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
Myelin loss is a severe pathological hallmark common to a number of neurodegenerative diseases, including multiple sclerosis (MS). Demyelination in the central nervous system appears in the form of lesions affecting both white and gray matter structures. The functional consequences of demyelination on neuronal network and brain function are not well understood. Current therapeutic strategies for ameliorating the course of such diseases usually focus on promoting remyelination, but the effectiveness of these approaches strongly depends on the timing in relation to the disease state. In this study, we sought to characterize the time course of sensory and behavioral alterations induced by de- and remyelination to establish a rational for the use of remyelination strategies. By taking advantage of animal models of general and focal demyelination, we tested the consequences of myelin loss on the functionality of the auditory thalamocortical system: a well-studied neuronal network consisting of both white and gray matter regions. We found that general demyelination was associated with a permanent loss of the tonotopic cortical organization in vivo, and the inability to induce tone-frequency-dependent conditioned behaviors, a status persisting after remyelination. Targeted, focal lysolecithin-induced lesions in the white matter fiber tract, but not in the gray matter regions of cortex, were fully reversible at the morphological, functional and behavioral level. These findings indicate that remyelination of white and gray matter lesions have a different functional regeneration potential, with the white matter being able to regain full functionality while cortical gray matter lesions suffer from permanently altered network function. Therefore therapeutic interventions aiming for remyelination have to consider both region- and time-dependent strategies.
Asunto(s)
Corteza Cerebral/fisiopatología , Enfermedades Desmielinizantes/fisiopatología , Red Nerviosa/fisiopatología , Inmunidad Adaptativa , Animales , Conducta Animal , Cuprizona , Enfermedades Desmielinizantes/inducido químicamente , Enfermedades Desmielinizantes/psicología , Electrodos Implantados , Sustancia Gris/patología , Lisofosfatidilcolinas , Ratones , Ratones Endogámicos C57BL , Vaina de Mielina/patología , Recuperación de la Función , Sensación , Sustancia Blanca/patologíaRESUMEN
As part of the extended amygdala network, the bed nucleus of the stria terminalis (BNST) was shown to be critically involved in processing sustained fear responses to diffuse and unpredictable threats. However, neuronal activity patterns in relation to sustained components of the fear response remain elusive, so far. We used a fear training paradigm with unpredictable pairing of conditioned and unconditioned stimuli allowing distinction between phasic and sustained components of conditioned fear, and recorded single units in the anterolateral part of the BNST (BNSTal) in freely behaving mice. An objective, non-biased cluster-analysis was performed for each identified single unit on specific waveform-, activity-, stimulus-dependent and LFP-related parameters. The analysis revealed three distinct neuronal subpopulations of biphasic-, sustained fear on- and fear off-neurons. Results show that activities of biphasic- and sustained fear on-neurons temporally coincide with the shift from phasic to sustained components of the fear response. Presentation of non-conditioned auditory stimuli resulted in a variety of neuronal responses in BNSTal with no indication of biphasic response profiles. It is suggested that fear conditioning sharpens neuronal response profiles in BNSTal with biphasic-cells signaling phasic and sustained fear. These results confirm the pivotal role of BNST in processing sustained fear on the neuronal level, thereby complementing pharmacological experimental animal and human imaging data.
Asunto(s)
Condicionamiento Clásico/fisiología , Miedo/fisiología , Neuronas/fisiología , Núcleos Septales/fisiología , Animales , Conducta Animal/fisiología , Fenómenos Electrofisiológicos , Masculino , Ratones , Ratones Endogámicos C57BLRESUMEN
Although various types of ion channels are known to have an impact on human T cell effector functions, their exact mechanisms of influence are still poorly understood. The patch clamp technique is a well-established method for the investigation of ion channels in neurons and T cells. However, small cell sizes and limited selectivity of pharmacological blockers restrict the value of this experimental approach. Building a realistic T cell computer model therefore can help to overcome these kinds of limitations as well as reduce the overall experimental effort. The computer model introduced here was fed off ion channel parameters from literature and new experimental data. It is capable of simulating the electrophysiological behaviour of resting and activated human CD4(+) T cells under basal conditions and during extracellular acidification. The latter allows for the very first time to assess the electrophysiological consequences of tissue acidosis accompanying most forms of inflammation.
