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ABSTRACT: Induced pluripotent stem cells (iPSCs) have enabled the generation of various difficult-to-access cell types such as human nociceptors. A key challenge associated with human iPSC-derived nociceptors (hiPSCdNs) is their prolonged functional maturation. While numerous studies have addressed the expression of classic neuronal markers and ion channels in hiPSCdNs, the temporal development of key signaling cascades regulating nociceptor activity has remained largely unexplored. In this study, we used an immunocytochemical high-content imaging approach alongside electrophysiological staging to assess metabotropic and ionotropic signaling of large scale-generated hiPSCdNs across 70 days of in vitro differentiation. During this period, the resting membrane potential became more hyperpolarized, while rheobase, action potential peak amplitude, and membrane capacitance increased. After 70 days, hiPSCdNs exhibited robust physiological responses induced by GABA, pH shift, ATP, and capsaicin. Direct activation of protein kinase A type II (PKA-II) through adenylyl cyclase stimulation with forskolin resulted in PKA-II activation at all time points. Depolarization-induced activation of PKA-II emerged after 35 days of differentiation. However, effective inhibition of forskolin-induced PKA-II activation by opioid receptor agonists required 70 days of in vitro differentiation. Our results identify a pronounced time difference between early expression of functionally important ion channels and emergence of regulatory metabotropic sensitizing and desensitizing signaling only at advanced stages of in vitro cultivation, suggesting an independent regulation of ionotropic and metabotropic signaling. These data are relevant for devising future studies into the development and regulation of human nociceptor function and for defining time windows suitable for hiPSCdN-based drug discovery.
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Células Madre Pluripotentes Inducidas , Humanos , Analgésicos Opioides , Colforsina/farmacología , Nocicepción , Células Receptoras Sensoriales , Canales IónicosRESUMEN
Gangliogliomas (GGs), composed of dysmorphic neurons and neoplastic astroglia, represent the most frequent tumor entity associated with chronic recurrent epileptic seizures. So far, a systematic analysis of potential differences in neurochemical profiles of dysmorphic tumoral neurons as well as neurons of the peritumoral microenvironment (PTME) was hampered by the inability to unequivocally differentiate between the distinct neuronal components in human GG biopsies. Here, we have applied a novel GG mouse model that allows to clearly resolve the neurochemical profiles of GG-intrinsic versus PTME neurons. For this purpose, glioneuronal tumors in mice were induced by intraventricular in utero electroporation (IUE) of piggyBac-based plasmids for BRAFV600E and activated Akt (AktT308D/S473D, further referred to as AktDD) and analyzed neurochemically by immunocytochemistry against specific marker proteins. IUE of BRAFV600E/AktDD in mice resulted in tumors with the morphological features of human GGs. Our immunocytochemical analysis revealed a strong reduction of GABAARα1 immunoreactivity in the tumor compared to the PTME. In contrast, the extent of NMDAR1 immunoreactivity in the tumor appeared comparable to the PTME. Interestingly, tumor cells maintained the potential to express both receptors. Fittingly, the abundance of the presynaptic vesicular neurotransmitter transporters VGLUT1 and VGAT was also decreased in the tumor. Additionally, the fraction of parvalbumin and somatostatin nonneoplastic interneurons was reduced. In conclusion, changes in the levels of key proteins in neurotransmitter signaling suggest a loss of synapses and may thereby lead to neuronal network alterations in mouse GGs.
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Neoplasias Encefálicas , Epilepsia , Ganglioglioma , Humanos , Ratones , Animales , Ganglioglioma/complicaciones , Ganglioglioma/metabolismo , Ganglioglioma/patología , Convulsiones , Neuronas/metabolismo , Epilepsia/complicaciones , Neoplasias Encefálicas/complicaciones , Neoplasias Encefálicas/metabolismo , Neoplasias Encefálicas/patología , Microambiente TumoralRESUMEN
Maintaining an appropriate balance between excitation and inhibition is critical for neuronal information processing. Cortical neurons can cell-autonomously adjust the inhibition they receive to individual levels of excitatory input, but the underlying mechanisms are unclear. We describe that Ste20-like kinase (SLK) mediates cell-autonomous regulation of excitation-inhibition balance in the thalamocortical feedforward circuit, but not in the feedback circuit. This effect is due to regulation of inhibition originating from parvalbumin-expressing interneurons, while inhibition via somatostatin-expressing interneurons is unaffected. Computational modeling shows that this mechanism promotes stable excitatory-inhibitory ratios across pyramidal cells and ensures robust and sparse coding. Patch-clamp RNA sequencing yields genes differentially regulated by SLK knockdown, as well as genes associated with excitation-inhibition balance participating in transsynaptic communication and cytoskeletal dynamics. These data identify a mechanism for cell-autonomous regulation of a specific inhibitory circuit that is critical to ensure that a majority of cortical pyramidal cells participate in information coding.
