RESUMEN
OBJECTIVE: Epileptic spikes and seizures seem present early in the disease process of Alzheimer's disease (AD). However, it is unclear how soluble and insoluble amyloid beta (Aß) and tau proteins affect seizure development in vivo. We aim to contribute to this field by assessing the vulnerability to 6 Hz corneal kindling of young female mice from two well-characterized transgenic AD models and by testing their responsiveness to selected antiseizure drugs (ASDs). METHODS: We used 7-week-old triple transgenic (3xTg) mice that have both amyloid and tau mutations, and amyloid precursor protein Swedish/presenillin 1 dE9 (APP/PS1) mice, bearing only amyloid-related mutations. We assessed the absence of plaques via immunohistochemistry and analyzed the concentrations of both soluble and insoluble forms of Aß1-42 and total tau (t-tau) in brain hippocampal and prefrontal cortical tissue. Seven-week-old mice of the different genotypes were subjected to the 6 Hz corneal kindling model. After kindling acquisition, we tested the anticonvulsant effects of three marketed ASDs (levetiracetam, brivaracetam, and lamotrigine) in fully kindled mice. RESULTS: No Aß plaques were present in either genotype. Soluble Aß1-42 levels were increased in both AD genotypes, whereas insoluble Aß1-42 concentrations were only elevated in APP/PS1 mice compared with their respective controls. Soluble and insoluble forms of t-tau were increased in 3xTg mice only. 3xTg and APP/PS1 mice displayed more severe seizures induced by 6 Hz corneal kindling from the first stimulation onward and were more rapidly kindled compared with control mice. In fully kindled AD mice, ASDs had less-pronounced anticonvulsive effects compared with controls. SIGNIFICANCE: Mutations increasing Aß only or both Aß and tau in the brain enhance susceptibility for seizures and kindling in mice. The effect of ASDs on seizures measured by the Racine scale is less pronounced in both investigated AD models and suggests that seizures of young AD mice are more difficult to treat.
Asunto(s)
Enfermedad de Alzheimer , Proteínas tau , Enfermedad de Alzheimer/tratamiento farmacológico , Enfermedad de Alzheimer/genética , Péptidos beta-Amiloides/metabolismo , Precursor de Proteína beta-Amiloide/genética , Precursor de Proteína beta-Amiloide/metabolismo , Animales , Anticonvulsivantes/farmacología , Anticonvulsivantes/uso terapéutico , Modelos Animales de Enfermedad , Femenino , Lamotrigina , Levetiracetam , Ratones , Ratones Transgénicos , Convulsiones , Proteínas tau/metabolismoRESUMEN
Accumulating evidence shows a key function for astrocytic connexin43 (Cx43) signaling in epilepsy. However, the lack of experimental distinction between Cx43 gap junction channels (GJCs) and hemichannels (HCs) has impeded the identification of the exact contribution of either channel configurations to epilepsy. We therefore investigated whether TAT-Gap19, a Cx mimetic peptide that inhibits Cx43 HCs but not the corresponding Cx43 GJCs, influences experimentally induced seizures in rodents. Dye uptake experiments in acute hippocampal slices of mice demonstrated that astroglial Cx43 HCs open in response to the chemoconvulsant pilocarpine and this was inhibited by TAT-Gap19. In vivo, pilocarpine-induced seizures as well as the accompanying increase in D-serine microdialysate levels were suppressed by Cx43 HC inhibition. Moreover, the anticonvulsant action of TAT-Gap19 was reversed by exogenous D-serine administration, suggesting that Cx43 HC inhibition protects against seizures by lowering extracellular D-serine levels. The anticonvulsive properties of Cx43 HC inhibition were further confirmed in electrical seizure mouse models, i.e. an acute 6 Hertz (Hz) model of refractory seizures and a chronic 6 Hz corneal kindling model. Collectively, these results indicate that Cx43 HCs play a role in seizures and underscore their potential as a novel and druggable target in epilepsy treatment.