Asunto(s)
Simulación por Computador , Enfermedad , Fenómenos Electrofisiológicos , Salud , Linfocitos T/citología , Linfocitos T CD4-Positivos/metabolismo , Calcio/metabolismo , Cationes , Humanos , Concentración de Iones de Hidrógeno , Activación del Canal Iónico , Canales Iónicos/metabolismo , Potenciales de la Membrana , Modelos Biológicos , Potasio/metabolismo , Médula Espinal/metabolismoRESUMEN
KEY POINTS: During the behavioural states of sleep and wakefulness thalamocortical relay neurons fire action potentials in high frequency bursts or tonic sequences, respectively. The modulation of specific K(+) channel types, termed TASK and TREK, allows these neurons to switch between the two modes of activity. In this study we show that the signalling lipids phosphatidylinositol 4,5-bisphosphate (PIP2) and diacylglycerol (DAG), which are components of their membrane environment, switch on and shut off TREK and TASK channels, respectively. These channel modulations contribute to a better understanding of the molecular basis of the effects of neurotransmitters such as ACh which are released by the brainstem arousal system. The present report introduces PIP2 and DAG as new elements of signal transduction in the thalamus. The activity of two-pore domain potassium channels (K2P ) regulates the excitability and firing modes of thalamocortical (TC) neurons. In particular, the inhibition of two-pore domain weakly inwardly rectifying K(+) channel (TWIK)-related acid-sensitive K(+) (TASK) channels and TWIK-related K(+) (TREK) channels, as a consequence of the stimulation of muscarinic ACh receptors (MAChRs) which are coupled to phosphoinositide-specific phospholipase C (PLCß), induces a shift from burst to tonic firing. By using a whole cell patch-clamp approach, the contribution of the membrane-bound second messenger molecules phosphatidylinositol 4,5-bisphosphate (PIP2 ) and diacylglycerol (DAG) acting downstream of PLCß was probed. The standing outward current (ISO ) was used to monitor the current through TASK and TREK channels in TC neurons. By exploiting different manoeuvres to change the intracellular PIP2 level in TC neurons, we here show that the scavenging of PIP2 (by neomycin) results in an increased muscarinic effect on ISO whereas increased availability of PIP2 (inclusion to the patch pipette; histone-based carrier) decreased muscarinic signalling. The degree of muscarinic inhibition specifically depends on phosphatidylinositol phosphate (PIP) and PIP2 but no other phospholipids (phosphatidic acid, phosphatidylserine). The use of specific blockers revealed that PIP2 is targeting TREK but not TASK channels. Furthermore, we demonstrate that the inhibition of TASK channels is induced by the application of the DAG analogue 1-oleoyl-2-acetyl-sn-glycerol (OAG). Under current clamp conditions the activation of MAChRs and PLCß as well as the application of OAG resulted in membrane depolarization, while PIP2 application via histone carrier induced a hyperpolarization. These results demonstrate a differential role of PIP2 and DAG in K2P channel modulation in native neurons which allows a fine-tuned inhibition of TREK (via PIP2 depletion) and TASK (via DAG) channels following MAChR stimulation.
Asunto(s)
Diglicéridos/fisiología , Fosfatidilinositol 4,5-Difosfato/fisiología , Canales de Potasio de Dominio Poro en Tándem/fisiología , Tálamo/fisiología , Animales , Femenino , Masculino , Ratones Endogámicos C57BL , Ratones Noqueados , Proteínas del Tejido Nervioso , Neuronas/fisiología , Ratas Long-Evans , Fosfolipasas de Tipo C/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
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
A variety of ion channels are supposed to orchestrate the homoeostatic volume regulation in T lymphocytes. However, the relative contribution of different potassium channels to the osmotic volume regulation and in particular to the regulatory volume decrease (RVD) in T cells is far from clear. This study explores a putative role of the newly identified K(2P) channels (TASK1, TASK2, TASK3 and TRESK) along with the voltage-gated potassium channel K(V)1.3 and the calcium-activated potassium channel K(Ca)3.1 in the RVD of murine T lymphocytes, using genetic and pharmacological approaches. K(2P) channel knockouts exerted profound effects on the osmotic properties of murine T lymphocytes, as revealed by reduced water and RVD-related solute permeabilities. Moreover, both genetic and pharmacological data proved a key role of K(V)1.3 and TASK2 channels in the RVD of murine T cells exposed to hypotonic saline. Our experiments demonstrate a leading role of potassium channels in the osmoregulation of T lymphocytes under different conditions. In summary, the present study sheds new light on the complex and partially redundant network of potassium channels involved in the basic physiological process of the cellular volume homeostasis and extends the repertoire of potassium channels by the family of K(2P) channels.