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Células PiramidalesRESUMEN
Dendrites of hippocampal CA1 pyramidal cells amplify clustered glutamatergic input by activation of voltage-gated sodium channels and N-methyl-D-aspartate receptors (NMDARs). NMDAR activity depends on the presence of NMDAR co-agonists such as D-serine, but how co-agonists influence dendritic integration is not well understood. Using combinations of whole-cell patch clamp, iontophoretic glutamate application, two-photon excitation fluorescence microscopy and glutamate uncaging in acute rat and mouse brain slices we found that exogenous D-serine reduced the threshold of dendritic spikes and increased their amplitude. Triggering an astrocytic mechanism controlling endogenous D-serine supply via endocannabinoid receptors (CBRs) also increased dendritic spiking. Unexpectedly, this pathway was activated by pyramidal cell activity primarily in the theta range, which required HCN channels and astrocytic CB1Rs. Therefore, astrocytes close a positive and frequency-dependent feedback loop between pyramidal cell activity and their integration of dendritic input. Its disruption in mice led to an impairment of spatial memory, which demonstrated its behavioral relevance.
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Astrocitos , Región CA1 Hipocampal , Dendritas , Aprendizaje Espacial , Animales , Ratones , Ratas , Astrocitos/fisiología , Dendritas/fisiología , Ácido Glutámico/metabolismo , Células Piramidales/fisiología , Receptores de N-Metil-D-Aspartato/metabolismo , Serina/metabolismo , Aprendizaje Espacial/fisiología , Región CA1 Hipocampal/fisiologíaRESUMEN
OBJECTIVE: Inflammation of brain structures, in particular the hippocampal formation, can induce neuronal degeneration and be associated with increased excitability manifesting as propensity for repetitive seizures. An increase in the abundance of individual proinflammatory molecules including interleukin 1 beta has been observed in brain tissue samples of patients with pharmacoresistant temporal lobe epilepsy (TLE) and corresponding animal models. The NLRP3-inflammasome, a cytosolic protein complex, acts as a key regulator in proinflammatory innate immune signalling. Upon activation, it leads to the release of interleukin 1 beta and inflammation-mediated neurodegeneration. Transient brain insults, like status epilepticus (SE), can render hippocampi chronically hyperexcitable and induce segmental neurodegeneration. The underlying mechanisms are referred to as epileptogenesis. Here, we have tested the hypothesis that distinct NLRP3-dependent transcript and protein signalling dynamics are induced by SE and whether they differ between two classical SE models. We further correlated the association of NLRP3-related transcript abundance with convulsive activity in human TLE hippocampi of patients with and without associated neurodegenerative damage. METHODS: Hippocampal mRNA- and protein-expression of NLRP3 and associated signalling molecules were analysed longitudinally in pilocarpine- and kainic acid-induced SE TLE mouse models. Complementarily, we studied NLRP3 inflammasome-associated transcript patterns in epileptogenic hippocampi with different damage patterns of pharmacoresistant TLE patients that had undergone epilepsy surgery for seizure relief. RESULTS: Pilocarpine- and kainic acid-induced SE elicit distinct hippocampal Nlrp3-associated molecular signalling. Transcriptional activation of NLRP3 pathway elements is associated with seizure activity but independent of the particular neuronal damage phenotype in KA-induced and in human TLE hippocampi. SIGNIFICANCE: These data suggest highly dynamic inflammasome signalling in SE-induced TLE and highlight a vicious cycle associated with seizure activity. Our results provide promising perspectives for the inflammasome signalling pathway as a target for anti-epileptogenic and -convulsive therapeutic strategies. The latter may even applicable to a particularly broad spectrum of TLE patients with currently pharmacoresistant disease.
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Epilepsia del Lóbulo Temporal , Proteína con Dominio Pirina 3 de la Familia NLR , Enfermedades Neuroinflamatorias , Estado Epiléptico , Animales , Modelos Animales de Enfermedad , Epilepsia del Lóbulo Temporal/inducido químicamente , Epilepsia del Lóbulo Temporal/patología , Hipocampo/metabolismo , Humanos , Inflamasomas/metabolismo , Interleucina-1beta/metabolismo , Ácido Kaínico , Ratones , Proteína con Dominio Pirina 3 de la Familia NLR/genética , Proteína con Dominio Pirina 3 de la Familia NLR/metabolismo , Enfermedades Neuroinflamatorias/patología , Pilocarpina , Convulsiones/metabolismo , Estado Epiléptico/inducido químicamente , Estado Epiléptico/patologíaRESUMEN
The size and structure of the dendritic arbor play important roles in determining how synaptic inputs of neurons are converted to action potential output. The regulatory mechanisms governing the development of dendrites, however, are insufficiently understood. The evolutionary conserved Ste20/Hippo kinase pathway has been proposed to play an important role in regulating the formation and maintenance of dendritic architecture. A key element of this pathway, Ste20-like kinase (SLK), regulates cytoskeletal dynamics in non-neuronal cells and is strongly expressed throughout neuronal development. However, its function in neurons is unknown. We show that, during development of mouse cortical neurons, SLK has a surprisingly specific role for proper elaboration of higher, ≥ third-order dendrites both in male and in female mice. Moreover, we demonstrate that SLK is required to maintain excitation-inhibition balance. Specifically, SLK knockdown caused a selective loss of inhibitory synapses and functional inhibition after postnatal day 15, whereas excitatory neurotransmission was unaffected. Finally, we show that this mechanism may be relevant for human disease, as dysmorphic neurons within human cortical malformations revealed significant loss of SLK expression. Overall, the present data identify SLK as a key regulator of both dendritic complexity during development and inhibitory synapse maintenance.SIGNIFICANCE STATEMENT We show that dysmorphic neurons of human epileptogenic brain lesions have decreased levels of the Ste20-like kinase (SLK). Decreasing SLK expression in mouse neurons revealed that SLK has essential functions in forming the neuronal dendritic tree and in maintaining inhibitory connections with neighboring neurons.