Asunto(s)
Anticonvulsivantes/farmacología , Astrocitos/efectos de los fármacos , Conexina 43/metabolismo , Fragmentos de Péptidos/metabolismo , Adenosina Trifosfato/farmacología , Animales , Astrocitos/metabolismo , Conexinas/metabolismo , Uniones Comunicantes/efectos de los fármacos , Uniones Comunicantes/metabolismo , Hipocampo/efectos de los fármacos , Hipocampo/metabolismo , Ratones Transgénicos , Transducción de Señal/efectos de los fármacosRESUMEN
The development of the cerebral cortex is a complex process that requires the generation, migration, and differentiation of neurons. Interfering with any of these steps can impair the establishment of connectivity and, hence, function of the adult brain. Neurotransmitter receptors have emerged as critical players to regulate these biological steps during brain maturation. Among them, α2 subunit-containing glycine receptors (GlyRs) regulate cortical neurogenesis and the present work demonstrates the long-term consequences of their genetic disruption on neuronal connectivity in the postnatal cerebral cortex. Our data indicate that somatosensory cortical neurons of Glra2 knockout mice (Glra2KO) have more dendritic branches with an overall increase in total spine number. These morphological defects correlate with a disruption of the excitation/inhibition balance, thereby increasing network excitability and enhancing susceptibility to epileptic seizures after pentylenetetrazol tail infusion. Taken together, our findings show that the loss of embryonic GlyRα2 ultimately impairs the formation of cortical circuits in the mature brain.
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Corteza Cerebral/embriología , Corteza Cerebral/metabolismo , Neuronas/metabolismo , Receptores de Glicina/metabolismo , Animales , Corteza Cerebral/citología , Modelos Animales de Enfermedad , Inmunohistoquímica , Masculino , Potenciales de la Membrana/fisiología , Ratones Endogámicos C57BL , Ratones Noqueados , Vías Nerviosas/citología , Vías Nerviosas/embriología , Vías Nerviosas/metabolismo , Neuronas/citología , Técnicas de Placa-Clamp , Pentilenotetrazol , Receptores de Glicina/genética , Convulsiones/metabolismo , Técnicas de Cultivo de TejidosRESUMEN
Parkinson's disease (PD) is a neurodegenerative disorder, characterized by a loss of dopamine (DA) neurons in the substantia nigra pars compacta (SNc). Caloric restriction (CR) has been shown to exert ghrelin-dependent neuroprotective effects in the 1-methyl-4-phenyl-1,2,3,6-tetrathydropyridine (MPTP)-based animal model for PD. We here investigated whether CR is neuroprotective in the lactacystin (LAC) mouse model for PD, in which proteasome disruption leads to the destruction of the DA neurons of the SNc, and whether this effect is mediated via the ghrelin receptor. Adult male ghrelin receptor wildtype (WT) and knockout (KO) mice were maintained on an ad libitum (AL) diet or on a 30% CR regimen. After 3 weeks, LAC was injected unilaterally into the SNc, and the degree of DA neuron degeneration was evaluated 1 week later. In AL mice, LAC injection significanty reduced the number of DA neurons and striatal DA concentrations. CR protected against DA neuron degeneration following LAC injection. However, no differences were observed between ghrelin receptor WT and KO mice. These results indicate that CR can protect the nigral DA neurons from toxicity related to proteasome disruption; however, the ghrelin receptor is not involved in this effect.
Asunto(s)
Acetilcisteína/análogos & derivados , Restricción Calórica , Neuronas Dopaminérgicas/efectos de los fármacos , Neuronas Dopaminérgicas/metabolismo , Fármacos Neuroprotectores , Receptores de Ghrelina/metabolismo , Acetilcisteína/administración & dosificación , Acetilcisteína/farmacología , Factores de Edad , Animales , Recuento de Células , Masculino , Ratones , Ratones Noqueados , Receptores de Ghrelina/genética , Sustancia Negra/efectos de los fármacos , Sustancia Negra/metabolismo , Sustancia Negra/patologíaRESUMEN
Within the hippocampus, the major somatostatin (SRIF) receptor subtype, the sst2A receptor, is localized at postsynaptic sites of the principal neurons where it modulates neuronal activity. Following agonist exposure, this receptor rapidly internalizes and recycles slowly through the trans-Golgi network. In epilepsy, a high and chronic release of somatostatin occurs, which provokes, in both rat and human tissue, a decrease in the density of this inhibitory receptor at the cell surface. The insulin-regulated aminopeptidase (IRAP) is involved in vesicular trafficking and shares common regional distribution with the sst2A receptor. In addition, IRAP ligands display anticonvulsive properties. We therefore sought to assess by in vitro and in vivo experiments in hippocampal rat tissue whether IRAP ligands could regulate the trafficking of the sst2A receptor and, consequently, modulate limbic seizures. Using pharmacological and cell biological approaches, we demonstrate that IRAP ligands accelerate the recycling of the sst2A receptor that has internalized in neurons in vitro or in vivo. Most importantly, because IRAP ligands increase the density of this inhibitory receptor at the plasma membrane, they also potentiate the neuropeptide SRIF inhibitory effects on seizure activity. Our results further demonstrate that IRAP is a therapeutic target for the treatment of limbic seizures and possibly for other neurological conditions in which downregulation of G-protein-coupled receptors occurs. SIGNIFICANCE STATEMENT: The somatostatin type 2A receptor (sst2A) is localized on principal hippocampal neurons and displays anticonvulsant properties. Following agonist exposure, however, this receptor rapidly internalizes and recycles slowly. The insulin-regulated aminopeptidase (IRAP) is involved in vesicular trafficking and shares common regional distribution with the sst2A receptor. We therefore assessed by in vitro and in vivo experiments whether IRAP could regulate the trafficking of this receptor. We demonstrate that IRAP ligands accelerate sst2A recycling in hippocampal neurons. Because IRAP ligands increase the density of sst2A receptors at the plasma membrane, they also potentiate the effects of this inhibitory receptor on seizure activity. Our results further demonstrate that IRAP is a therapeutic target for the treatment of limbic seizures.
