels to this signaling cascade. By the use of spadin and A293 as specific blockers, we identify TWIK-related K(+) (TREK)-1 channel as new targets and confirm TWIK-related acid-sensitve K(+) (TASK)-1 channels as known effectors of muscarinic signaling in TC neurons. These findings were confirmed using a high affinity blocker of TASK-3 and TREK-1, namely, tetrahexylammonium chloride. It was found that the effect of muscarinic stimulation was inhibited by M(1)AChR-(pirenzepine, MT-7) and M(3)AChR-specific (4-DAMP) antagonists, phosphoinositide-specific PLCß (PI-PLC) inhibitors (U73122, ET-18-OCH(3)), but not the phosphatidylcholine-specific PLC (PC-PLC) blocker D609. By comparison, depleting guanosine-5'-triphosphate (GTP) in the intracellular milieu nearly completely abolished the effect of MAChR stimulation. The block of TASK and TREK channels was accompanied by a reduction of the muscarinic effect on I (SO). Current-clamp recordings revealed a membrane depolarization following MAChR stimulation, which was sufficient to switch TC neurons from burst to tonic firing under control conditions but not during block of M(1)AChR/M(3)AChR and in the absence of intracellular GTP. These findings point to a critical role of G proteins and PLC as well as TASK and TREK channels in the muscarinic modulation of thalamic activity modes.
Asunto(s)
Potenciales de Acción/fisiología , Neuronas Colinérgicas/fisiología , Transducción de Señal/fisiología , Sueño/fisiología , Tálamo/fisiología , Potenciales de Acción/efectos de los fármacos , Animales , Neuronas Colinérgicas/efectos de los fármacos , Fenómenos Electrofisiológicos/efectos de los fármacos , Fenómenos Electrofisiológicos/fisiología , Subunidades alfa de la Proteína de Unión al GTP Gq-G11/genética , Expresión Génica/genética , Guanosina Difosfato/análogos & derivados , Guanosina Difosfato/farmacología , Guanosina Trifosfato/antagonistas & inhibidores , Guanosina Trifosfato/metabolismo , Concentración de Iones de Hidrógeno , Núcleos Talámicos Laterales/citología , Núcleos Talámicos Laterales/fisiología , Potenciales de la Membrana/efectos de los fármacos , Potenciales de la Membrana/fisiología , Muscarina/farmacología , Agonistas Muscarínicos/farmacología , Antagonistas Muscarínicos/farmacología , Proteínas del Tejido Nervioso , Oxotremorina/análogos & derivados , Oxotremorina/farmacología , Técnicas de Placa-Clamp , Fosfolipasa C beta/antagonistas & inhibidores , Fosfolipasa C beta/genética , Fosfolipasa C beta/metabolismo , Canales de Potasio de Dominio Poro en Tándem/antagonistas & inhibidores , Canales de Potasio de Dominio Poro en Tándem/genética , Canales de Potasio de Dominio Poro en Tándem/metabolismo , Ratas , Ratas Long-Evans , Receptor Muscarínico M1/agonistas , Receptor Muscarínico M1/antagonistas & inhibidores , Receptor Muscarínico M1/genética , Receptor Muscarínico M1/metabolismo , Receptor Muscarínico M3/antagonistas & inhibidores , Receptor Muscarínico M3/genética , Receptor Muscarínico M3/metabolismo , Transducción de Señal/efectos de los fármacos , Tálamo/citología , Tionucleótidos/farmacologíaRESUMEN
Hyperpolarization-activated cyclic nucleotide-gated cation (HCN) channels represent the molecular substrate of the hyperpolarization-activated inward current (I(h)). Although these channels act as pacemakers for the generation of rhythmic activity in the thalamocortical network during sleep and epilepsy, their developmental profile in the thalamus is not yet fully understood. Here we combined electrophysiological, immunohistochemical, and mathematical modeling techniques to examine HCN gene expression and I(h) properties in thalamocortical relay (TC) neurons of the dorsal part of the lateral geniculate nucleus (dLGN) in an epileptic (WAG/Rij) compared to a non-epileptic (ACI) rat strain. Recordings of TC neurons between postnatal day (P) 7 and P90 in both rat strains revealed that I(h) was characterized by higher current density, more hyperpolarized voltage dependence, faster activation kinetics, and reduced cAMP-sensitivity in epileptic animals. All four HCN channel isoforms (HCN1-4) were detected in dLGN, and quantitative analyses revealed a developmental increase of protein expression of HCN1, HCN2, and HCN4 but a decrease of HCN3. HCN1 was expressed at higher levels in WAG/Rij rats, a finding that was correlated with increased expression of the interacting proteins filamin A (FilA) and tetratricopeptide repeat-containing Rab8b-interacting protein (TRIP8b). Analysis of a simplified computer model of the thalamic network revealed that the alterations of I(h) found in WAG/Rij rats compensate each other in a way that leaves I(h) availability constant, an effect that ensures unaltered cellular burst activity and thalamic oscillations. These data indicate that during postnatal developmental the hyperpolarizing shift in voltage dependency (resulting in less current availability) is compensated by an increase in current density in WAG/Rij thereby possibly limiting the impact of I(h) on epileptogenesis. Because HCN3 is expressed higher in young versus older animals, HCN3 likely does not contribute to alterations in I(h) in older animals.