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Corteza Cerebral/metabolismo , Dendritas/genética , Inhibición Neural/genética , Proteínas Serina-Treonina Quinasas/genética , Sinapsis/genética , Transmisión Sináptica/fisiología , Adolescente , Adulto , Anciano , Animales , Corteza Cerebral/patología , Niño , Preescolar , Dendritas/metabolismo , Dendritas/patología , Femenino , Células HEK293 , Humanos , Masculino , Ratones , Persona de Mediana Edad , Proteínas Serina-Treonina Quinasas/metabolismo , Sinapsis/metabolismo , Sinapsis/patología , Adulto JovenRESUMEN
OBJECTIVE: Many antiseizure drugs (ASDs) act on voltage-dependent sodium channels, and the molecular basis of these effects is well established. In contrast, how ASDs act on the level of neuronal networks is much less understood. METHODS: In the present study, we determined the effects of eslicarbazepine (S-Lic) on different types of inhibitory neurons, as well as inhibitory motifs. Experiments were performed in hippocampal slices from both sham-control and chronically epileptic pilocarpine-treated rats. RESULTS: We found that S-Lic causes an unexpected reduction of feed-forward inhibition in the CA1 region at high concentrations (300 µM), but not at lower concentrations (100 µM). Concurrently, 300 but not 100 µM S-Lic significantly reduced maximal firing rates in putative feed-forward interneurons located in the CA1 stratum radiatum of sham-control and epileptic animals. In contrast, feedback inhibition was not inhibited by S-Lic. Instead, application of S-Lic, in contrast to previous data for other drugs like carbamazepine (CBZ), resulted in a lasting potentiation of feedback inhibitory post-synaptic currents (IPSCs) only in epileptic and not in sham-control animals, which persisted after washout of S-Lic. We hypothesized that this plasticity of inhibition might rely on anti-Hebbian potentiation of excitatory feedback inputs onto oriens-lacunosum moleculare (OLM) interneurons, which is dependent on Ca2+ -permeable α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors. Indeed, we show that blocking Ca2+ -permeable AMPA receptors completely prevents upmodulation of feedback inhibition. SIGNIFICANCE: These results suggest that S-Lic affects inhibitory circuits in the CA1 hippocampal region in unexpected ways. In addition, ASD actions may not be sufficiently explained by acute effects on their target channels, rather, it may be necessary to take plasticity of inhibitory circuits into account.
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Anticonvulsivantes/farmacología , Región CA1 Hipocampal/efectos de los fármacos , Dibenzazepinas/farmacología , Epilepsia/fisiopatología , Interneuronas/efectos de los fármacos , Inhibición Neural/efectos de los fármacos , Células Piramidales/efectos de los fármacos , Adamantano/análogos & derivados , Adamantano/farmacología , Animales , Región CA1 Hipocampal/metabolismo , Región CA1 Hipocampal/fisiopatología , Calcio/metabolismo , Modelos Animales de Enfermedad , Relación Dosis-Respuesta a Droga , Epilepsia/inducido químicamente , Retroalimentación Fisiológica/efectos de los fármacos , Hipocampo/efectos de los fármacos , Hipocampo/metabolismo , Hipocampo/fisiopatología , Potenciales Postsinápticos Inhibidores/efectos de los fármacos , Interneuronas/metabolismo , Potenciación a Largo Plazo , Agonistas Muscarínicos/toxicidad , Plasticidad Neuronal , Neuronas/efectos de los fármacos , Neuronas/metabolismo , Pilocarpina/toxicidad , Ratas , Receptores AMPA/antagonistas & inhibidores , Receptores AMPA/metabolismoRESUMEN
Precise genome editing in combination with viral delivery systems provides a valuable tool for neuroscience research. Traditionally, the role of genes in neuronal circuits has been addressed by overexpression or knock-out/knock-down systems. However, those techniques do not manipulate the endogenous loci and therefore have limitations. Those constraints include that many genes exhibit extensive alternative splicing, which can be regulated by neuronal activity. This complexity cannot be easily reproduced by overexpression of one protein variant. The CRISPR activation and interference/inhibition systems (CRISPRa/i) directed to promoter sequences can modulate the expression of selected target genes in a highly specific manner. This strategy could be particularly useful for the overexpression of large proteins and for alternatively spliced genes, e.g., for studying large ion channels known to be affected in ion channelopathies in a variety of neurological diseases. Here, we demonstrate the feasibility of a newly developed CRISPRa/i toolbox to manipulate the promoter activity of the Cacna1h gene. Impaired, function of the low-voltage-activated T-Type calcium channel CaV3.2 is involved in genetic/mutational as well as acquired/transcriptional channelopathies that emerge with epileptic seizures. We show CRISPR-induced activation and inhibition of the Cacna1h locus in NS20Y cells and primary cortical neurons, as well as activation in mouse organotypic slice cultures. In future applications, the system offers the intriguing perspective to study functional effects of gain-of-function or loss-of-function variations in the Cacna1h gene in more detail. A better understanding of CaV3.2 channelopathies might result in a major advancement in the pharmacotherapy of CaV3.2 channelopathy diseases.