Asunto(s)
Cistinil Aminopeptidasa/metabolismo , Hipocampo/metabolismo , Receptores de Somatostatina/metabolismo , Convulsiones/metabolismo , Convulsiones/prevención & control , Animales , Células CHO , Cricetinae , Cricetulus , Humanos , Sistema Límbico/metabolismo , Masculino , Ratones , Transporte de Proteínas/fisiología , Ratas , Ratas WistarRESUMEN
Ghrelin has anticonvulsant and neuroprotective effects in models of chemoconvulsant-induced seizures and status epilepticus. In this study we investigated whether deletion of the ghrelin receptor could alter the kindling process in the 6 Hz corneal kindling model and whether ghrelin receptor ligands possess anticonvulsant effects in fully kindled mice. Ghrelin receptor wild-type and knockout mice were electrically stimulated at a subconvulsive current twice daily via corneal electrodes until they reached the fully kindled state. Mice lacking the ghrelin receptor showed similar seizure severity during kindling acquisition as well as in the maintenance phase when compared to their wild-type littermates. Subsequently we proceeded by investigating possible anticonvulsant effects of the ghrelin receptor ligands in the acute 6 Hz seizure model and the fully 6 Hz kindled mice. The ghrelin receptor agonist JMV-1843 decreased the seizure severity score both in acutely 6 Hz stimulated mice and in fully kindled ghrelin receptor wild-type mice, but not in fully kindled ghrelin receptor knockout mice. No effect on seizure severity was observed following the ghrelin receptor antagonist JMV-2959 in both models. This finding indicates that JMV-1843 exerts an anticonvulsant effect in kindled mice via the ghrelin receptor.
Asunto(s)
Anticonvulsivantes/uso terapéutico , Córnea/inervación , Excitación Neurológica/efectos de los fármacos , Receptores de Ghrelina/agonistas , Estado Epiléptico/tratamiento farmacológico , Animales , Modelos Animales de Enfermedad , Estimulación Eléctrica/efectos adversos , Glicina/análogos & derivados , Glicina/uso terapéutico , Indoles , Excitación Neurológica/fisiología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Oligopéptidos/uso terapéutico , Receptores de Ghrelina/deficiencia , Receptores de Ghrelina/genética , Estado Epiléptico/genética , Triazoles/uso terapéutico , Triptófano/análogos & derivadosRESUMEN
Objectives: Epileptiform activity and seizures are present in patients with Alzheimer's disease (AD) and genetic animal models of AD. Amyloid beta 1-42 (Aß1-42) oligomers are thought to be crucial in AD and can cause neuronal hyperexcitability in vitro. However, it is unclear whether these Aß1-42 oligomers cause the increased seizure susceptibility in vivo in people with AD and in AD animal models, nor via which mechanisms it would do so. We investigated this question by injecting Aß1-42 oligomers intracerebrally in mice and assessed its impact on seizure susceptibility. Materials and methods: We performed a single intracerebral injection of synthetic Aß1-42 oligomers or scrambled Aß1-42 in NMRI mice in three different cohorts and subjected them to an i.v. infusion of a chemoconvulsant. We evoked the seizures 1.5 h, 1 week, or 3 weeks after the intracerebral injection of Aß1-42 oligomers, covering also the timepoints and injection locations that were used by others in similar experimental set-ups. Results: With a thioflavine T assay and transmission electron microscopy we confirmed that Aß1-42 monomers spontaneously aggregated to oligomers. We did not find an effect of Aß1-42 oligomers on susceptibility to seizures - evoked 1.5 h, 1 week or 3 weeks - after their intracerebral injection. Significance: The lack of effect of Aß1-42 oligomers on seizure susceptibility in our experiments contrasts with recent findings in similar experimental set-ups. Contradicting conclusions are frequent in experiments with Aß1-42 and they are often attributed to subtle differences in the various aggregation forms of the Aß1-42 used in different experiments. We confirmed the presence of Aß1-42 oligomers with state-of-the-art methods but cannot ascertain that the protein aggregates we used are identical to those used by others. Whether our findings or those previously published best represent the role of Aß1-42 oligomers on seizures in AD remains unclear.