Asunto(s)
Tamaño de la Célula , Canal de Potasio Kv1.3/metabolismo , Canales de Potasio de Dominio Poro en Tándem/metabolismo , Linfocitos T/metabolismo , Equilibrio Hidroelectrolítico , Animales , Tamaño de la Célula/efectos de los fármacos , Células Cultivadas , Soluciones Hipotónicas , Canal de Potasio Kv1.3/antagonistas & inhibidores , Ratones , Ratones Endogámicos C3H , Ratones Endogámicos C57BL , Ratones Noqueados , Modelos Biológicos , Proteínas del Tejido Nervioso/metabolismo , Bloqueadores de los Canales de Potasio/farmacología , Canales de Potasio/metabolismo , Canales de Potasio de Dominio Poro en Tándem/antagonistas & inhibidores , Canales de Potasio de Dominio Poro en Tándem/deficiencia , Canales de Potasio de Dominio Poro en Tándem/genética , Solución Salina Hipertónica , Linfocitos T/efectos de los fármacos , Factores de Tiempo , Equilibrio Hidroelectrolítico/efectos de los fármacosRESUMEN
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
Glatiramer acetate (GA) is a synthetic, random, basic copolymer capable of modulating adaptive T cell responses. In animal models of various inflammatory and degenerative central nervous system disorders, GA-induced T cells cross the blood-brain barrier, secrete high levels of anti-inflammatory cytokines and neurotrophins, and thus both reduce neuronal damage and promote neurogenesis. Recently, it has been suggested that GA itself may permeate the (impaired) blood-brain-barrier and directly protect neurons under conditions of inflammation-mediated neurodegeneration. To test this hypothesis, we examined the direct effects of GA on neuronal functionality and T cell-mediated neuronal apoptosis in culture, acute brain slices, and focal experimental autoimmune encephalomyelitis. GA caused a depolarization of the resting membrane potential and led to an immediate impairment of action potential generation in neurons. Moreover, GA-incubated neurons underwent dose-dependent apoptosis. Apoptosis of ovalbumin peptide-loaded major histocompatibility complex class I-expressing neurons induced by ovalbumin-specific effector T cells could be reduced by pre-incubation of T cells, but not neurons with GA. Similar results could be found using acute brain slices. In focal experimental autoimmune encephalomyelitis, lesion size and neuronal apoptosis could be limited by pretreating rats with GA, whereas intracerebral GA application into the inflammatory lesion had no effect on neuronal survival. Our data suggest that GA attenuates adaptive pro-inflammatory T cell responses, but does not exert direct neuroprotective effects.
Asunto(s)
Inflamación/patología , Neuronas/efectos de los fármacos , Péptidos/farmacología , Linfocitos T/efectos de los fármacos , Animales , Muerte Celular/efectos de los fármacos , Muerte Celular/inmunología , Células Cultivadas , Citoprotección/efectos de los fármacos , Citoprotección/inmunología , Regulación hacia Abajo/efectos de los fármacos , Regulación hacia Abajo/inmunología , Evaluación Preclínica de Medicamentos , Embrión de Mamíferos , Encefalomielitis Autoinmune Experimental/inmunología , Encefalomielitis Autoinmune Experimental/metabolismo , Encefalomielitis Autoinmune Experimental/patología , Acetato de Glatiramer , Inflamación/inmunología , Inflamación/metabolismo , Mediadores de Inflamación/metabolismo , Potenciales de la Membrana/efectos de los fármacos , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Neuronas/inmunología , Neuronas/metabolismo , Neuronas/patología , Ratas , Ratas Transgénicas , Linfocitos T/inmunología , Linfocitos T/metabolismoRESUMEN
OBJECTIVE: Activation of T cells critically depends on potassium channels. We here characterize the impact of K(2P)5.1 (KCNK5; TASK2), a member of the 2-pore domain family of potassium channels, on T-cell function and demonstrate its putative relevance in a T-cell-mediated autoimmune disorder, multiple sclerosis (MS). METHODS: Expression of K(2P)5.1 was investigated on RNA and protein level in different immune cells and in MS patients' biospecimens (peripheral blood mononuclear cells, cerebrospinal fluid cells, brain tissue specimen). Functional consequences of K(2P)5.1 expression were analyzed using pharmacological modulation, small interfering RNA (siRNA), overexpression, electrophysiological recordings, and computer modeling. RESULTS: Human T cells constitutively express K(2P)5.1. After T-cell activation, a significant and time-dependent upregulation of K(2P)5.1 channel expression was observed. Pharmacological blockade of K(2P)5.1 or knockdown with siRNA resulted in reduced T-cell functions, whereas overexpression of K(2P)5.1 had the opposite effect. Electrophysiological recordings of T cells clearly dissected K(2P)5.1-mediated effects from other potassium channels. The pathophysiological relevance of these findings was demonstrated by a significant K(2P)5.1 upregulation in CD4(+) and CD8(+) T cells in relapsing/remitting MS (RRMS) patients during acute relapses as well as higher levels on CD8(+) T cells of clinically isolated syndrome, RRMS, and secondary progressive multiple sclerosis patients during clinically stable disease. T cells in the cerebrospinal fluid from MS patients exhibit significantly elevated K(2P)5.1 levels. Furthermore, K(2P)5.1-positive T cells can be found in inflammatory lesions in MS tissue specimens. INTERPRETATION: Selective targeting of K(2P)5.1 may hold therapeutic promise for MS and putatively other T-cell-mediated disorders.