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OBJECTIVE: Limbic encephalitis (LE) comprises a spectrum of inflammatory changes in affected brain structures including the presence of autoantibodies and lymphoid cells. However, the potential of distinct lymphocyte subsets alone to elicit key clinicopathological sequelae of LE potentially inducing temporal lobe epilepsy (TLE) with chronic spontaneous seizures and hippocampal sclerosis (HS) is unresolved. METHODS: Here, we scrutinized pathogenic consequences emerging from CD8+ T cells targeting hippocampal neurons by recombinant adeno-associated virus-mediated expression of the model-autoantigen ovalbumin (OVA) in CA1 neurons of OT-I/RAG1-/- mice (termed "OVA-CD8+ LE model"). RESULTS: Viral-mediated antigen transfer caused dense CD8+ T cell infiltrates confined to the hippocampal formation starting on day 5 after virus transduction. Flow cytometry indicated priming of CD8+ T cells in brain-draining lymph nodes preceding hippocampal invasion. At the acute model stage, the inflammatory process was accompanied by frequent seizure activity and impairment of hippocampal memory skills. Magnetic resonance imaging scans at day 7 of the OVA-CD8+ LE model revealed hippocampal edema and blood-brain barrier disruption that converted into atrophy until day 40. CD8+ T cells specifically targeted OVA-expressing, SIINFEKL-H-2Kb -positive CA1 neurons and caused segmental apoptotic neurodegeneration, astrogliosis, and microglial activation. At the chronic model stage, mice exhibited spontaneous recurrent seizures and persisting memory deficits, and the sclerotic hippocampus was populated with CD8+ T cells escorted by NK cells. INTERPRETATION: These data indicate that a CD8+ T-cell-initiated attack of distinct hippocampal neurons is sufficient to induce LE converting into TLE-HS. Intriguingly, the role of CD8+ T cells exceeds neurotoxic effects and points to their major pathogenic role in TLE following LE. ANN NEUROL 2021;89:666-685.
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Linfocitos T CD8-positivos/patología , Epilepsia del Lóbulo Temporal/etiología , Epilepsia del Lóbulo Temporal/patología , Encefalitis Límbica/complicaciones , Encefalitis Límbica/patología , Animales , Barrera Hematoencefálica/patología , Región CA1 Hipocampal/patología , Epilepsia del Lóbulo Temporal/psicología , Hipocampo/patología , Proteínas de Homeodominio/genética , Encefalitis Límbica/psicología , Ganglios Linfáticos/patología , Imagen por Resonancia Magnética , Trastornos de la Memoria/etiología , Trastornos de la Memoria/psicología , Ratones Endogámicos C57BL , Ratones Noqueados , Neuronas/patología , Ovalbúmina/genética , Ovalbúmina/inmunología , Fragmentos de Péptidos/genética , Convulsiones/genética , Convulsiones/patologíaRESUMEN
Transgenic Cre-recombinase expressing mouse lines are widely used to express fluorescent proteins and opto-/chemogenetic actuators, making them a cornerstone of modern neuroscience. The investigation of interneurons in particular has benefitted from the ability to genetically target specific cell types. However, the specificity of some Cre driver lines has been called into question. Here, we show that nonspecific expression in a subset of hippocampal neurons can have substantial nonspecific functional effects in a somatostatin-Cre (SST-Cre) mouse line. Nonspecific targeting of CA3 pyramidal cells caused large optogenetically evoked excitatory currents in remote brain regions. Similar, but less severe patterns of nonspecific expression were observed in a widely used SST-IRES-Cre line, when crossed with a reporter mouse line. Viral transduction on the other hand yielded more specific expression but still resulted in nonspecific expression in a minority of pyramidal layer cells. These results suggest that a careful analysis of specificity is mandatory before the use of Cre driver lines for opto- or chemogenetic manipulation approaches.
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Región CA3 Hipocampal/citología , Región CA3 Hipocampal/metabolismo , Integrasas/biosíntesis , Interneuronas/metabolismo , Optogenética/métodos , Somatostatina/biosíntesis , Animales , Región CA3 Hipocampal/química , Expresión Génica , Integrasas/análisis , Integrasas/genética , Interneuronas/química , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Somatostatina/análisis , Somatostatina/genéticaRESUMEN
BACKGROUND: Neuropathic pain resulting from peripheral nerve lesions is a common medical condition, but current analgesics are often insufficient. The identification of key molecules involved in pathological pain processing is a prerequisite for the development of new analgesic drugs. Hyperexcitability of nociceptive DRG-neurons due to regulation of voltage-gated ion-channels is generally assumed to contribute strongly to neuropathic pain. There is increasing evidence, that T-type Ca2+-currents and in particular the Cav3.2 T-type-channel isoform play an important role in neuropathic pain, but experimental results are contradicting. PURPOSE: To clarify the role of T-type Ca2+-channels and in particular the Cav3.2 T-type-channel isoform in neuropathic pain. METHODS: The effect of partial sciatic nerve ligation (PNL) on pain behavior and the properties of T-type-currents in nociceptive DRG-neurons was tested in wild-type and Cav3.2-deficient mice. RESULTS: In wild-type mice, PNL of the sciatic nerve caused neuropathic pain and an increase of T-type Ca2+-currents in capsaicin-responsive neurons, while capsaicin-unresponsive neurons were unaffected. Pharmacological experiments revealed that this upregulation was due to an increase of a Ni2+-resistant Ca2+-current component, inconsistent with Cav3.2 up-regulation. Moreover, following PNL Cav3.2-deficient mice showed neuropathic pain behavior and an increase of T-Type Ca2+-currents indistinguishable to that of PNL treated wild-type mice. CONCLUSION: These data suggest that PNL induces an upregulation of T-Type Ca2+-currents in capsaicin-responsive DRG-neurons mediated by an increase of a Ni2+-insensitive current component (possibly Cav3.1 or Cav3.3). These findings provide relevance for the development of target specific analgesic drugs.