RESUMEN
Two closely related glycogen synthase kinase-3 (GSK-3) isoforms have been identified in mammals: GSK-3α and GSK-3ß. GSK-3ß is the most prominent in the central nervous system and was previously shown to control neuronal excitability. We previously demonstrated that indirubin and its structural analogue and the nonselective GSK-3 inhibitor BIO-acetoxime exerted anticonvulsant effects in acute seizure models in zebrafish, mice, and rats. We here examined for the first time the anticonvulsant effect of TCS2002, a specific and potent inhibitor of GSK-3ß, in two models for limbic seizures: the pilocarpine rat model for focal seizures and the acute 6 Hz corneal mouse model for refractory seizures. Next, we additionally used the 6 Hz kindling model to establish differences in seizure susceptibility and seizure progression in mice that either overexpress human GSK-3ß (GSK-3ß OE) or lack GSK-3ß (GSK-3ß-/-) in neurons. We demonstrate that TCS2002 exerts anticonvulsant actions against pilocarpine- and 6 Hz-evoked seizures. Compared to wild-type littermates, GSK-3ß OE mice are less susceptible to seizures but are more rapidly kindled. Interestingly, compared to GSK-3ß+/+ mice, neuronal GSK-3ß-/- mice show increased susceptibility to 6 Hz-induced seizures. These contrasting observations suggest compensatory neurodevelopmental mechanisms that alter seizure susceptibility in GSK-3ß OE and GSK-3ß-/- mice. Although the pronounced anticonvulsant effects of selective and acute GSK-3ß inhibition in the 6 Hz model identify GSK-3ß as a potential drug target for pharmacoresistant seizures, our data on the sustained disruption of GSK-3ß activity in the transgenic mice suggest a role for GSK-3 in kindling and warrants further research into the long-term effects of selective pharmacological GSK-3ß inhibition.
Asunto(s)
Anticonvulsivantes , Pilocarpina , Animales , Anticonvulsivantes/farmacología , Anticonvulsivantes/uso terapéutico , Modelos Animales de Enfermedad , Glucógeno Sintasa Quinasa 3 , Glucógeno Sintasa Quinasa 3 beta , Mamíferos , Ratones , Pilocarpina/toxicidad , Isoformas de Proteínas , Ratas , Convulsiones/inducido químicamente , Convulsiones/tratamiento farmacológico , Pez CebraRESUMEN
Manipulating firing-rate neuronal homeostasis, which enables neurons to regulate their intrinsic excitability, offers an attractive opportunity to prevent seizures. However, to date, no drug-based interventions have been reported that manipulate this type of neuronal homeostatic mechanism. Here, we used a combination of Drosophila and mouse, and, in the latter, both a pentylenetetrazole (PTZ)-induced seizure model and an electrically induced seizure model for refractory seizures to evaluate the anticonvulsant efficacy of a novel class of anticonvulsant compounds, based on 4-tert-butyl-benzaldehyde (4-TBB). The mode of action included increased expression of the firing rate homeostatic regulator Pumilio (PUM). Knockdown of pum expression, in Drosophila, blocked anticonvulsive effects of 4-TBB, while analysis of validated PUM targets in mouse brain revealed significant reductions following exposure to this compound. A structure-activity study identified the active parts of the molecule and, further, showed that the pyrazole analogue demonstrates highest efficacy, being active against both PTZ-induced and electrically induced seizures. This study provides a proof of principle that anticonvulsant effects can be achieved through regulation of firing rate neuronal homeostasis and identifies a possible chemical compound for future development.