Asunto(s)
Esclerosis Múltiple/fisiopatología , Canales de Potasio de Dominio Poro en Tándem/metabolismo , Linfocitos T/fisiología , Encéfalo/metabolismo , Complejo CD3/metabolismo , Linfocitos T CD4-Positivos/efectos de los fármacos , Linfocitos T CD4-Positivos/fisiología , Linfocitos T CD8-positivos/efectos de los fármacos , Linfocitos T CD8-positivos/fisiología , Línea Celular , Líquido Cefalorraquídeo , Humanos , Leucocitos Mononucleares/metabolismo , Potenciales de la Membrana/efectos de los fármacos , Potenciales de la Membrana/fisiología , Bloqueadores de los Canales de Potasio/farmacología , Canales de Potasio de Dominio Poro en Tándem/antagonistas & inhibidores , Canales de Potasio de Dominio Poro en Tándem/genética , ARN Mensajero/metabolismo , Linfocitos T/efectos de los fármacos , Regulación hacia ArribaRESUMEN
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
Cytotoxic CD8(+) T cells are considered important effector cells contributing to neuronal damage in inflammatory and degenerative CNS disorders. Using time-lapse video microscopy and two-photon imaging in combination with whole-cell patch-clamp recordings, we here show that major histocompatibility class I (MHC I)-restricted neuronal antigen presentation and T cell receptor specificity determine CD8(+) T-cell locomotion and neuronal damage in culture and hippocampal brain slices. Two separate functional consequences result from a direct cell-cell contact between antigen-presenting neurons and antigen-specific CD8(+) T cells. (1) An immediate impairment of electrical signaling in single neurons and neuronal networks occurs as a result of massive shunting of the membrane capacitance after insertion of channel-forming perforin (and probably activation of other transmembrane conductances), which is paralleled by an increase of intracellular Ca(2+) levels (within <10 min). (2) Antigen-dependent neuronal apoptosis may occur independently of perforin and members of the granzyme B cluster (within approximately 1 h), suggesting that extracellular effects can substitute for intracellular delivery of granzymes by perforin. Thus, electrical silencing is an immediate consequence of MHC I-restricted interaction of CD8(+) T cells with neurons. This mechanism is clearly perforin-dependent and precedes, but is not causally linked, to neuronal cell death.
Asunto(s)
Antígenos CD8/metabolismo , Comunicación Celular/fisiología , Neuronas/fisiología , Perforina/metabolismo , Linfocitos T/fisiología , Animales , Calcio/metabolismo , Muerte Celular/fisiología , Movimiento Celular/fisiología , Células Cultivadas , Capacidad Eléctrica , Genes MHC Clase I/fisiología , Granzimas/genética , Granzimas/metabolismo , Hipocampo/fisiología , Técnicas In Vitro , Espacio Intracelular/fisiología , Potenciales de la Membrana/fisiología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Vías Nerviosas/fisiología , Perforina/genética , Factores de TiempoRESUMEN
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
Hyperpolarization-activated cyclic nucleotide-gated cation (HCN) channels have important functions in controlling neuronal excitability and generating rhythmic oscillatory activity. The role of tetratricopeptide repeat-containing Rab8b-interacting protein (TRIP8b) in regulation of hyperpolarization-activated inward current, I h, in the thalamocortical system and its functional relevance for the physiological thalamocortical oscillations were investigated. A significant decrease in I h current density, in both thalamocortical relay (TC) and cortical pyramidal neurons was found in TRIP8b-deficient mice (TRIP8b-/-). In addition basal cAMP levels in the brain were found to be decreased while the availability of the fast transient A-type K+ current, I A, in TC neurons was increased. These changes were associated with alterations in intrinsic properties and firing patterns of TC neurons, as well as intrathalamic and thalamocortical network oscillations, revealing a significant increase in slow oscillations in the delta frequency range (0.5-4 Hz) during episodes of active-wakefulness. In addition, absence of TRIP8b suppresses the normal desynchronization response of the EEG during the switch from slow-wave sleep to wakefulness. It is concluded that TRIP8b is necessary for the modulation of physiological thalamocortical oscillations due to its direct effect on HCN channel expression in thalamus and cortex and that mechanisms related to reduced cAMP signaling may contribute to the present findings.