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OBJECTIVE: Pharmacoresistance is a problem affecting â¼30% of chronic epilepsy patients. An understanding of the mechanisms of pharmacoresistance requires a precise understanding of how antiepileptic drugs interact with their targets in control and epileptic tissue. Although the effects of (S)-licarbazepine (S-Lic) on sodium channel fast inactivation are well understood and have revealed maintained activity in epileptic tissue, it is not known how slow inactivation processes are affected by S-Lic in epilepsy. METHODS: We have used voltage clamp recordings in isolated dentate granule cells (DGCs) and cortical pyramidal neurons of control versus chronically epileptic rats (pilocarpine model of epilepsy) and in DGCs isolated from hippocampal specimens from temporal lobe epilepsy patients to examine S-Lic effects on sodium channel slow inactivation. RESULTS: S-Lic effects on entry into and recovery from slow inactivation were negligible, even at high concentrations of S-Lic (300 µmol/L). Much more pronounced S-Lic effects were observed on the voltage dependence of slow inactivation, with significant effects at 100 µmol/L S-Lic in DGCs from control and epileptic rats or temporal lobe epilepsy patients. For none of these effects of S-Lic could we observe significant differences either between sham-control and epileptic rats, or between human DGCs and the two animal groups. S-Lic was similarly effective in cortical pyramidal neurons from sham-control and epileptic rats. Finally, we show in expression systems that S-Lic effects on slow inactivation voltage dependence are only observed in Nav 1.2 and Nav 1.6 subunits, but not in Nav 1.1 and Nav 1.3 subunits. SIGNIFICANCE: From these data, we conclude that a major mechanism of action of S-Lic is an effect on slow inactivation, primarily through effects on slow inactivation voltage dependence of Nav 1.2 and Nav 1.6 channels. Second, we demonstrate that this main effect of S-Lic is maintained in both experimental and human epilepsy and applies to principal neurons of different brain areas.
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Anticonvulsivantes/farmacología , Giro Dentado/patología , Dibenzazepinas/farmacología , Epilepsia/patología , Neuronas/efectos de los fármacos , Canales de Sodio/fisiología , Adulto , Análisis de Varianza , Animales , Anticonvulsivantes/uso terapéutico , Biofisica , Células Cultivadas , Dibenzazepinas/uso terapéutico , Modelos Animales de Enfermedad , Relación Dosis-Respuesta a Droga , Estimulación Eléctrica , Epilepsia/inducido químicamente , Femenino , Humanos , Técnicas In Vitro , Masculino , Potenciales de la Membrana/efectos de los fármacos , Persona de Mediana Edad , Técnicas de Placa-Clamp , Pilocarpina/toxicidad , Ratas , Ratas WistarRESUMEN
Despite the development of numerous novel anticonvulsant drugs, â¼30% of epilepsy patients remain refractory to antiepileptic drugs (AEDs). Many established and novel AEDs reduce hyperexcitability via voltage- and use-dependent inhibition of voltage-gated Na+ channels. For the widely used anticonvulsant carbamazepine (CBZ), use-dependent block of Na+ channels is significantly reduced both in experimental and human epilepsy. However, the molecular underpinnings of this potential cellular mechanism for pharmacoresistance have remained enigmatic.Here, we describe the mechanism that leads to the emergence of CBZ-resistant Na+ channels. We focused on the endogenous polyamine system, which powerfully modulates Na+ channels in a use-dependent manner. We had shown previously that the intracellular polyamine spermine is reduced in chronic epilepsy, resulting in increased persistent Na+ currents. Because spermine and CBZ both bind use-dependently in spatial proximity within the Na+ channel pore, we hypothesized that spermine loss might also be related to diminished CBZ response. Using the pilocarpine model of refractory epilepsy in male rats and whole-cell patch-clamp recordings, we first replicated the reduction of use-dependent block by CBZ in chronically epileptic animals. We then substituted intracellular spermine via the patch pipette in different concentrations. Under these conditions, we found that exogenous spermine significantly rescues use-dependent block of Na+ channels by CBZ. These findings indicate that an unexpected modulatory mechanism, depletion of intracellular polyamines, leads both to increased persistent Na+ currents and to diminished CBZ sensitivity of Na+ channels. These findings could lead to novel strategies for overcoming pharmacoresistant epilepsy that target the polyamine system.SIGNIFICANCE STATEMENT Pharmacoresistant epilepsy affects â¼18 million people worldwide, and intense efforts have therefore been undertaken to uncover the underlying molecular and cellular mechanisms. One of the key known candidate mechanisms of pharmacoresistance has been a loss of use-dependent Na+ channel block by the anticonvulsant carbamazepine (CBZ), both in human and experimental epilepsies. Despite intense scrutiny, the molecular mechanisms underlying this phenomenon have not been elucidated. We now show that a loss of intracellular spermine in chronic epilepsy is a major causative factor leading to the development of CBZ-resistant Na+ currents. This finding can be exploited both for the screening of anticonvulsants in expression systems, and for novel strategies to overcome pharmacoresistance that target the polyamine system.
