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1.
Toxicol Appl Pharmacol ; 426: 115643, 2021 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-34265354

RESUMO

The chemical threat agent tetramethylenedisulfotetramine (TETS) is a γ-aminobutyric acid type A receptor (GABA AR) antagonist that causes life threatening seizures. Currently, there is no specific antidote for TETS intoxication. TETS-induced seizures are typically treated with benzodiazepines, which function as nonselective positive allosteric modulators (PAMs) of synaptic GABAARs. The major target of TETS was recently identified as the GABAAR α2ß3γ2 subtype in electrophysiological studies using recombinantly expressed receptor combinations. Here, we tested whether these in vitro findings translate in vivo by comparing the efficacy of GABAAR subunit-selective PAMs in reducing TETS-induced seizure behavior in larval zebrafish. We tested PAMs targeting α1, α2, α2/3/5, α6, ß2/3, ß1/2/3, and δ subunits and compared their efficacy to the benzodiazepine midazolam (MDZ). The data demonstrate that α2- and α6-selective PAMs (SL-651,498 and SB-205384, respectively) were effective at mitigating TETS-induced seizure-like behavior. Combinations of SB-205384 and MDZ or SL-651,498 and 2-261 (ß2/3-selective) mitigated TETS-induced seizure-like behavior at concentrations that did not elicit sedating effects in a photomotor behavioral assay, whereas MDZ alone caused sedation at the concentration required to stop seizure behavior. Isobologram analyses suggested that SB-205384 and MDZ interacted in an antagonistic fashion, while the effects of SL-651,498 and 2-261 were additive. These results further elucidate the molecular mechanism by which TETS induces seizures and provide mechanistic insight regarding specific countermeasures against this chemical convulsant.


Assuntos
Hidrocarbonetos Aromáticos com Pontes , Convulsivantes , Moduladores GABAérgicos/farmacologia , Hipnóticos e Sedativos/farmacologia , Subunidades Proteicas/fisiologia , Receptores de GABA-A/fisiologia , Convulsões/induzido quimicamente , Animais , Comportamento Animal/efeitos dos fármacos , Larva , Locomoção/efeitos dos fármacos , Midazolam/farmacologia , Subunidades Proteicas/genética , Receptores de GABA-A/genética , Convulsões/fisiopatologia , Peixe-Zebra
2.
Epilepsia ; 62(7): 1677-1688, 2021 07.
Artigo em Inglês | MEDLINE | ID: mdl-34080183

RESUMO

OBJECTIVE: The kainic acid (KA)-induced status epilepticus (SE) model in rats is a well-defined model of epileptogenesis. This model closely recapitulates many of the clinical and pathological characteristics of human temporal lobe epilepsy (TLE) that arise following SE or another neurological insult. Spontaneous recurrent seizures (SRS) in TLE can present after a latent period following a neurological insult (traumatic brain injury, SE event, viral infection, etc.). Moreover, this model is suitable for preclinical studies to evaluate the long-term process of epileptogenesis and screen putative disease-modifying/antiepileptogenic agents. The burden of human TLE is highly variable, similar to the post-KA SE rat model. In this regard, this model may have broad translational relevance. This report thus details the pharmacological characterization and methodological refinement of a moderate-throughput drug screening program using the post-KA-induced SE model of epileptogenesis in male Sprague Dawley rats to identify potential agents that may prevent or modify the burden of SRS. Specifically, we sought to demonstrate whether our protocol could prevent the development of SRS or lead to a reduced frequency/severity of SRS. METHODS: Rats were administered either everolimus (2-3 mg/kg po) beginning 1, 2, or 24 h after SE onset, or phenobarbital (60 mg/kg ip) beginning 1 h after SE onset. All treatments were administered once/day for 5-7 days. Rats in all studies (n = 12/treatment dose/study) were then monitored intermittently by video-electroencephalography (2 weeks on, 2 weeks off, 2 weeks on epochs) to determine latency to onset of SRS and disease burden. RESULTS: Although no adverse side effects were observed in our studies, no treatment significantly modified disease or prevented the presentation of SRS by 6 weeks after SE onset. SIGNIFICANCE: Neither phenobarbital nor everolimus administered at several time points after SE onset prevented the development of SRS. Nonetheless, we demonstrate a practical and moderate-throughput screen for potential antiepileptogenic agents in a rat model of TLE.