Asunto(s)
Canales Catiónicos Regulados por Nucleótidos Cíclicos/metabolismo , Epilepsia/metabolismo , Cuerpos Geniculados/metabolismo , Neuronas/metabolismo , Animales , Canales Catiónicos Regulados por Nucleótidos Cíclicos/genética , Epilepsia/genética , Epilepsia/fisiopatología , Expresión Génica , Cuerpos Geniculados/fisiopatología , Ratas , Especificidad de la EspecieRESUMEN
Transient A-type K+ currents (I(A)) are known to influence the firing pattern of a number of thalamic cell types, but have not been investigated in intralaminar thalamocortical (TC) relay neurons yet. We therefore combined whole-cell patch-clamp techniques, PCR analysis, and immunohistochemistry to investigate the voltage-dependent and pharmacological properties of I(A) and to determine its molecular basis in TC neurons from the centrolateral, paracentral, and centromedial thalamic nuclei. I(A) revealed half-maximal (V (h)) activation and inactivation at about -17 and -67 mV, respectively. At a concentration of 5-10 mM 4-aminopyridine (4-AP) completely blocked I(A). Furthermore, I(A) was nearly unaffected by two sea anemone toxins (blood depressing substances 1 and 2, BDS1 and BDS2; 6-8% block at a concentration of 1 µM) but strongly sensitive to the K(V)4 channel blocker Heteropoda venatoria toxin 2 (HpTx2; about 45% block at a concentration of 5 µM). PCR screening revealed the expression of K(V)4.1-4.3, with strongest expression for K(V)4.2 and weak expression for K(V)4.1. Accordingly K(V)4.1 was not detected in immunohistochemical staining. Furthermore, K(V)4.2 and K(V)4.3 revealed mainly dendritic and somatic staining, respectively. Together with current clamp recordings, these findings point to a scenario where the fast transient I(A) in intralaminar TC neurons has a depolarized threshold at potentials negative to -50 mV, is substantially generated by K(V)4.2 and K(V)4.3 channels, allows prominent burst firing at hyperpolarized potentials, prevents the generation of high-threshold potentials, generates a delayed onset of firing at more depolarized potentials, and allows fast tonic firing.
Asunto(s)
Neuronas/fisiología , Canales de Potasio/fisiología , Tálamo/fisiología , 4-Aminopiridina/farmacología , Animales , Técnicas de Placa-Clamp , Canales de Potasio/efectos de los fármacos , Canales de Potasio/metabolismo , Ratas , Tálamo/citologíaRESUMEN
Hyperpolarization-activated cyclic nucleotide-gated cation (HCN) channels are the molecular substrate of the hyperpolarization-activated inward current (I(h)). Because the developmental profile of HCN channels in the thalamus is not well understood, we combined electrophysiological, molecular, immunohistochemical, EEG recordings in vivo, and computer modeling techniques to examine HCN gene expression and I(h) properties in rat thalamocortical relay (TC) neurons in the dorsal part of the lateral geniculate nucleus and the functional consequence of this maturation. Recordings of TC neurons revealed an approximate sixfold increase in I(h) density between postnatal day 3 (P3) and P106, which was accompanied by significantly altered current kinetics, cAMP sensitivity, and steady-state activation properties. Quantification on tissue levels revealed a significant developmental decrease in cAMP. Consequently the block of basal adenylyl cyclase activity was accompanied by a hyperpolarizing shift of the I(h) activation curve in young but not adult rats. Quantitative analyses of HCN channel isoforms revealed a steady increase of mRNA and protein expression levels of HCN1, HCN2, and HCN4 with reduced relative abundance of HCN4. Computer modeling in a simplified thalamic network indicated that the occurrence of rhythmic delta activity, which was present in the EEG at P12, differentially depended on I(h) conductance and modulation by cAMP at different developmental states. These data indicate that the developmental increase in I(h) density results from increased expression of three HCN channel isoforms and that isoform composition and intracellular cAMP levels interact in determining I(h) properties to enable progressive maturation of rhythmic slow-wave sleep activity patterns.