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Anticonvulsivantes/farmacología , Carbamazepina/farmacología , Epilepsia Refractaria/metabolismo , Epilepsia Refractaria/fisiopatología , Espermina/metabolismo , Animales , Poliaminas Biogénicas/metabolismo , Resistencia a Medicamentos/fisiología , Hipocampo/efectos de los fármacos , Hipocampo/metabolismo , Hipocampo/fisiopatología , Masculino , Técnicas de Cultivo de Órganos , Ratas , Ratas WistarRESUMEN
Guillain-Barré syndrome (GBS) is an acute, immune-mediated polyradiculoneuropathy characterized by rapidly progressive paresis and sensory disturbances. Moderate to severe and often intractable neuropathic pain is a common symptom of GBS, but its underlying mechanisms are unknown. Pathology of GBS is classically attributed to demyelination of large, myelinated peripheral fibers. However, there is increasing evidence that neuropathic pain in GBS is associated with impaired function of small, unmyelinated, nociceptive fibers. We therefore examined the functional properties of small DRG neurons, the somata of nociceptive fibers, in a rat model of GBS (experimental autoimmune neuritis=EAN). EAN rats developed behavioral signs of neuropathic pain. This was accompanied by a significant shortening of action potentials due to a more rapid repolarization and an increase in repetitive firing in a subgroup of capsaicin-responsive DRG neurons. Na+ current measurements revealed a significant increase of the fast TTX-sensitive current and a reduction of the persistent TTX-sensitive current component. These changes of Na+ currents may account for the significant decrease in AP duration leading to an overall increase in excitability and are therefore possibly directly linked to pathological pain behavior. Thus, like in other animal models of neuropathic and inflammatory pain, Na+ channels seem to be crucially involved in the pathology of GBS and may constitute promising targets for pain modulating pharmaceuticals.
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Ganglios Espinales/fisiopatología , Neuralgia/fisiopatología , Neuritis Autoinmune Experimental/fisiopatología , Nociceptores/fisiología , Animales , Células Cultivadas , Fenómenos Electrofisiológicos/fisiología , Ganglios Espinales/patología , Masculino , Neuralgia/patología , Neuritis Autoinmune Experimental/patología , Nociceptores/patología , Dimensión del Dolor/métodos , Ratas , Ratas Endogámicas LewRESUMEN
OBJECTIVE: In human epilepsy, pharmacoresistance to antiepileptic drug therapy is a major problem affecting ~30% of patients with epilepsy. Many classical antiepileptic drugs target voltage-gated sodium channels, and their potent activity in inhibiting high-frequency firing has been attributed to their strong use-dependent blocking action. In chronic epilepsy, a loss of use-dependent block has emerged as a potential cellular mechanism of pharmacoresistance for anticonvulsants acting on voltage-gated sodium channels. The anticonvulsant drug lacosamide (LCM) also targets sodium channels, but has been shown to preferentially affect sodium channel slow inactivation processes, in contrast to most other anticonvulsants. METHODS: We used whole-cell voltage clamp recordings in acutely isolated cells to investigate the effects of LCM on transient Na+ currents. Furthermore, we used whole-cell current clamp recordings to assess effects on repetitive action potential firing in hippocampal slices. RESULTS: We show here that LCM exerts its effects primarily via shifting the slow inactivation voltage dependence to more hyperpolarized potentials in hippocampal dentate granule cells from control and epileptic rats, and from patients with epilepsy. It is important to note that this activity of LCM was maintained in chronic experimental and human epilepsy. Furthermore, we demonstrate that the efficacy of LCM in inhibiting high-frequency firing is undiminished in chronic experimental and human epilepsy. SIGNIFICANCE: Taken together, these results show that LCM exhibits maintained efficacy in chronic epilepsy, in contrast to conventional use-dependent sodium channel blockers such as carbamazepine. They also establish that targeting slow inactivation may be a promising strategy for overcoming target mechanisms of pharmacoresistance.