Assuntos
Anticonvulsivantes/uso terapêutico , Epilepsia do Lobo Temporal/prevenção & controle , Everolimo/uso terapêutico , Fenobarbital/uso terapêutico , Animais , Anticonvulsivantes/efeitos adversos , Peso Corporal , Convulsivantes , Efeitos Psicossociais da Doença , Modelos Animais de Doenças , Composição de Medicamentos , Descoberta de Drogas , Avaliação Pré-Clínica de Medicamentos , Eletroencefalografia , Epilepsia do Lobo Temporal/induzido quimicamente , Everolimo/efeitos adversos , Ensaios de Triagem em Larga Escala , Ácido Caínico , Masculino , Fenobarbital/efeitos adversos , Ratos , Ratos Sprague-Dawley , Convulsões/prevenção & controle , Pesquisa Médica Translacional
3.
Epilepsia ; 62(7): 1569-1583, 2021 07.
Artigo em Inglês | MEDLINE | ID: mdl-33955001

RESUMO

OBJECTIVE: Growing evidence suggests that dysfunctional astrocytes are crucial players in the development of mesial temporal lobe epilepsy (MTLE). Using a mouse model closely recapitulating key alterations of chronic human MTLE with hippocampal sclerosis, here we asked whether death of astrocytes contributes to the initiation of the disease and investigated potential underlying molecular mechanisms. METHODS: Antibody staining was combined with confocal imaging and semiquantitative real-time polymerase chain reaction analysis to identify markers of different cellular death mechanisms between 4 h and 3 days after epilepsy induction. RESULTS: Four hours after kainate-mediated induction of status epilepticus (SE), we found a significant reduction in the density of astrocytes in the CA1 stratum radiatum (SR) of the ipsilateral hippocampus. This reduction was transient, as within the next 3 days, astrocyte cell numbers recovered to the initial values, which was accompanied by enhanced proliferation. Four hours after SE induction, a small proportion of astrocytes in the ipsilateral CA1 SR expressed autophagy-related genes and proteins, whereas we did not find astrocytes positive for cleaved caspase 3 or terminal deoxynucleotide transferase-mediated deoxyuridine triphosphate nick-end labeling, ruling out apoptosis-related astrocytic death. Importantly, at the same early time point post-SE, many astrocytes in the ipsilateral CA1 SR showed strong expression of genes encoding pro-necroptosis factors, including receptor-interacting protein kinase 3 (RIPK3) and mixed lineage kinase domain-like protein (MLKL). Phosphorylation of MLKL (pMLKL), formation of necrosome complexes composed of RIPK3 and pMLKL, and translocation of pMLKL to the nucleus and to the plasma membrane were often observed in astrocytes of the ipsilateral hippocampus 4 h post-SE. SIGNIFICANCE: The present study revealed that astrocytes die shortly after induction of SE. Our expression data and immunohistochemistry suggest that necroptosis and autophagy contribute to astrocytic death. These findings help to better understand how dysfunctional and pathological remodeling of astrocytes contributes to the initiation of temporal lobe epilepsy.


Assuntos
Astrócitos/patologia , Região CA1 Hipocampal/patologia , Morte Celular , Epilepsia/patologia , Animais , Autofagia/genética , Caspase 3/genética , Contagem de Células , Proliferação de Células , Convulsivantes , Epilepsia/induzido quimicamente , Ácido Caínico , Masculino , Camundongos , Microglia/patologia , Proteínas Quinases/genética , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , Proteína Serina-Treonina Quinases de Interação com Receptores/genética , Estado Epiléptico/induzido quimicamente , Estado Epiléptico/patologia
4.
Epilepsia ; 62(7): 1505-1517, 2021 07.
Artigo em Inglês | MEDLINE | ID: mdl-33979453

RESUMO

OBJECTIVE: One of the challenges in treating patients with drug-resistant epilepsy is that the mechanisms of seizures are unknown. Most current interventions are based on the assumption that epileptic activity recruits neurons and progresses by synaptic transmission. However, several experimental studies have shown that neural activity in rodent hippocampi can propagate independently of synaptic transmission. Recent studies suggest these waves are self-propagating by electric field (ephaptic) coupling. In this study, we tested the hypothesis that neural recruitment during seizures can occur by electric field coupling. METHODS: 4-Aminopyridine was used in both in vivo and in vitro preparation to trigger seizures or epileptiform activity. A transection was made in the in vivo hippocampus and in vitro hippocampal and cortical slices to study whether the induced seizure activity can recruit neurons across the gap. A computational model was built to test whether ephaptic coupling alone can account for neural recruitment across the transection. The model prediction was further validated by in vitro experiments. RESULTS: Experimental results show that electric fields generated by seizure-like activity in the hippocampus both in vitro and in vivo can recruit neurons locally and through a transection of the tissue. The computational model suggests that the neural recruitment across the transection is mediated by electric field coupling. With in vitro experiments, we show that a dielectric material can block the recruitment of epileptiform activity across a transection, and that the electric fields measured within the gap are similar to those predicted by model simulations. Furthermore, this nonsynaptic neural recruitment is also observed in cortical slices, suggesting that this effect is robust in brain tissue. SIGNIFICANCE: These results indicate that ephaptic coupling, a nonsynaptic mechanism, can underlie neural recruitment by a small electric field generated by seizure activity and could explain the low success rate of surgical transections in epilepsy patients.