Asunto(s)
Relojes Biológicos/fisiología , Corteza Cerebral/metabolismo , Canales Catiónicos Regulados por Nucleótidos Cíclicos/biosíntesis , Regulación del Desarrollo de la Expresión Génica/fisiología , Canales Iónicos/biosíntesis , Neuronas/metabolismo , Canales de Potasio/biosíntesis , Tálamo/metabolismo , Animales , Animales Recién Nacidos , Corteza Cerebral/crecimiento & desarrollo , Canales Catiónicos Regulados por Nucleótidos Cíclicos/genética , Canales Regulados por Nucleótidos Cíclicos Activados por Hiperpolarización , Canales Iónicos/genética , Vías Nerviosas/crecimiento & desarrollo , Vías Nerviosas/metabolismo , Neuronas/fisiología , Canales de Potasio/genética , Isoformas de Proteínas/biosíntesis , Ratas , Ratas Sprague-Dawley , Tálamo/crecimiento & desarrolloRESUMEN
T-type Ca(2+) current-dependent burst firing of thalamic neurons is thought to be involved in the hyper-synchronous activity observed during absence seizures. Here we investigate the correlation between the expression of T-channel coding genes (alpha1G, -H, -I), T-type Ca(2+) current, and the T-current-dependent low threshold Ca(2+) spike in three functionally distinct thalamic nuclei (lateral geniculate nucleus; centrolateral nucleus; reticular nucleus) in a rat model of absence epilepsy, the WAG/Rij rats, and a non-epileptic control strain, the ACI rats. The lateral geniculate nucleus and centrolateral nucleus were found to primarily express alpha1G and alpha1I, while the reticular thalamic nucleus expressed alpha1H and alpha1I. Expression was higher in WAG/Rij when compared to ACI. The T-type Ca(2+) current properties matched the predictions derived from the expression pattern analysis. Current density was larger in all nuclei of WAG/Rij rats when compared to ACI and correlated with LTS size and the minimum LTS generating slope, while T-type Ca(2+) current voltage dependency correlated with the LTS onset potential.
Asunto(s)
Canales de Calcio Tipo T , Calcio/metabolismo , Epilepsia Tipo Ausencia/metabolismo , Neuronas/fisiología , Isoformas de Proteínas , Tálamo/citología , Potenciales de Acción/fisiología , Animales , Canales de Calcio Tipo T/genética , Canales de Calcio Tipo T/metabolismo , Modelos Animales de Enfermedad , Epilepsia Tipo Ausencia/genética , Epilepsia Tipo Ausencia/fisiopatología , Femenino , Humanos , Activación del Canal Iónico , Masculino , Técnicas de Placa-Clamp , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , ARN Mensajero/metabolismo , Ratas , Ratas Endogámicas , Bloqueadores de los Canales de Sodio/metabolismo , Tetrodotoxina/metabolismo , Tálamo/metabolismoRESUMEN
Channel blocking, anti-oscillatory, and anti-epileptic effects of clinically used anti-absence substances (ethosuximide, valproate) and the T-type Ca2+ current (IT) blocker mibefradil were tested by analyzing membrane currents in acutely isolated local circuit interneurons and thalamocortical relay (TC) neurons, slow intrathalamic oscillations in brain slices, and spike and wave discharges (SWDs) occurring in vivo in Wistar Albino Glaxo rats from Rijswijk (WAG/Rij). Substance effects in vitro were compared between WAG/Rij and a non-epileptic control strain, the ACI rats. Ethosuximide (ETX) and valproate were found to block IT in acutely isolated thalamic neurons. Block of IT by therapeutically relevant ETX concentrations (0.25-0.75 mM) was stronger in WAG/Rij, although the maximal effect at saturating concentrations (>or=10 mM) was stronger in ACI. Ethosuximide delayed the onset of the low threshold Ca2+ spike (LTS) of neurons recorded in slice preparations. Mibefradil (>or=2 microM) completely blocked IT and the LTS, dampened evoked thalamic oscillations, and attenuated SWDs in vivo. Computational modeling demonstrated that the complete effect of ETX can be replicated by a sole reduction of IT. However, the necessary degree of IT reduction was not induced by therapeutically relevant ETX concentrations. A combined reduction of IT, the persistent sodium current, and the Ca2+ activated K+ current resulted in an LTS alteration resembling the experimental observations. In summary, these results support the hypothesis of IT reduction as part of the mechanism of action of anti-absence drugs and demonstrate the ability of a specific IT antagonist to attenuate rhythmic burst firing and SWDs.