Asunto(s)
Corteza Cerebral/fisiología , Proteínas de la Membrana/metabolismo , Vías Nerviosas/fisiología , Peroxinas/metabolismo , Tálamo/fisiología , Potenciales de Acción/genética , Adenina/análogos & derivados , Adenina/farmacología , Inhibidores de Adenilato Ciclasa/farmacología , Animales , Fármacos Cardiovasculares/farmacología , Corteza Cerebral/citología , AMP Cíclico/farmacología , GMP Cíclico/análogos & derivados , GMP Cíclico/farmacología , Femenino , Canales Regulados por Nucleótidos Cíclicos Activados por Hiperpolarización/fisiología , Masculino , Proteínas de la Membrana/genética , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Modelos Neurológicos , Peroxinas/genética , Pirimidinas/farmacología , Bloqueadores de los Canales de Sodio/farmacología , Tetrodotoxina/farmacología , Tionucleótidos/farmacologíaRESUMEN
Multiple sclerosis is characterized by intermingled episodes of de- and remyelination and the occurrence of white- and grey-matter damage. To mimic the randomly distributed pathophysiological brain lesions observed in MS, we assessed the impact of focal white and grey matter demyelination on thalamic function by directing targeted lysolecithin-induced lesions to the capsula interna (CI), the auditory cortex (A1), or the ventral medial geniculate nucleus (vMGN) in mice. Pathophysiological consequences were compared with those of cuprizone treatment at different stages of demyelination and remyelination. Combining single unit recordings and auditory stimulation in freely behaving mice revealed changes in auditory response profile and electrical activity pattern in the thalamus, depending on the region of the initial insult and the state of remyelination. Cuprizone-induced general demyelination significantly diminished vMGN neuronal activity and frequency-specific responses. Targeted lysolecithin-induced lesions directed either to A1 or to vMGN revealed a permanent impairment of frequency-specific responses, an increase in latency of auditory responses and a reduction in occurrence of burst firing in vMGN neurons. These findings indicate that demyelination of grey matter areas in the thalamocortical system permanently affects vMGN frequency specificity and the prevalence of bursting in the auditory thalamus.
Asunto(s)
Potenciales de Acción/fisiología , Enfermedades Desmielinizantes/patología , Tálamo/fisiopatología , Estimulación Acústica/métodos , Potenciales de Acción/efectos de los fármacos , Animales , Corteza Auditiva/efectos de los fármacos , Corteza Auditiva/fisiopatología , Cuprizona/toxicidad , Enfermedades Desmielinizantes/inducido químicamente , Enfermedades Desmielinizantes/tratamiento farmacológico , Enfermedades Desmielinizantes/fisiopatología , Modelos Animales de Enfermedad , Femenino , Lateralidad Funcional , Cuerpos Geniculados/patología , Gliosis/inducido químicamente , Gliosis/patología , Sustancia Gris/patología , Lisofosfatidilcolinas/farmacología , Ratones , Ratones Endogámicos C57BL , Inhibidores de la Monoaminooxidasa/toxicidad , Proteína Proteolipídica de la Mielina/metabolismo , Neuronas/efectos de los fármacos , Neuronas/fisiología , Psicoacústica , Tálamo/efectos de los fármacosRESUMEN
Alterations in cortical cellular organization, network functionality, as well as cognitive and locomotor deficits were recently suggested to be pathological hallmarks in multiple sclerosis and corresponding animal models as they might occur following demyelination. To investigate functional changes following demyelination in a well-defined, topographically organized neuronal network, in vitro and in vivo, we focused on the primary auditory cortex (A1) of mice in the cuprizone model of general de- and remyelination. Following myelin loss in this model system, the spatiotemporal propagation of incoming stimuli in A1 was altered and the hierarchical activation of supra- and infragranular cortical layers was lost suggesting a profound effect exerted on neuronal network level. In addition, the response latency in field potential recordings and voltage-sensitive dye imaging was increased following demyelination. These alterations were accompanied by a loss of auditory discrimination abilities in freely behaving animals, a reduction of the nuclear factor-erythroid 2-related factor-2 (Nrf-2) protein in the nucleus in histological staining and persisted during remyelination. To find new strategies to restore demyelination-induced network alteration in addition to the ongoing remyelination, we tested the cytoprotective potential of dimethyl fumarate (DMF). Therapeutic treatment with DMF during remyelination significantly modified spatiotemporal stimulus propagation in the cortex, reduced the cognitive impairment, and prevented the demyelination-induced decrease in nuclear Nrf-2. These results indicate the involvement of anti-oxidative mechanisms in regulating spatiotemporal cortical response pattern following changes in myelination and point to DMF as therapeutic compound for intervention.