Assuntos
Campos Eletromagnéticos , Epilepsia/fisiopatologia , Recrutamento Neurofisiológico , 4-Aminopiridina , Animais , Córtex Cerebral/fisiopatologia , Simulação por Computador , Convulsivantes , Epilepsia/diagnóstico , Feminino , Hipocampo/fisiopatologia , Masculino , Camundongos Transgênicos , Modelos Neurológicos , Valor Preditivo dos Testes , Ratos , Ratos Sprague-Dawley , Convulsões/diagnóstico , Convulsões/fisiopatologia , Transmissão Sináptica
5.
Epilepsia ; 62(7): 1701-1714, 2021 07.
Artigo em Inglês | MEDLINE | ID: mdl-34002378

RESUMO

OBJECTIVE: Early life seizures (ELSs) alter activity-dependent maturation of neuronal circuits underlying learning and memory. The pathophysiological mechanisms underpinning seizure-induced cognitive impairment are not fully understood, and critical variables such as sex and dynamic brain states with regard to cognitive outcomes have not been explored. We hypothesized that in comparison to control (CTL) rats, ELS rats would exhibit deficits in spatial cognition correlating with impaired dynamic neural signal coordination between the hippocampus and medial prefrontal cortex (mPFC). METHODS: Male and female rat pups were given 50 flurothyl-induced seizures over 10 days starting at postnatal Day 15. As adults, spatial cognition was tested through active avoidance on a rotating arena. Microwire tetrodes were implanted in the mPFC and CA1 subfield. Single cells and local field potentials were recorded and analyzed in each region during active avoidance and sleep. RESULTS: ELS males exhibited avoidance impairments, whereas female rats were unaffected. During avoidance, hippocampus-mPFC coherence was higher in CTL females than CTL males across bandwidths. In comparison to CTL males, ELS male learners exhibit increased coherence within theta bandwidth as well as altered burst-timing in mPFC cell activity. Hippocampus-mPFC coherence levels are predictive of cognitive outcome in the active avoidance spatial task. SIGNIFICANCE: Spatial cognitive outcome post-ELS is sex-dependent, as females fare better than males. ELS males that learn the task exhibit increased mPFC coherence levels at low-theta frequency, which may compensate for ELS effects on mPFC cell timing. These results suggest that coherence may serve as a biomarker for spatial cognitive outcome post-ELS and emphasize the significance of analyzing sex and dynamic cognition as variables in understanding seizure effects on the developing brain.


Assuntos
Encéfalo/patologia , Hipocampo/patologia , Rede Nervosa/patologia , Córtex Pré-Frontal/patologia , Convulsões/patologia , Animais , Aprendizagem da Esquiva , Encéfalo/fisiopatologia , Região CA1 Hipocampal/patologia , Cognição , Convulsivantes , Eletrodos Implantados , Eletroencefalografia , Feminino , Flurotila , Masculino , Aprendizagem em Labirinto , Ratos , Ratos Sprague-Dawley , Convulsões/induzido quimicamente , Convulsões/psicologia , Caracteres Sexuais , Sono , Percepção Espacial , Ritmo Teta
6.
Eur J Pharmacol ; 901: 174068, 2021 Jun 15.
Artigo em Inglês | MEDLINE | ID: mdl-33798600

RESUMO

Innovative therapeutic strategies are highly needed to tackle the major medical needs of epilepsy, like prevention of epilepsy development in at-risk individuals, treatment of severe and drug-resistant forms, control of co-morbidities. The Neural Regeneration Peptide NRP2945 (a peptidomimetic analogue of the human CAPS-2 protein) has been recently found to exert many potentially anti-epileptic effects, for example increased neuronal survival and differentiation. In the present study, we tested the effects of NRP2945 on the development of epilepsy (epileptogenesis) and on chronic, spontaneous seizures, by using the pilocarpine model of temporal lobe epilepsy. We found that NRP2945 exerts a robust anti-epileptogenic effect, reducing the frequency of spontaneous seizures, exerting a significant neuroprotective effect and attenuating anxiety-like behaviors and cognitive impairment. These effects appear to depend on modulation of the epileptogenesis process and not on seizure suppression, because NRP2945 did not reduce frequency or duration of spontaneous seizures when administered to already epileptic animals. These findings may form the basis for a preventive therapy for individuals at-risk of developing epilepsy.


Assuntos
Anticonvulsivantes/uso terapêutico , Epilepsia do Lobo Temporal/tratamento farmacológico , Oligopeptídeos/uso terapêutico , Animais , Ansiedade/tratamento farmacológico , Ansiedade/psicologia , Comportamento Animal/efeitos dos fármacos , Convulsivantes/uso terapêutico , Epilepsia do Lobo Temporal/induzido quimicamente , Epilepsia do Lobo Temporal/psicologia , Masculino , Fármacos Neuroprotetores/farmacologia , Fármacos Neuroprotetores/uso terapêutico , Pilocarpina , Ratos , Ratos Sprague-Dawley , Reconhecimento Psicológico/efeitos dos fármacos , Convulsões/tratamento farmacológico , Convulsões/etiologia
7.
J Neurosci ; 41(20): 4367-4377, 2021 05 19.
Artigo em Inglês | MEDLINE | ID: mdl-33827934