Asunto(s)
Anticonvulsivantes/farmacología , Bloqueadores de los Canales de Calcio/farmacología , Canales de Calcio Tipo T/fisiología , Epilepsia Tipo Ausencia/patología , Interneuronas/efectos de los fármacos , Tálamo/patología , Animales , Animales Recién Nacidos , Anticonvulsivantes/uso terapéutico , Modelos Animales de Enfermedad , Relación Dosis-Respuesta a Droga , Relación Dosis-Respuesta en la Radiación , Interacciones Farmacológicas , Estimulación Eléctrica/métodos , Electroencefalografía , Epilepsia Tipo Ausencia/tratamiento farmacológico , Etosuximida/farmacología , Potenciales de la Membrana/efectos de los fármacos , Potenciales de la Membrana/fisiología , Potenciales de la Membrana/efectos de la radiación , Mibefradil/farmacología , Técnicas de Placa-Clamp/métodos , Ratas , Ratas Endogámicas ACIRESUMEN
The role of hyperpolarization-activated, cyclic nucleotide-modulated (HCN) channel isoforms and hyperpolarization-activated cation current (Ih) for seizure-related burst firing in thalamocortical (TC) neurons was investigated in a rat genetic model of absence epilepsy [Wistar Albino Glaxo rats, bred in Rijswijk (WAG/Rij)]. Burst discharges in TC neurons locked to seizure activity in vivo were prolonged during blockade of Ih by Cs+ and ZD7288 (4-ethylphenylamino-1,2-dimethyl-6-methylaminopyrimidinium chloride). In vitro analyses revealed a hyperpolarizing shift of half-maximal Ih activation (Vh) in WAG/Rij (Vh = -93.2 mV) compared with nonepileptic controls [August x Copenhagen-Irish (ACI) (Vh = -88.0 mV)]. This effect is explained by a shift of the responsiveness of Ih to cAMP toward higher concentrations in TC neurons from WAG/Rij, as revealed by application of 8-bromo-cAMP and the phosphodiesterase inhibitor IBMX. During blockade of adenylyl cyclase activity, Ih activation was similar in the two strains, whereas the difference in cAMP responsiveness persisted, thereby voting against different ambient cAMP levels between strains. Increasing the intracellular cAMP level and shifting Ih activation led to a change from burst to tonic firing mode in WAG/Rij but not in ACI rats. Furthermore, HCN1 expression was significantly increased on mRNA and protein levels, with no changes in HCN2-4 expression. In conclusion, there is an increase in HCN1 expression in the epileptic thalamus, associated with a decrease in cAMP responsiveness of Ih in TC neurons and resulting impairment to control the shift from burst to tonic firing, which, in turn, will prolong burst activity after recruitment of Ih during absence seizures.