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Acetamidas/uso terapéutico , Anticonvulsivantes/uso terapéutico , Epilepsia/tratamiento farmacológico , Potenciales de la Membrana/efectos de los fármacos , Canales de Sodio/efectos de los fármacos , Canales de Sodio/metabolismo , Acetamidas/farmacología , Adulto , Análisis de Varianza , Animales , Anticonvulsivantes/farmacología , Biofisica , Células Cultivadas , Modelos Animales de Enfermedad , Relación Dosis-Respuesta a Droga , Estimulación Eléctrica , Epilepsia/inducido químicamente , Hipocampo/citología , Hipocampo/efectos de los fármacos , Hipocampo/fisiología , Humanos , Técnicas In Vitro , Lacosamida , Agonistas Muscarínicos/toxicidad , Neuronas/efectos de los fármacos , Técnicas de Placa-Clamp , Pilocarpina/toxicidad , Ratas WistarRESUMEN
Dendritic voltage-gated ion channels profoundly shape the integrative properties of neuronal dendrites. In epilepsy, numerous changes in dendritic ion channels have been described, all of them due to either their altered transcription or phosphorylation. In pilocarpine-treated chronically epileptic rats, we describe a novel mechanism that causes an increased proximal dendritic persistent Na(+) current (INaP). We demonstrate using a combination of electrophysiology and molecular approaches that the upregulation of dendritic INaP is due to a relief from polyamine-dependent inhibition. The polyamine deficit in hippocampal neurons is likely caused by an upregulation of the degrading enzyme spermidine/spermine acetyltransferase. Multiphoton glutamate uncaging experiments revealed that the increase in dendritic INaP causes augmented dendritic summation of excitatory inputs. These results establish a novel post-transcriptional modification of ion channels in chronic epilepsy and may provide a novel avenue for treatment of temporal lobe epilepsy. SIGNIFICANCE STATEMENT: In this paper, we describe a novel mechanism that causes increased dendritic persistent Na(+) current. We demonstrate using a combination of electrophysiology and molecular approaches that the upregulation of persistent Na(+) currents is due to a relief from polyamine-dependent inhibition. The polyamine deficit in hippocampal neurons is likely caused by an upregulation of the degrading enzyme spermidine/spermine acetyltransferase. Multiphoton glutamate uncaging experiments revealed that the increase in dendritic persistent Na current causes augmented dendritic summation of excitatory inputs. We believe that these results establish a novel post-transcriptional modification of ion channels in chronic epilepsy.
Asunto(s)
Región CA1 Hipocampal/patología , Dendritas/fisiología , Regulación hacia Abajo/fisiología , Canales de Sodio/fisiología , Espermina/metabolismo , Estado Epiléptico/patología , Potenciales de Acción/efectos de los fármacos , Potenciales de Acción/genética , Análisis de Varianza , Animales , Dendritas/efectos de los fármacos , Modelos Animales de Enfermedad , Regulación hacia Abajo/efectos de los fármacos , Humanos , Técnicas In Vitro , Masculino , Agonistas Muscarínicos/toxicidad , Pilocarpina/toxicidad , ARN Mensajero/metabolismo , Ratas , Ratas Wistar , Bloqueadores de los Canales de Sodio/farmacología , Canales de Sodio/efectos de los fármacos , Estadísticas no Paramétricas , Estado Epiléptico/inducido químicamente , Sinaptofisina/metabolismo , Tetrodotoxina/farmacología , Regulación hacia Arriba/efectos de los fármacos , Regulación hacia Arriba/fisiologíaRESUMEN
Temporal lobe epilepsy (TLE) is the most common focal seizure disorder in adults. In many patients, transient brain insults, including status epilepticus (SE), are followed by a latent period of epileptogenesis, preceding the emergence of clinical seizures. In experimental animals, transcriptional upregulation of CaV3.2 T-type Ca(2+)-channels, resulting in an increased propensity for burst discharges of hippocampal neurons, is an important trigger for epileptogenesis. Here we provide evidence that the metal-regulatory transcription factor 1 (MTF1) mediates the increase of CaV3.2 mRNA and intrinsic excitability consequent to a rise in intracellular Zn(2+) that is associated with SE. Adeno-associated viral (rAAV) transfer of MTF1 into murine hippocampi leads to increased CaV3.2 mRNA. Conversely, rAAV-mediated expression of a dominant-negative MTF1 abolishes SE-induced CaV3.2 mRNA upregulation and attenuates epileptogenesis. Finally, data from resected human hippocampi surgically treated for pharmacoresistant TLE support the Zn(2+)-MTF1-CaV3.2 cascade, thus providing new vistas for preventing and treating TLE.
Asunto(s)
Canales de Calcio Tipo T/genética , Proteínas de Unión al ADN/metabolismo , Epilepsia del Lóbulo Temporal/genética , Estado Epiléptico/genética , Factores de Transcripción/metabolismo , Zinc/metabolismo , Animales , Canales de Calcio Tipo T/metabolismo , Proteínas de Unión al ADN/genética , Epilepsia del Lóbulo Temporal/metabolismo , Regulación de la Expresión Génica , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Ratas , Estado Epiléptico/metabolismo , Factores de Transcripción/genética , Factor de Transcripción MTF-1RESUMEN
The large isoforms of the Rab3 interacting molecule (RIM) family, RIM1α/ß and RIM2α/ß, have been shown to be centrally involved in mediating presynaptic active zone function. The RIM protein family contains two additional small isoforms, RIM3γ and RIM4γ, which are composed only of the RIM-specific C-terminal C2B domain and varying N-terminal sequences and whose function remains to be elucidated. Here, we report that both, RIM3γ and RIM4γ, play an essential role for the development of neuronal arborization and of dendritic spines independent of synaptic function. γ-RIM knock-down in rat primary neuronal cultures and in vivo resulted in a drastic reduction in the complexity of neuronal arborization, affecting both axonal and dendritic outgrowth, independent of the time point of γ-RIM downregulation during dendrite development. Rescue experiments revealed that the phenotype is caused by a function common to both γ-RIMs. These findings indicate that γ-RIMs are involved in cell biological functions distinct from the regulation of synaptic vesicle exocytosis and play a role in the molecular mechanisms controlling the establishment of dendritic complexity and axonal outgrowth.