RESUMO

Early-life inflammatory stress increases seizure susceptibility later in life. However, possible sex- and age-specific differences and the associated mechanisms are largely unknown. C57BL/6 mice were bred in house, and female and male pups were injected with lipopolysaccharide (LPS; 100 µg/kg, i.p.) or vehicle control (saline solution) at postnatal day 14 (P14). Seizure threshold was assessed in response to pentylenetetrazol (1% solution, i.v.) in adolescence (∼P40) and adulthood (∼P60). We found that adult, but not adolescent, mice treated with LPS displayed ∼34% lower seizure threshold compared with controls. Females and males showed similar increased seizure susceptibility, suggesting that altered brain excitability was age dependent, but not sex dependent. Whole-cell recordings revealed no differences in excitatory synaptic activity onto CA1 pyramidal neurons from control or neonatally inflamed adolescent mice of either sex. However, adult mice of both sexes previously exposed to LPS displayed spontaneous EPSC frequency approximately twice that of controls, but amplitude was unchanged. Although these changes were not associated with alterations in dendritic spines or in the NMDA/AMPA receptor ratio, they were linked to an increased glutamate release probability from Schaffer collateral, but not temporoammonic pathway. This glutamate increase was associated with reduced activity of presynaptic GABAB receptors and was independent of the endocannabinoid-mediated suppression of excitation. Our new findings demonstrate that early-life inflammation leads to long-term increased hippocampal excitability in adult female and male mice associated with changes in glutamatergic synaptic transmission. These alterations may contribute to enhanced vulnerability of the brain to subsequent pathologic challenges such as epileptic seizures.SIGNIFICANCE STATEMENT Adult physiology has been shown to be affected by early-life inflammation. Our data reveal that early-life inflammation increases excitatory synaptic transmission onto hippocampal CA1 pyramidal neurons in an age-dependent manner through disrupted presynaptic GABAB receptor activity on Schaffer collaterals. This hyperexcitability was seen only in adult, and not in adolescent, animals of either sex. The data suggest a maturation process, independent of sex, in the priming action of early-life inflammation and highlight the importance of studying mature brains to reveal cellular changes associated with early-life interventions.


Assuntos
Inflamação/fisiopatologia , Células Piramidais/fisiologia , Convulsões/fisiopatologia , Transmissão Sináptica/fisiologia , Animais , Convulsivantes/toxicidade , Potenciais Pós-Sinápticos Excitadores/fisiologia , Feminino , Hipocampo/efeitos dos fármacos , Hipocampo/fisiopatologia , Inflamação/induzido quimicamente , Lipopolissacarídeos/toxicidade , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Pentilenotetrazol/toxicidade , Convulsões/induzido quimicamente
8.
J Pharm Pharmacol ; 73(1): 93-97, 2021 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-33791806

RESUMO

OBJECTIVES: The erythrinan alkaloids erythravine and 11α-hydroxy-erythravine from Erythrina verna (Vell.) have been extensively investigated for their anxiolytic and anticonvulsant effects. Both are structurally similar to the erythrartine that also exhibit anxiolytic effects, but there is no report on its anticonvulsant potential. Since some anxiolytic drugs can be useful in the management of epileptic seizures, we investigated whether erythrartine could prevent seizures induced by different chemoconvulsants. METHODS: Experiments were performed using different concentrations of erythrartine injected via intracerebroventricular in rats submitted to pilocarpine, kainic acid, pentylenetetrazol or picrotoxin-induced seizures. Moreover, the rotarod test was performed to verify the effects of erythrartine on animal motor coordination. RESULTS: Our data showed for the first time that erythrartine prevented the occurrence of seizures induced by all of the chemoconvulsants tested and did not affect locomotor performance neither produced sedative effect on animals. CONCLUSION: Obtained results validate the ethnopharmacological significance of E. verna and provide new information on erythrartine, another erythrinian alkaloid of biotechnological and medicinal interest.


Assuntos
Alcaloides/uso terapêutico , Anticonvulsivantes/uso terapêutico , Erythrina/química , Fitoterapia , Extratos Vegetais/uso terapêutico , Convulsões/prevenção & controle , Alcaloides/farmacologia , Animais , Ansiolíticos/farmacologia , Ansiolíticos/uso terapêutico , Anticonvulsivantes/farmacologia , Convulsivantes , Modelos Animais de Doenças , Epilepsia/tratamento farmacológico , Masculino , Extratos Vegetais/farmacologia , Ratos Wistar , Convulsões/induzido quimicamente
9.
Biochim Biophys Acta Mol Basis Dis ; 1867(6): 166128, 2021 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-33722745