Asunto(s)
Corteza Cerebral/citología , Epilepsia Tipo Ausencia/metabolismo , Canales Iónicos/metabolismo , Neuronas/metabolismo , Tálamo/citología , 1-Metil-3-Isobutilxantina/farmacología , 8-Bromo Monofosfato de Adenosina Cíclica/farmacología , Potenciales de Acción/efectos de los fármacos , Adenina/análogos & derivados , Adenina/farmacología , Animales , Northern Blotting/métodos , Cesio/farmacología , Cloruros/farmacología , Canales Catiónicos Regulados por Nucleótidos Cíclicos , Modelos Animales de Enfermedad , Relación Dosis-Respuesta a Droga , Relación Dosis-Respuesta en la Radiación , Interacciones Farmacológicas , Estimulación Eléctrica/métodos , Electroencefalografía/métodos , Inhibidores Enzimáticos/farmacología , Epilepsia Tipo Ausencia/genética , Epilepsia Tipo Ausencia/fisiopatología , Femenino , Regulación de la Expresión Génica/efectos de los fármacos , Regulación de la Expresión Génica/fisiología , Canales Regulados por Nucleótidos Cíclicos Activados por Hiperpolarización , Inmunohistoquímica/métodos , Hibridación in Situ/métodos , Técnicas In Vitro , Masculino , Potenciales de la Membrana/efectos de los fármacos , Potenciales de la Membrana/fisiología , Potenciales de la Membrana/efectos de la radiación , Microinyecciones , Técnicas de Placa-Clamp/métodos , Canales de Potasio , Isoformas de Proteínas/metabolismo , Pirimidinas/farmacología , ARN Mensajero/biosíntesis , Ratas , Ratas Mutantes , Ratas Wistar , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/métodosRESUMEN
Homogeneous IgG fractions were obtained by chromatography of the sera of patients with multiple sclerosis (MS) on Protein G-Sepharose under conditions that remove non-specifically bound proteins. These IgGs contained several chelated metals, the relative amount of which decreases in the order: Fe>or=Ca>Cu>or=Zn>or=Mg>or=Mn>or=Pb>or=Co>or=Ni. In contrast to homogeneous IgGs of healthy individuals, Abs of MS patients effectively hydrolyzed human myelin basic protein (MBP). A minor metal-dependent fraction was obtained by chromatography of highly purified IgGs from MS patient on Chelex-100. This IgG fraction did not hydrolyze human MBP in the absence of Me(2+) ions but was activated after addition of Me(2+) ions: Mg(2+)>Mn(2+)>Cu(2+)>Ca(2+). Proteolytic activities of IgGs from other MS patients were also activated by other metal ions (Ni(2+), Fe(2+), Co(2+), Zn(2+), Pb(2+), and Co(2+)) and especially Ni(2+). Ni(2+)-activated IgGs were separated into distinct MBP-hydrolyzing fractions by chromatography on HiTraptrade mark Chelating Sepharose charged with Ni(2+). Detection of Mg(2+)-dependent proteolytic activity in the SDS-PAGE area corresponding only to IgG provided direct evidence that IgG from sera of MS patients possesses metal-dependent human MBP-hydrolyzing activity. Observed properties of MS abzymes distinguish them from other known mammalian metalloproteases and demonstrate their pronounced catalytic diversity. Metal-dependent IgGs from MS patients represent the first example of abzymes with metal-dependent proteolytic activity.
Asunto(s)
Anticuerpos Catalíticos/metabolismo , Metales Alcalinotérreos/farmacología , Metales Pesados/farmacología , Esclerosis Múltiple/inmunología , Proteína Básica de Mielina/metabolismo , Adolescente , Adulto , Anticuerpos Catalíticos/farmacología , Femenino , Humanos , Hidrólisis , Masculino , Persona de Mediana Edad , Esclerosis Múltiple/sangre , Proteína Básica de Mielina/química , Proteína Básica de Mielina/efectos de los fármacosRESUMEN
Although hyperpolarization-activated cyclic nucleotide-gated cation (HCN) channels and the corresponding h-current (Ih) have been shown to fundamentally shape the activity pattern in the thalamocortical network, little is known about their function in local circuit GABAergic interneurons (IN) of the dorsal part of the lateral geniculate nucleus (dLGN). By combining electrophysiological, molecular biological, immunohistochemical and cluster analysis, we characterized the properties of Ih and the expression profile of HCN channels in IN. Passive and active electrophysiological properties of IN differed. Two subclasses of IN were resolved by unsupervised cluster analysis. Small cells were characterized by depolarized resting membrane potentials (RMP), stronger anomalous rectification, higher firing frequency of faster action potentials (APs), appearance of rebound bursting, and higher Ih current density compared to the large IN. The depolarization exerted by sustained HCN channel activity facilitated neuronal firing. In addition to cyclic nucleotides, Ih in IN was modulated by PIP2 probably based on the abundant expression of the HCN3 isoform. Furthermore, only IN with larger cell diameters expressed neuronal nitric oxide synthase (nNOS). It is discussed that Ih in IN is modulated by neurotransmitters present in the thalamus and that the specific properties of Ih in these cells closely reflect their modulatory options.