Asunto(s)
Corteza Auditiva/patología , Dimetilfumarato/uso terapéutico , Inmunosupresores/uso terapéutico , Esclerosis Múltiple/tratamiento farmacológico , Factor 2 Relacionado con NF-E2/metabolismo , Remielinización/efectos de los fármacos , Animales , Ansiedad , Corteza Auditiva/diagnóstico por imagen , Escala de Evaluación de la Conducta , Cuprizona/farmacología , Dimetilfumarato/administración & dosificación , Modelos Animales de Enfermedad , Estimulación Eléctrica , Inmunosupresores/administración & dosificación , Locomoción/efectos de los fármacos , Ratones , Ratones Endogámicos C57BL , Esclerosis Múltiple/inducido químicamente , Esclerosis Múltiple/diagnóstico por imagen , Red Nerviosa/diagnóstico por imagen , Red Nerviosa/fisiopatología , Plasticidad Neuronal/efectos de los fármacos , Imagen de Colorante Sensible al VoltajeRESUMEN
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 simulation system NEURON is a common research tool for constructing structurally and functionally realistic models of neuronal systems. NEURON allows the development of simulations at any level of complexity, from subcellular components to single cells, cellular networks, and system-level models. Focusing on an in vitro cell model of a single, acutely isolated thalamic neuron, we used the simulation environment to address and to discuss the following questions in an undergraduate course: (i) Which parts are required to design a single compartment with passive electrical properties? (ii) Which components are necessary to model a single action potential or a train of action potentials? (iii) What can we learn from voltage-clamp and current-clamp experiments? (iv) What kind of cellular parameters are accessible from the modeling data? (v) What are the differences between single-compartment models and multi-compartment models? (vi) What are the advantages and disadvantages of artificial cell models? (vii) Can realistic modeling open up new strategies to discover the way that neurons process information?
RESUMEN
Olfactory ensheathing cells (OECs) are a specialised type of glial cells, supporting axon growth and guidance during development and regeneration of the olfactory nerve and the nerve layer of the olfactory bulb. We measured calcium signalling in OECs in olfactory bulb in-toto preparations using confocal and epifluorescence microscopy and the calcium indicator Fluo-4. We identified two subpopulations of olfactory bulb OECs: OECs in the outer sublamina of the nerve layer responded to purinergic neurotransmitters such as adenosine triphosphate with calcium transients, while OECs in the inner sublamina of the nerve layer did not respond to neurotransmitters. However, the latter generated spontaneous calcium waves that covered hundreds of cells. These calcium waves persisted in the presence of tetrodotoxin and in calcium-free saline, but were abolished after calcium store depletion with cyclopiazonic acid or inositol trisphosphate receptor blockage with 2-APB. Calcium waves could be triggered by laser photolysis of caged inositol trisphosphate. Blocking purinoceptors with PPADS had no effect on calcium wave propagation, whereas blocking gap junctions with carbenoxolone or meclofenamic acid entirely suppressed calcium waves. Increasing calcium buffer capacity in OECs with NP-EGTA ("caged" Ca(2+)) prevented calcium wave generation, and laser photolysis of NP-EGTA in a small group of OECs resulted in a calcium increase in the irradiated cells followed by a calcium wave. We conclude that calcium waves in OECs can be initiated by calcium-induced calcium release via InsP3 receptors and propagate through gap junctions, while purinergic signalling is not involved.