RESUMO

Neural precursor cell expressed developmentally down-regulated gene 4-like (NEDD4-2) encodes a ubiquitin E3 ligase that is involved in epileptogenesis with mechanisms needing further investigation. We constructed a novel Nedd4-2+/- mouse model with half level of both Nedd4-2 long and short isoforms in the brain. Nedd4-2 haploinsufficiency caused increased susceptibility and severity of pentylenetetrazole (PTZ)-induced seizures. Of the 3379 proteins identified by the hippocampal proteomic analysis, 55 were considered altered in Nedd4-2+/- mice compared with wild-type control, among which the inwardly rectifying K+ channel Kir4.1 was up-regulated by 1.83-fold. Kir4.1 was subsequently confirmed to be less ubiquitinated in response to comprised Nedd4-2 in mouse brains and C6 cells. Kir4.1 associated with Nedd4-2 through the threonine312-proline motif in the intracellular domain by target mutagenesis. Adaptor protein 14-3-3 facilitated Nedd4-2-mediated ubiquitination of Kir4.1. Our data consolidate the detailed molecular mechanism of Nedd4-2-mediated Kir4.1 ubiquitination, and provide a possible relationship between increased seizure susceptibility and impaired Kir4.1 ubiquitination in the brain.


Assuntos
Complexos Endossomais de Distribuição Requeridos para Transporte/metabolismo , Haploinsuficiência , Ubiquitina-Proteína Ligases Nedd4/fisiologia , Canais de Potássio Corretores do Fluxo de Internalização/metabolismo , Proteoma/metabolismo , Convulsões/etiologia , Ubiquitinação , Animais , Convulsivantes/toxicidade , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Pentilenotetrazol/toxicidade , Canais de Potássio Corretores do Fluxo de Internalização/genética , Proteoma/análise , Convulsões/metabolismo , Convulsões/patologia
10.
Biochim Biophys Acta Mol Basis Dis ; 1867(6): 166124, 2021 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-33727197

RESUMO

With an associated 20% death risk, epilepsy mainly involves seizures of an unpredictable and recurrent nature. This study was designed to evaluate the neuroprotective effects and underlying mechanisms of insulin on mitochondrial disruption, oxidative stress, cell apoptosis and neurological deficits after epilepsy seizures. Mice were exposed to repetitive injections of pentylenetetrazol at a dose of 37 mg per kg. The influence of insulin was assessed by many biochemical assays, histopathological studies and neurobehavioral experiments. The administration of insulin was proven to increase the latency of seizures while also decreasing their intensity. It also caused a reversal of mitochondrial dysfunction and ameliorated oxidative stress. Additionally, insulin pretreatment upregulated Bcl-2, downregulated Bax, and then played a neuroprotective role against hippocampal neuron apoptosis. Furthermore, when insulin was administered, SIRT1/PGC-1α/SIRT3 signals were activated, possibly due to the fact that insulin's neuroprotective and anti-mitochondrial damage characteristics added to its observed antiepileptic functions. Finally, insulin treatment is thus extremely valuable for effecting improvements in neurological functions, as has been estimated in a series of functional tests. In conclude, the results of this study consequently demonstrate insulin to have significant potential for future application in epilepsy management.


Assuntos
Regulação da Expressão Gênica/efeitos dos fármacos , Insulina/farmacologia , Mitocôndrias/efeitos dos fármacos , Fármacos Neuroprotetores/farmacologia , Estresse Oxidativo/efeitos dos fármacos , Pentilenotetrazol/toxicidade , Convulsões/tratamento farmacológico , Animais , Convulsivantes/toxicidade , Hipoglicemiantes/administração & dosagem , Hipoglicemiantes/farmacologia , Insulina/administração & dosagem , Masculino , Camundongos , Mitocôndrias/metabolismo , Mitocôndrias/patologia , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo/genética , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo/metabolismo , Convulsões/induzido quimicamente , Convulsões/patologia , Transdução de Sinais , Sirtuína 1/genética , Sirtuína 1/metabolismo , Sirtuína 3/genética , Sirtuína 3/metabolismo
11.
Toxicology ; 454: 152737, 2021 04 30.
Artigo em Inglês | MEDLINE | ID: mdl-33631299

RESUMO

Ureases are microbial virulence factors either because of the enzymatic release of ammonia or due to many other non-enzymatic effects. Here we studied two neurotoxic urease isoforms, Canatoxin (CNTX) and Jack Bean Urease (JBU), produced by the plant Canavalia ensiformis, whose mechanisms of action remain elusive. The neurotoxins provoke convulsions in rodents (LD50 ∼2 mg/kg) and stimulate exocytosis in cell models, affecting intracellular calcium levels. Here, electrophysiological and brain imaging techniques were applied to elucidate their mode of action. While systemic administration of the toxins causes tonic-clonic seizures in rodents, JBU injected into rat hippocampus induced spike-wave discharges similar to absence-like seizures. JBU reduced the amplitude of compound action potential from mouse sciatic nerve in a tetrodotoxin-insensitive manner. Hippocampal slices from CNTX-injected animals or slices treated in vitro with JBU failed to induce long term potentiation upon tetanic stimulation. Rat cortical synaptosomes treated with JBU released L-glutamate. JBU increased the intracellular calcium levels and spontaneous firing rate in rat hippocampus neurons. MicroPET scans of CNTX-injected rats revealed increased [18]Fluoro-deoxyglucose uptake in epileptogenesis-related areas like hippocampus and thalamus. Curiously, CNTX did not affect voltage-gated sodium, calcium or potassium channels currents, neither did it interfere on cholinergic receptors, suggesting an indirect mode of action that could be related to the ureases' membrane-disturbing properties. Understanding the neurotoxic mode of action of C. ensiformis ureases could help to unveil the so far underappreciated relevance of these toxins in diseases caused by urease-producing microorganisms, in which the human central nervous system is affected.