Asunto(s)
Anticonvulsivantes , Pentilenotetrazol , Animales , Anticonvulsivantes/farmacología , Anticonvulsivantes/uso terapéutico , Benzaldehídos/efectos adversos , Drosophila , Homeostasis , Ratones , Neuronas , Pentilenotetrazol/efectos adversos , Pirazoles/uso terapéutico , Convulsiones/tratamiento farmacológico , Convulsiones/prevención & controlRESUMEN
Currently, a high variety of analytical techniques to perform metabolomics is available. One of these techniques is capillary electrophoresis coupled to mass spectrometry (CE-MS), which has emerged as a rather strong analytical technique for profiling polar and charged compounds. This work aims to discover with CE-MS potential metabolic consequences of evoked seizures in plasma by using a 6Hz acute corneal seizure mouse model. CE-MS is an appealing technique because of its capability to handle very small sample volumes, such as the 10 µL plasma samples obtained using capillary microsampling in this study. After liquid-liquid extraction, the samples were analyzed with CE-MS using low-pH separation conditions, followed by data analysis and biomarker identification. Both electrically induced seizures showed decreased values of methionine, lysine, glycine, phenylalanine, citrulline, 3-methyladenine and histidine in mice plasma. However, a second provoked seizure, 13 days later, showed a less pronounced decrease of the mean concentrations of these plasma metabolites, demonstrated by higher fold change ratios. Other obtained markers that can be related to seizure activities based on literature data, are isoleucine, serine, proline, tryptophan, alanine, arginine, valine and asparagine. Most amino acids showed relatively stable plasma concentrations between the basal levels (Time point 1) and after the 13-day wash-out period (Time point 3), which suggests its effectiveness. Overall, this work clearly demonstrated the possibility of profiling metabolite consequences related to seizure activities of an intrinsically low amount of body fluid using CE-MS. It would be useful to investigate and validate, in the future, the known and unknown metabolites in different animal models as well as in humans.
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Modelos Animales de Enfermedad , Convulsiones/metabolismo , Enfermedad Aguda , Animales , Electroforesis Capilar , Análisis de los Mínimos Cuadrados , Masculino , Espectrometría de Masas , Ratones , Análisis Multivariante , Convulsiones/sangreRESUMEN
In view of the clinical need for new antiseizure drugs (ASDs) with novel modes of action, we used a zebrafish seizure model to screen the anticonvulsant activity of medicinal plants used by traditional healers in the Congo for the treatment of epilepsy, and identified a crude plant extract that inhibited pentylenetetrazol (PTZ)-induced seizures in zebrafish larvae. Zebrafish bioassay-guided fractionation of this anticonvulsant Fabaceae species, Indigofera arrecta, identified indirubin, a compound with known inhibitory activity of glycogen synthase kinase (GSK)-3, as the bioactive component. Indirubin, as well as the more potent and selective GSK-3 inhibitor 6-bromoindirubin-3'-oxime (BIO-acetoxime) were tested in zebrafish and rodent seizure assays. Both compounds revealed anticonvulsant activity in PTZ-treated zebrafish larvae, with electroencephalographic recordings revealing reduction of epileptiform discharges. Both indirubin and BIO-acetoxime also showed anticonvulsant activity in the pilocarpine rat model for limbic seizures and in the 6-Hz refractory seizure mouse model. Most interestingly, BIO-acetoxime also exhibited anticonvulsant actions in 6-Hz fully kindled mice. Our findings thus provide the first evidence for anticonvulsant activity of GSK-3 inhibition, thereby implicating GSK-3 as a potential therapeutic entry point for epilepsy. Our results also support the use of zebrafish bioassay-guided fractionation of antiepileptic medicinal plant extracts as an effective strategy for the discovery of new ASDs with novel mechanisms of action.