Asunto(s)
Potenciales de Acción , Neuronas GABAérgicas/clasificación , Neuronas GABAérgicas/fisiología , Cuerpos Geniculados/citología , Canales Regulados por Nucleótidos Cíclicos Activados por Hiperpolarización/análisis , Interneuronas/clasificación , Interneuronas/fisiología , Animales , RatonesRESUMEN
The thalamocortical network is characterized by rhythmic burst activity during natural sleep and tonic single-spike activity during wakefulness. The change between these two activity modes is partially governed by transmitters acting on leak K+ currents in the thalamus, although the nature of the constituting ion channels is not yet known. In the present study, the contribution of members of the two-pore domain K+ channel family to the leak current was investigated using whole-cell patch-clamp techniques and molecular biological techniques. RT-PCR and in situ hybridization revealed the expression of TWIK-related acid-sensitive K+ channel 1 (TASK 1) and TASK3 channels in the rat dLGN. Voltage-clamp recordings of thalamocortical relay neurons in slice preparations demonstrated the existence of a current component sensitive to the TASK channel blocker bupivacaine, which reversed at the presumed K+ equilibrium potential, showed outward rectification, and contributed approximately 40% to the standing outward current at depolarized values of the membrane potential (-28 mV). The pharmacological profile was indicative of TASK channels, in that the current was sensitive to changes in extracellular pH, reduced by muscarine and increased by halothane, and these effects were occluded by a near-maximal action of bupivacaine. Pharmacological manipulation of this current under current-clamp conditions resulted in a shift between burst and tonic firing modes. It is concluded that TASK1 and TASK3 channels contribute to the muscarine- and halothane-sensitive conductance in thalamocortical relay neurons, thereby contributing to the change in the activity mode of thalamocortical networks observed during the sleep-wake cycle and on application of inhalational anesthetics.
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Proteínas del Tejido Nervioso/metabolismo , Neuronas/fisiología , Canales de Potasio de Dominio Poro en Tándem , Canales de Potasio/metabolismo , Tálamo/fisiología , Anestésicos por Inhalación/farmacología , Animales , Bario/farmacología , Bupivacaína/farmacología , Cuerpos Geniculados/metabolismo , Halotano/farmacología , Hibridación in Situ , Técnicas In Vitro , Potenciales de la Membrana/efectos de los fármacos , Potenciales de la Membrana/fisiología , Proteínas del Tejido Nervioso/antagonistas & inhibidores , Proteínas del Tejido Nervioso/genética , Neuronas/efectos de los fármacos , Neuronas/metabolismo , Técnicas de Placa-Clamp , Potasio/metabolismo , Canales de Potasio/biosíntesis , Canales de Potasio/efectos de los fármacos , Canales de Potasio/genética , Ratas , Ratas Long-Evans , Receptores Muscarínicos/biosíntesis , Receptores Muscarínicos/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Sueño/fisiología , Tálamo/citología , Tálamo/metabolismo , Vías Visuales/fisiología , Vigilia/fisiologíaRESUMEN
BACKGROUND AND PURPOSE: The existence of functional K(v)7 channels in thalamocortical (TC) relay neurons and the effects of the K(+)-current termed M-current (I(M)) on thalamic signal processing have long been debated. Immunocytochemical evidence suggests their presence in this brain region. Therefore, we aimed to verify their existence, pharmacological properties and function in regulating activity in neurons of the ventrobasal thalamus (VB). EXPERIMENTAL APPROACH: Characterization of K(v)7 channels was performed by combining in vitro, in vivo and in silico techniques with a pharmacological approach. Retigabine (30 µM) and XE991 (20 µM), a specific K(v)7 channel enhancer and blocker, respectively, were applied in acute brain slices during electrophysiological recordings. The effects of intrathalamic injection of retigabine (3 mM, 300 nL) and/or XE991 (2 mM, 300 nL) were investigated in freely moving animals during hot-plate tests by recording behaviour and neuronal activity. KEY RESULTS: K(v)7.2 and K(v)7.3 subunits were found to be abundantly expressed in TC neurons of mouse VB. A slow K(+)-current with properties of IM was activated by retigabine and inhibited by XE991. K(v)7 channel activation evoked membrane hyperpolarization, a reduction in tonic action potential firing, and increased burst firing in vitro and in computational models. Single-unit recordings and pharmacological intervention demonstrated a specific burst-firing increase upon I(M) activation in vivo. A K(v)7 channel-mediated increase in pain threshold was associated with fewer VB units responding to noxious stimuli, and increased burst firing in responsive neurons. CONCLUSIONS AND IMPLICATIONS: K(v)7 channel enhancement alters somatosensory activity and may reflect an anti-nociceptive mechanism during acute pain processing.