Asunto(s)
Dendritas/fisiología , Proteínas de Transporte de Membrana/fisiología , Proteínas del Tejido Nervioso/fisiología , Neuronas/fisiología , Animales , Western Blotting , Supervivencia Celular/fisiología , Células Cultivadas , Cartilla de ADN/genética , Potenciales Postsinápticos Excitadores/genética , Potenciales Postsinápticos Excitadores/fisiología , Femenino , Silenciador del Gen , Vectores Genéticos/genética , Aparato de Golgi/fisiología , Aparato de Golgi/ultraestructura , Células HEK293 , Humanos , Inmunohistoquímica , Hibridación in Situ , Lentivirus/genética , Masculino , Proteínas de Transporte de Membrana/genética , Proteínas del Tejido Nervioso/genética , Ratas , Ratas Wistar , Fracciones Subcelulares/metabolismo , Sinapsis/fisiología , TransfecciónRESUMEN
PURPOSE: In chronic epilepsy, a substantial proportion of up to 30% of patients remain refractory to antiepileptic drugs (AEDs). An understanding of the mechanisms of pharmacoresistance requires precise knowledge of how AEDs interact with their targets. Many commonly used AEDs act on the transient and/or the persistent components of the voltage-gated Na(+) current (I(NaT) and I(NaP) , respectively). Lacosamide (LCM) is a novel AED with a unique mode of action in that it selectively enhances slow inactivation of fast transient Na(+) channels. Given that functional loss of accessory Na(+) channel subunits is a feature of a number of neurologic disorders, including epilepsy, we examined the effects of LCM versus carbamazepine (CBZ) on the persistent Na(+) current (I(NaP) ), in the presence and absence of accessory subunits within the channel complex. METHODS: Using patch-clamp recordings in intact hippocampal CA1 neurons of Scn1b null mice, I(NaP) was recorded using slow voltage ramps. Application of 100 µm CBZ or 300 µm LCM reduced the maximal I(NaP) conductance in both wild-type and control mice. KEY FINDINGS: As shown previously by our group in Scn1b null mice, CBZ induced a paradoxical increase of I(NaP) conductance in the subthreshold voltage range, resulting in an ineffective block of repetitive firing in Scn1b null neurons. In contrast, LCM did not exhibit such a paradoxical increase, and accordingly maintained efficacy in blocking repetitive firing in Scn1b null mice. SIGNIFICANCE: These results suggest that the novel anticonvulsant LCM maintains activity in the presence of impaired Na(+) channel ß(1) subunit expression and thus may offer an improved efficacy profile compared with CBZ in diseases associated with an impaired expression of ß sub-units as observed in epilepsy.
Asunto(s)
Acetamidas/farmacología , Potenciales de Acción/efectos de los fármacos , Carbamazepina/farmacología , Subunidad beta-1 de Canal de Sodio Activado por Voltaje/deficiencia , Potenciales de Acción/fisiología , Animales , Células Cultivadas , Lacosamida , Ratones , Ratones Congénicos , Ratones Endogámicos C57BL , Ratones Noqueados , Neuronas/efectos de los fármacos , Neuronas/fisiología , Subunidades de Proteína/deficiencia , Subunidades de Proteína/fisiología , Subunidad beta-1 de Canal de Sodio Activado por Voltaje/fisiologíaRESUMEN
To ensure operation of synaptic transmission within an appropriate dynamic range, neurons have evolved mechanisms of activity-dependent plasticity, including changes in presynaptic efficacy. The multidomain protein RIM1α is an integral component of the cytomatrix at the presynaptic active zone and has emerged as key mediator of presynaptically expressed forms of synaptic plasticity. We have therefore addressed the role of RIM1α in aberrant cellular plasticity and structural reorganization after an episode of synchronous neuronal activity pharmacologically induced in vivo [status epilepticus (SE)]. Post-SE, all animals developed spontaneous seizure events, but their frequency was dramatically increased in RIM1α-deficient mice (RIM1α(-/-)). We found that in wild-type mice (RIM1α(+/+)) SE caused an increase in paired-pulse facilitation in the CA1 region of the hippocampus to the level observed in RIM1α(-/-) mice before SE. In contrast, this form of short-term plasticity was not further enhanced in RIM1α-deficient mice after SE. Intriguingly, RIM1α(-/-) mice showed a unique pattern of selective hilar cell loss (i.e., endfolium sclerosis), which so far has not been observed in a genetic epilepsy animal model, as well as less severe astrogliosis and attenuated mossy fiber sprouting. These findings indicate that the decrease in release probability and altered short- and long-term plasticity as present in RIM1α(-/-) mice result in the formation of a hyperexcitable network but act in part neuroprotectively with regard to neuropathological alterations associated with epileptogenesis. In summary, our results suggest that presynaptic plasticity and proper function of RIM1α play an important part in a neuron's adaptive response to aberrant electrical activity.