Asunto(s)
Anticonvulsivantes/uso terapéutico , Epilepsia/tratamiento farmacológico , Epilepsia/enzimología , Glucógeno Sintasa Quinasa 3/antagonistas & inhibidores , Glucógeno Sintasa Quinasa 3/metabolismo , Inhibidores de Proteínas Quinasas/uso terapéutico , Animales , Anticonvulsivantes/farmacología , Indoles/farmacología , Indoles/uso terapéutico , Masculino , Ratones , Ratones Endogámicos C57BL , Inhibidores de Proteínas Quinasas/farmacología , Ratas , Ratas Wistar , Pez CebraRESUMEN
Long term treatment with L-3,4-dihydroxyphenylalanine (L-DOPA) is associated with several motor complications. Clinical improvement of this treatment is therefore needed. Lesions or high frequency stimulation of the hyperactive subthalamic nucleus (STN) in Parkinson's disease (PD), alleviate the motor symptoms and reduce dyskinesia, either directly and/or by allowing the reduction of the L-DOPA dose. N-methyl-D-aspartate (NMDA) receptor antagonists might have similar actions. However it remains elusive how the neurochemistry changes in the STN after a separate or combined administration of L-DOPA and a NMDA receptor antagonist. By means of in vivo microdialysis, the effect of L-DOPA and/or MK 801, on the extracellular dopamine (DA) and glutamate (GLU) levels was investigated for the first time in the STN of sham and 6-hydroxydopamine-lesioned rats. The L-DOPA-induced DA increase in the STN was significantly higher in DA-depleted rats compared to shams. MK 801 did not influence the L-DOPA-induced DA release in shams. However, MK 801 enhanced the L-DOPA-induced DA release in hemi-parkinson rats. Interestingly, the extracellular STN GLU levels remained unchanged after nigral degeneration. Furthermore, administration of MK 801 alone or combined with L-DOPA did not alter the STN GLU levels in both sham and DA-depleted rats. The present study does not support the hypothesis that DA-ergic degeneration influences the STN GLU levels neither that MK 801 alters the GLU levels in lesioned and non-lesioned rats. However, NMDA receptor antagonists could be used as a beneficial adjuvant treatment for PD by enhancing the therapeutic efficacy of l-DOPA at least in part in the STN.
Asunto(s)
Antiparkinsonianos/farmacología , Dopamina/metabolismo , Levodopa/farmacología , Trastornos Parkinsonianos/tratamiento farmacológico , Receptores de N-Metil-D-Aspartato/antagonistas & inhibidores , Núcleo Subtalámico/efectos de los fármacos , Animales , Modelos Animales de Enfermedad , Maleato de Dizocilpina/farmacología , Antagonistas de Aminoácidos Excitadores/farmacología , Espacio Extracelular/metabolismo , Lateralidad Funcional , Ácido Glutámico/metabolismo , Masculino , Microdiálisis , Oxidopamina , Trastornos Parkinsonianos/fisiopatología , Ratas Wistar , Receptores de N-Metil-D-Aspartato/metabolismo , Sustancia Negra/efectos de los fármacos , Sustancia Negra/fisiopatología , Núcleo Subtalámico/fisiopatologíaRESUMEN
Cortistatin (CST)-14, a neuropeptide that is structurally and functionally related to somatostatin-14 (SRIF) binds all five somatostatin receptor subtypes (sst1-sst5). Using in vivo microdialysis and telemetry-based electroencephalographic recordings, we provide the first experimental evidence for anticonvulsive effects of CST-14 in a pilocarpine-induced seizure model in rats and mice and for the involvement of sst2 and sst3 receptors in these anticonvulsant actions of CST-14. Both receptor subtypes are required for the anticonvulsant effects of CST-14 given that co-perfusion of a selective sst2 antagonist (cyanamid15486) or a selective sst3 antagonist (SST3-ODN-8) reversed anticonvulsant effect of CST-14, and this, independently of each other. Next, as the ghrelin receptor has been proposed as a target for the biological effects of CST-14, we used ghrelin receptor knockout mice and their wild type littermates to study the involvement of this receptor in the anticonvulsive actions of CST-14. Our results show a significant decrease in seizure duration in both genotypes when CST-14 treated mice were compared with corresponding control animals receiving only pilocarpine. In addition, this CST-14-induced decrease was comparable in both genotypes. We here thus provide the first evidence that ghrelin receptors are not involved in mediating anticonvulsant actions of CST-14 in vivo.