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Dolor Agudo/fisiopatología , Canales de Potasio KCNQ/metabolismo , Tálamo/efectos de los fármacos , Potenciales de Acción , Animales , Antracenos/farmacología , Encéfalo/efectos de los fármacos , Encéfalo/metabolismo , Carbamatos/farmacología , Modelos Animales de Enfermedad , Canal de Potasio KCNQ2/metabolismo , Canal de Potasio KCNQ3/metabolismo , Ratones , Ratones Endogámicos C57BL , Neuronas/efectos de los fármacos , Umbral del Dolor/fisiología , Fenilendiaminas/farmacología , Tálamo/metabolismoRESUMEN
Rats of the Wistar Albino Glaxo/Rij (WAG/Rij) strain show symptoms resembling human absence epilepsy. Thalamocortical neurons of WAG/Rij rats are characterized by an increased HCN1 expression, a negative shift in I h activation curve, and an altered responsiveness of I h to cAMP. We cloned HCN1 channels from rat thalamic cDNA libraries of the WAG/Rij strain and found an N-terminal deletion of 37 amino acids. In addition, WAG-HCN1 has a stretch of six amino acids, directly following the deletion, where the wild-type sequence (GNSVCF) is changed to a polyserine motif. These alterations were found solely in thalamus mRNA but not in genomic DNA. The truncated WAG-HCN1 was detected late postnatal in WAG/Rij rats and was not passed on to rats obtained from pairing WAG/Rij and non-epileptic August Copenhagen Irish rats. Heterologous expression in Xenopus oocytes revealed 2.2-fold increased current amplitude of WAG-HCN1 compared to rat HCN1. While WAG-HCN1 channels did not have altered current kinetics or changed regulation by protein kinases, fluorescence imaging revealed a faster and more pronounced surface expression of WAG-HCN1. Using co-expression experiments, we found that WAG-HCN1 channels suppress heteromeric HCN2 and HCN4 currents. Moreover, heteromeric channels of WAG-HCN1 with HCN2 have a reduced cAMP sensitivity. Functional studies revealed that the gain-of-function of WAG-HCN1 is not caused by the N-terminal deletion alone, thus requiring a change of the N-terminal GNSVCF motif. Our findings may help to explain previous observations in neurons of the WAG/Rij strain and indicate that WAG-HCN1 may contribute to the genesis of absence seizures in WAG/Rij rats.
RESUMEN
IgG and IgM antibodies from the sera of patients with multiple sclerosis (MS) were found to possess amylolytic activity hydrolyzing alpha-(1-->4)-glucosyl linkages of maltooligosaccharides, glycogen, and several artificial substrates. Individual IgM fractions isolated from 54 analyzed patients with the clinically definite diagnoses of MS had approximately three orders of magnitude higher specific amylolytic activity than that for healthy donors, whereas IgG from only a few patients had high amylolytic activity. Strict criteria were used to prove that the amylolytic activity of IgMs and IgGs is their intrinsic property and is not due to any enzyme contamination. Fab fragments produced from IgM and IgG fractions of the MS patients displayed the same amylolytic activity. IgMs from various patients demonstrated different modes of action in hydrolyzing maltooligosaccharides.
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Anticuerpos Catalíticos/metabolismo , Inmunoglobulina G/metabolismo , Inmunoglobulina M/metabolismo , Esclerosis Múltiple/enzimología , Esclerosis Múltiple/inmunología , Adulto , Amilasas/metabolismo , Anticuerpos Catalíticos/sangre , Anticuerpos Catalíticos/química , Electroforesis en Gel de Poliacrilamida , Femenino , Humanos , Hidrólisis , Inmunoglobulina G/sangre , Inmunoglobulina G/química , Inmunoglobulina M/sangre , Inmunoglobulina M/química , Masculino , Persona de Mediana Edad , Esclerosis Múltiple/sangre , Oligosacáridos/metabolismoRESUMEN
Adenylyl cyclases (ACs) synthesize the second messenger cyclic AMP (cAMP) which influences the function of multiple ion channels. Former studies point to a malfunction of cAMP-dependent ion channel regulation in thalamocortical relay neurons that contribute to the development of the absence epileptic phenotype of a rat genetic model (WAG/Rij). Here, we provide detailed information about the thalamic gene and protein expression of Ca(2+)/calmodulin-activated AC isoforms in rat thalamus. Data from WAG/Rij were compared to those from non-epileptic controls (August-Copenhagen Irish rats) to elucidate whether differential expression of ACs contributes to the dysregulation of thalamocortical activity. At one postnatal stage (P21), we found the gene expression of two specific Ca(2+)-activated AC isoforms (AC-1 and AC-3) to be significantly down-regulated in epileptic tissue, and we identified the isoform AC-1 to be the most prominent one in both strains. However, Western blot data and analysis of enzymatic AC activity revealed no differences between the two strains. While basal AC activity was low, cAMP production was boosted by application of a forskolin derivative up to sevenfold. Despite previous hints pointing to a major contribution of ACs, the presented data show that there is no apparent causality between AC activity and the occurrence of the epileptic phenotype.