Assuntos
Canavalia/química , Síndromes Neurotóxicas/etiologia , Proteínas de Plantas/toxicidade , Toxinas Biológicas/toxicidade , Urease/toxicidade , Animais , Convulsivantes/isolamento & purificação , Convulsivantes/toxicidade , Feminino , Masculino , Camundongos , Sistema Nervoso/efeitos dos fármacos , Sistema Nervoso/patologia , Síndromes Neurotóxicas/fisiopatologia , Proteínas de Plantas/isolamento & purificação , Ratos , Ratos Wistar , Toxinas Biológicas/isolamento & purificação , Urease/isolamento & purificação , Xenopus laevis
12.
Biomed Pharmacother ; 137: 111354, 2021 May.
Artigo em Inglês | MEDLINE | ID: mdl-33561642

RESUMO

Following the high treatment gap and massive impact of epilepsy on global health particularly in low- and middle-income countries, our study aims to investigate cryptolepine, the major alkaloid of Cryptolepis sanguinolenta as well as its solid-lipid nanoparticle formulation for potential antiseizure activity. Cryptolepine was isolated and a solid-lipid formulation was prepared. Antiseizure activity of Solid-Lipid Nanoparticle formulation of cryptolepine (SLN-CRYP) was investigated using Pentylenetetrazole (PTZ)-induced model of seizure-like behaviors in Zebrafish with 2.5 and 5 mg/kg each of cryptolepine and SLN-CRYP. Drug receptor binding and permeability of the compound across the Blood Brain Barrier (BBB) were also assessed. SLN formulation of cryptolepine increased its permeability to the BBB from 0.32 × 10-6 cm/s to 10.81 × 10-6 cm/s. 2.5 and 5 mg/kg of SLN-CRYP significantly reduced mean seizure score (P = 0.0018; F(6, 63) = 23.52) and significantly increased (P < 0.0001; F(6, 63) = 65.41) latency to onset of seizures. The total distance swam by fish administered with 2.5 and 5 mg/kg of SLN-CRYP was significantly (P < 0.000; F(6, 63) = 161.9) decreased. 5 mg/kg of cryptolepine also significantly decreased swimming distance. Cryptolepine exhibited inhibitory modulation of human voltage-gated calcium channels (Cav1.2), H1-receptor, Peripheral Benzodiazepine Receptor and Sigma 2 receptor with a high Ki values of 6133.38 nM and 2945.0 nM, indicating less potent antagonism on Cav1.2 and Sigma 2 receptors compared to Nifedipine and Haloperidol respectively. This study reveals that the solid-lipid nanoparticle formulation of cryptolepine improves its BBB permeability and hence antiseizure activity.


Assuntos
Anticonvulsivantes/química , Anticonvulsivantes/farmacologia , Alcaloides Indólicos/química , Alcaloides Indólicos/farmacologia , Nanopartículas , Quinolinas/química , Quinolinas/farmacologia , Animais , Anticonvulsivantes/administração & dosagem , Barreira Hematoencefálica , Convulsivantes , Cryptolepis/química , Composição de Medicamentos , Alcaloides Indólicos/administração & dosagem , Masculino , Atividade Motora/efeitos dos fármacos , Pentilenotetrazol , Quinolinas/administração & dosagem , Receptores de Droga/metabolismo , Convulsões/induzido quimicamente , Convulsões/prevenção & controle , Natação , Peixe-Zebra
13.
Ann Neurol ; 89(5): 1023-1035, 2021 05.
Artigo em Inglês | MEDLINE | ID: mdl-33604927

RESUMO

OBJECTIVE: Sudden unexpected death in epilepsy (SUDEP) is an unpredictable and devastating comorbidity of epilepsy that is believed to be due to cardiorespiratory failure immediately after generalized convulsive seizures. METHODS: We performed cardiorespiratory monitoring of seizure-induced death in mice carrying either a p.Arg1872Trp or p.Asn1768Asp mutation in a single Scn8a allele-mutations identified from patients who died from SUDEP-and of seizure-induced death in pentylenetetrazole-treated wild-type mice. RESULTS: The primary cause of seizure-induced death for all mice was apnea, as (1) apnea began during a seizure and continued for tens of minutes until terminal asystole, and (2) death was prevented by mechanical ventilation. Fatal seizures always included a tonic phase that was coincident with apnea. This tonic phase apnea was not sufficient to produce death, as it also occurred during many nonfatal seizures; however, all seizures that were fatal had tonic phase apnea. We also made the novel observation that continuous tonic diaphragm contraction occurred during tonic phase apnea, which likely contributes to apnea by preventing exhalation, and this was only fatal when breathing did not resume after the tonic phase ended. Finally, recorded seizures from a patient with developmental epileptic encephalopathy with a previously undocumented SCN8A likely pathogenic variant (p.Leu257Val) revealed similarities to those of the mice, namely, an extended tonic phase that was accompanied by apnea. INTERPRETATION: We conclude that apnea coincident with the tonic phase of a seizure, and subsequent failure to resume breathing, are the determining events that cause seizure-induced death in Scn8a mutant mice. ANN NEUROL 2021;89:1023-1035.