Asunto(s)
Anticonvulsivantes/uso terapéutico , Neuropéptidos/uso terapéutico , Péptidos Cíclicos/uso terapéutico , Receptores de Ghrelina/fisiología , Receptores de Somatostatina/fisiología , Convulsiones/tratamiento farmacológico , Animales , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Pilocarpina/toxicidad , Ratas , Ratas Wistar , Receptores de Ghrelina/antagonistas & inhibidores , Receptores de Ghrelina/deficiencia , Receptores de Somatostatina/antagonistas & inhibidores , Convulsiones/inducido químicamente , Convulsiones/metabolismoRESUMEN
Ghrelin is a pleiotropic neuropeptide that has been recently implicated in epilepsy. Animal studies performed to date indicate that ghrelin has anticonvulsant properties; however, its mechanism of anticonvulsant action is unknown. Here we show that the anticonvulsant effects of ghrelin are mediated via the growth hormone secretagogue receptor (GHSR). To our surprise, however, we found that the GHSR knockout mice had a higher seizure threshold than their wild-type littermates when treated with pilocarpine. Using both in vivo and in vitro models, we further discovered that inverse agonism and desensitization/internalization of the GHSR attenuate limbic seizures in rats and epileptiform activity in hippocampal slices. This constitutes a novel mechanism of anticonvulsant action, whereby an endogenous agonist reduces the activity of a constitutively active receptor.
Asunto(s)
Anticonvulsivantes/uso terapéutico , Ghrelina/uso terapéutico , Sistema Límbico/efectos de los fármacos , Receptores de Ghrelina/metabolismo , Convulsiones/tratamiento farmacológico , Convulsiones/patología , Análisis de Varianza , Animales , Anticonvulsivantes/farmacología , Calcio/metabolismo , Modelos Animales de Enfermedad , Susceptibilidad a Enfermedades , Relación Dosis-Respuesta a Droga , Interacciones Farmacológicas , Estimulación Eléctrica , Potenciales Postsinápticos Excitadores/efectos de los fármacos , Potenciales Postsinápticos Excitadores/genética , Proteínas Fluorescentes Verdes/genética , Células HEK293 , Hipocampo/citología , Humanos , Técnicas In Vitro , Sistema Límbico/fisiopatología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Microdiálisis , Agonistas Muscarínicos/toxicidad , Neuronas/efectos de los fármacos , Técnicas de Placa-Clamp , Pilocarpina/toxicidad , Piperidinas/uso terapéutico , Pirazoles/uso terapéutico , Ratas , Ratas Wistar , Receptores de Ghrelina/agonistas , Receptores de Ghrelina/deficiencia , Convulsiones/genética , Índice de Severidad de la Enfermedad , Especificidad de la Especie , Transfección , Ácido gamma-Aminobutírico/metabolismoRESUMEN
Neuropeptides seem to play an important role when the CNS is challenged. In order to obtain better insights into the central peptidergic effects, it is essential to monitor their concentration in the brain. Quantification of neuropeptides in dialysates is challenging due to their low extracellular concentrations (low pM range), their low microdialysis efficiencies, the need for acceptable temporal resolution, the small sample volumes, the complexity of the matrix and the tendency of peptides to stick to glass and polymeric materials. The quantification of neuropeptides in dialysates therefore necessitates the use of very sensitive nano-LC-MS/MS methods. A number of LC-MS/MS and microdialysis parameters need to be optimized to achieve maximal sensitivity. The optimized and validated methods can be used to investigate the in vivo neuropeptide release during pathological conditions, in this way initiating new and immense challenges for the development of new drugs.
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Encéfalo/metabolismo , Cromatografía Liquida/métodos , Neuropéptidos/metabolismo , Espectrometría de Masas en Tándem/métodos , Animales , Cromatografía Liquida/instrumentación , Humanos , Microdiálisis , Miniaturización , Espectrometría de Masas en Tándem/instrumentaciónRESUMEN
Somatostatin-14 (SRIF) is a potent anticonvulsant in rodent models of limbic seizures in which the hippocampus is its major site of action. However, the distribution of hippocampal sst receptors and their role in the anticonvulsant effects of SRIF remain controversial. Moreover, striking differences have been described between mice and rats. In rats, sst(2) but not sst(1) receptors play a critical role in the anticonvulsant effects of SRIF. At present, the role of rat sst(3) and sst(4) receptors in these anticonvulsive effects remains unknown. Here we demonstrate in vivo anticonvulsive actions of rat hippocampal sst(3) and sst(4) receptors. Using microdialysis and telemetry-based electroencephalographic recordings we show that intrahippocampal administration of the sst(2) agonist L-779,976 (500 nM), the sst(3) agonist L-796,778 (100 nM) or the sst(4) agonist L-803,087 (100 nM) protects rats against focal pilocarpine-induced seizures. SRIF (1 µM)-, sst(3)- and sst(4)-mediated anticonvulsive actions are reversed by the selective sst(2) receptor antagonist cyanamid 154806 (100 nM). Moreover, the selective sst(3) antagonist SST3-ODN-8 (100 nM) blocks the sst(4)-mediated anticonvulsant effect. Sst(3) antagonism does not reverse the sst(2)- or SRIF-mediated anticonvulsant effects. Our findings provide the first in vivo evidence for potent anticonvulsive properties of sst(3) and sst(4) receptors in the rat hippocampus. Nevertheless, selective sst(2) receptor antagonism prevented these sst(3)- or sst(4) receptor-mediated anticonvulsant effects, suggesting a functional cooperation with rat hippocampal sst(2) receptors.