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Adenilil Ciclasas/genética , Epilepsia Tipo Ausencia/enzimología , Epilepsia Tipo Ausencia/genética , Tálamo/enzimología , Tálamo/fisiología , Adenilil Ciclasas/metabolismo , Animales , Corteza Cerebral/citología , Corteza Cerebral/enzimología , AMP Cíclico/metabolismo , Modelos Animales de Enfermedad , Epilepsia Tipo Ausencia/fisiopatología , Regulación Enzimológica de la Expresión Génica/fisiología , Vías Nerviosas/citología , Vías Nerviosas/enzimología , Fenotipo , Ratas , Ratas Endogámicas , Ratas Mutantes , Tálamo/citologíaRESUMEN
Neuronal high-voltage-activated (HVA) Ca(2+) channels are rapidly inactivated by a mechanism that is termed Ca(2+)-dependent inactivation (CDI). In this study we have shown that ß-adrenergic receptor (ßAR) stimulation inhibits CDI in rat thalamocortical (TC) relay neurons. This effect can be blocked by inhibition of cAMP-dependent protein kinase (PKA) with a cell-permeable inhibitor (myristoylated protein kinase inhibitor-(14-22)-amide) or A-kinase anchor protein (AKAP) St-Ht31 inhibitory peptide, suggesting a critical role of these molecules downstream of the receptor. Moreover, inhibition of protein phosphatases (PP) with okadaic acid revealed the involvement of phosphorylation events in modulation of CDI after ßAR stimulation. Double fluorescence immunocytochemistry and pull down experiments further support the idea that modulation of CDI in TC neurons via ßAR stimulation requires a protein complex consisting of Ca(V)1.2, PKA and proteins from the AKAP family. All together our data suggest that AKAPs mediate targeting of PKA to L-type Ca(2+) channels allowing their phosphorylation and thereby modulation of CDI.
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Canales de Calcio Tipo L/química , Calcio/química , Receptores Adrenérgicos beta/metabolismo , Animales , Células COS , Corteza Cerebral/patología , Chlorocebus aethiops , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Inmunohistoquímica/métodos , Neuronas/metabolismo , Ácido Ocadaico/farmacología , Fosforilación , Ratas , Ratas Long-Evans , Transducción de Señal , Tálamo/patología , Distribución TisularRESUMEN
In thalamocortical relay (TC) neurons, G-protein-coupled receptors play an important part in the control of activity modes. A conditional Gα(q) knockout on the background of a constitutive Gα(11) knockout (Gα(q)/Gα(11) (-/-)) was used to determine the contribution of Gq/G11 family G-proteins to metabotropic serotonin (5-HT) and glutamate (Glu) function in the dorsal part of the lateral geniculate nucleus (dLGN). In control mice, current clamp recordings showed that α-m-5-HT induced a depolarization of V(rest) which was sufficient to suppress burst firing. This depolarization was concentration-dependent (100 µM: +6 ± 1 mV, n = 10; 200 µM: +10 ± 1 mV, n = 7) and had a conditioning effect on the activation of other Gα(q)-mediated pathways. The depolarization was significantly reduced in Gα(q)/Gα(11) (-/-) (100 µM: 3 ± 1 mV, n = 11; 200 µM: 5 ± 1 mV, n = 6) and was apparently insufficient to suppress burst firing. Activating Gα(q)-coupled muscarinic receptors affected the magnitude of α-m-5-HT-induced effects in a reciprocal manner. Furthermore, the depolarizing effect of mGluR1 agonists was significantly reduced in Gα(q)/Gα(11) (-/-) mice. Immunohistochemical stainings revealed binding of 5-HT(2C)R- and mGluR1α-, but not of 5-HT(2A)R-specific antibodies in the dLGN of Gα(q)/Gα(11) (-/-) mice. In conclusion, these findings demonstrate that transmitters of ascending brainstem fibers and corticofugal fibers both signal via a central element in the form of Gq/G11-mediated pathways to control activity modes in the TC system.