Assuntos
Apneia/complicações , Epilepsia/complicações , Morte Súbita Inesperada na Epilepsia , Animais , Convulsivantes , Diafragma/fisiopatologia , Eletroencefalografia , Eletromiografia , Feminino , Humanos , Lactente , Masculino , Camundongos , Canal de Sódio Disparado por Voltagem NAV1.6/genética , Pentilenotetrazol , Gravidez , Respiração Artificial , Mecânica Respiratória
14.
Metab Brain Dis ; 36(4): 571-579, 2021 04.
Artigo em Inglês | MEDLINE | ID: mdl-33559804

RESUMO

Epilepsy has been associated with several behavioral changes such as depression and anxiety while some antiepileptic drugs can precipitate psychiatric conditions in patients. This study evaluated the ameliorative effect of creatine on seizure severity and behavioral changes in pentylenetetrazole (PTZ) kindled mice. Mice were kindled by administering sub-convulsive doses of PTZ (35 mg/kg i.p.) at interval of 48 h. The naïve group (n = 7) constituted group 1, while successfully kindled mice were randomly assigned to five groups (n = 7). Group II served as vehicle treated group; groups III-V were treated with creatine 75, 150, and 300 mg/kg/day, p.o; Group V was given 25 mg/kg/day of phenytoin p.o. The treatment was for 15 consecutive days. The intensity of convulsion was scored according to a seven-point scale ranging from stage 0-7. Tail suspension test (TST) and Elevated plus maze (EPM) were utilized to assess depression and anxiety-like behavior respectively. After behavioral evaluation on day 15th, their brain was isolated and assayed for catalase, superoxide dismutase, reduced glutathione, and malondialdehyde. There was a significant (p < 0.05) reduction in the seizure scores, anxiety and depression-like behaviors in mice from the 5th day of treatment. The antioxidant assays revealed significant (p < 0.05) increase in catalase and reduced glutathione, and significant (p < 0.05) reduction in lipid peroxidation in treated mice. This study provides evidence for the seizure reducing property of creatine and its ameliorating potential on anxiety and depressive-like behaviors that follows seizure episodes.


Assuntos
Ansiedade/tratamento farmacológico , Creatina/uso terapêutico , Depressão/tratamento farmacológico , Pentilenotetrazol/toxicidade , Convulsões/tratamento farmacológico , Índice de Gravidade de Doença , Animais , Ansiedade/induzido quimicamente , Ansiedade/metabolismo , Convulsivantes/toxicidade , Creatina/farmacologia , Depressão/induzido quimicamente , Depressão/metabolismo , Relação Dose-Resposta a Droga , Glutationa/metabolismo , Peroxidação de Lipídeos/efeitos dos fármacos , Peroxidação de Lipídeos/fisiologia , Malondialdeído/metabolismo , Camundongos , Convulsões/induzido quimicamente , Convulsões/metabolismo
15.
Exp Neurol ; 335: 113512, 2021 01.
Artigo em Inglês | MEDLINE | ID: mdl-33098872

RESUMO

α-actinin-2 (α-actn-2) is an F-actin-crosslinking protein, localized in dendritic spines. In vitro studies suggested that it is involved in spinogenesis, morphogenesis, actin organization, cell migration and anchoring of the NR1 subunit of the N-methyl-D-aspartate (NMDA) receptors in dendritic spines. However, little is known regarding its function in vivo. We examined the levels of α-actn-2 expression within the dentate gyrus (DG) during the development of chronic limbic seizures (epileptogenesis) induced by pilocarpine in rats. In this model, plasticity of the DG glutamatergic granule cells including spine loss, spinogenesis, morphogenesis, neo-synaptogenesis, aberrant migration, and alterations of NMDA receptors have been well characterized. We showed that α-actn-2 immunolabeling was reduced in the inner molecular layer at 1-2 weeks post-status epilepticus (SE), when granule cell spinogenesis and morphogenesis occur. This low level persisted at the chronic stage when new functional synapses are established. This decreased of α-actn-2 protein is concomitant with the recovery of drebrin A (DA), another actin-binding protein, at the chronic stage. Indeed, we demonstrated in cultured cells that in contrast to DA, α-actn-2 did not protect F-actin destabilization and DA inhibited α-actn-2 binding to F-actin. Such alteration could affect the anchoring of NR1 in dendritic spines. Furthermore, we showed that the expression of α-actn-2 and NR1 are co-down-regulated in membrane fractions of pilocarpine animals at chronic stage. Last, we showed that α-actn-2 is expressed in migrating newly born granule cells observed within the hilus of pilocarpine-treated rats. Altogether, our results suggest that α-actn-2 is not critical for the structural integrity and stabilization of granule cell dendritic spines. Instead, its expression is regulated when spinogenesis and morphogenesis occur and within migrating granule cells. Our data also suggest that the balance between α-actn-2 and DA expression levels may modulate NR1 anchoring within dendritic spines.