Asunto(s)
Anticonvulsivantes/uso terapéutico , Hipocampo/efectos de los fármacos , Hipocampo/metabolismo , Receptores de Somatostatina/metabolismo , Convulsiones/tratamiento farmacológico , Convulsiones/patología , Amidas/farmacología , Amidas/uso terapéutico , Análisis de Varianza , Animales , Cromatografía Liquida , Modelos Animales de Enfermedad , Relación Dosis-Respuesta a Droga , Interacciones Farmacológicas , Electroencefalografía , Indoles/farmacología , Indoles/uso terapéutico , Masculino , Microdiálisis , Nitrobencenos/farmacología , Nitrobencenos/uso terapéutico , Pilocarpina/efectos adversos , Ratas , Ratas Wistar , Convulsiones/inducido químicamente , Somatostatina/uso terapéuticoRESUMEN
Somatostatin-14 (SRIF-14) exerts anticonvulsive effects in several rat seizure models, generally attributed to sst(2) receptor activation. Whereas sst(1) immunoreactivity has been localized to both polymorphic interneurons and principal cells in the rat hippocampus, its potential role as an inhibitory autoreceptor or as a receptor involved in mediating anticonvulsive actions remains unknown. We showed that intrahippocampal administration of the sst(1) antagonist SRA880 (1 microM) induced a robust increase in hippocampal SST-14 levels without affecting gamma-aminobutyric acid levels in conscious rats, indicating that the sst(1) receptor acts as an inhibitory autoreceptor. SRA880 did not affect seizure severity and did not reverse the anticonvulsive action of SRIF-14 (1 microM) against pilocarpine-induced seizures, suggesting that hippocampal sst(1) receptors are not involved in the anticonvulsive effects of SRIF-14.
Asunto(s)
Autorreceptores/metabolismo , Hipocampo/metabolismo , Piperazinas/farmacología , Quinolinas/farmacología , Receptores de Somatostatina/metabolismo , Convulsiones/metabolismo , Somatostatina/metabolismo , Animales , Cromatografía Liquida , Hipocampo/química , Hipocampo/efectos de los fármacos , Masculino , Microdiálisis/métodos , Microinyecciones/métodos , Agonistas Muscarínicos , Pilocarpina , Piperazinas/administración & dosificación , Quinolinas/administración & dosificación , Ratas , Ratas Wistar , Receptores de Somatostatina/agonistas , Convulsiones/inducido químicamente , Somatostatina/efectos de los fármacos , Ácido gamma-Aminobutírico/metabolismoRESUMEN
Reliable well-characterised animal models of seizures are necessary in order to better understand the underlying pathophysiological mechanisms as well as to screen potential anticonvulsant drugs. We currently use the focal pilocarpine model as an acute limbic seizure model. Due to breeding problems at the vendor, and apparent changes in pilocarpine-induced seizure susceptibility, we were forced to change breeding locations and vendors over a period of 2 years. Male Wistar rats were either purchased from two breeding locations of Charles River Laboratories (France and Germany), or obtained from Harlan Laboratories (The Netherlands). In the present retrospective study we evaluated the impact of these vendor changes on ketamine dosing to establish anaesthesia, on pilocarpine-induced seizure susceptibility, and on basal extracellular hippocampal noradrenaline, dopamine, serotonin, gamma-amino butyric acid, and glutamate levels of all pilocarpine-treated rats included in our studies. Significant differences were present in all of the parameters analyzed. This study clearly illustrates that intrastrain differences do exist from one vendor/breeding location to another, or even between rats from the same breeding location.