Assuntos
Actinina/biossíntese , Movimento Celular/genética , Espinhas Dendríticas , Giro Denteado/fisiopatologia , Plasticidade Neuronal/genética , Convulsões/fisiopatologia , Actinina/genética , Actinas/metabolismo , Animais , Convulsivantes , Masculino , Neurogênese/genética , Neuropeptídeos/metabolismo , Pilocarpina , Ratos , Ratos Wistar , Receptores de GABA/metabolismo , Convulsões/induzido quimicamente , Sinapses
16.
Pharmacol Rep ; 73(1): 296-302, 2021 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-33210244

RESUMO

BACKGROUND: The antiseizure drugs commonly used as first- and second-line treatments for neonatal seizures display poor efficacy. Thus, drug mechanisms of action that differ from these typical agents might provide better seizure control. Perampanel, an AMPA-receptor antagonist, and brivaracetam, a SV2A ligand, might fill that role. METHODS: We utilized methyl-6,7-dimethoxy-4-ethyl-beta-carboline-3-carboxylate (DMCM) to evoke seizures in rats to assess the efficacy of perampanel and brivaracetam treatment in clinically relevant doses. RESULTS: In postnatal day (P)10 rats, neither perampanel nor brivaracetam suppressed seizure activity. By contrast, in P21 rats, both drugs decreased the severity of seizures. This effect was evident at the 20 and 40 mg/kg doses of brivaracetam and at the 0.9 and 2.7 mg/kg doses of perampanel. CONCLUSIONS: These data indicate that while the efficacy of these drugs may be limited for neonatal seizures, their efficacy increases over early postnatal development.


Assuntos
Envelhecimento/fisiologia , Anticonvulsivantes/farmacologia , Carbolinas , Convulsivantes , Nitrilas/farmacologia , Piridonas/farmacologia , Pirrolidinonas/farmacologia , Convulsões/prevenção & controle , Animais , Feminino , Ratos , Ratos Sprague-Dawley , Convulsões/induzido quimicamente
17.
Mol Pharmacol ; 99(1): 78-91, 2021 01.
Artigo em Inglês | MEDLINE | ID: mdl-33109687

RESUMO

Tetramethylenedisulfotetramine (TETS) is a so-called "caged" convulsant that is responsible for thousands of accidental and malicious poisonings. Similar to the widely used GABA receptor type A (GABAA) antagonist picrotoxinin, TETS has been proposed to bind to the noncompetitive antagonist (NCA) site in the pore of the receptor channel. However, the TETS binding site has never been experimentally mapped, and we here set out to gain atomistic level insights into how TETS inhibits the human α 2 ß 3 γ 2 GABAA receptor. Using the Rosetta molecular modeling suite, we generated three homology models of the α 2 ß 3 γ 2 receptor in the open, desensitized, and closed/resting state. Three different ligand-docking algorithms (RosettaLigand, Glide, and Swissdock) identified two possible TETS binding sites in the channel pore. Using a combination of site-directed mutagenesis, electrophysiology, and modeling to probe both sites, we demonstrate that TETS binds at the T6' ring in the closed/resting-state model, in which it shows perfect space complementarity and forms hydrogen bonds or makes hydrophobic interactions with all five pore-lining threonine residues of the pentameric receptor. Mutating T6' in either the α 2 or ß 3 subunit reduces the IC50 of TETS by ∼700-fold in whole-cell patch-clamp experiments. TETS is thus interacting at the NCA site in the pore of the GABAA receptor at a location that is overlapping but not identical to the picrotoxinin binding site. SIGNIFICANCE STATEMENT: Our study identifies the binding site of the highly toxic convulsant tetramethylenedisulfotetramine (TETS), which is classified as a threat agent by the World Health Organization. Using a combination of homology protein modeling, ligand docking, site-directed mutagenesis, and electrophysiology, we show that TETS is binding in the pore of the α2ß3γ2 GABA receptor type A receptor at the so-called T6' ring, wherein five threonine residues line the permeation pathway of the pentameric receptor channel.


Assuntos
Hidrocarbonetos Aromáticos com Pontes/metabolismo , Convulsivantes/metabolismo , Receptores de GABA-A/metabolismo , Sítios de Ligação/efeitos dos fármacos , Sítios de Ligação/fisiologia , Hidrocarbonetos Aromáticos com Pontes/química , Convulsivantes/química , Relação Dose-Resposta a Droga , Humanos , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Receptores de GABA-A/química
19.
Nat Neurosci ; 24(1): 19-23, 2021 01.
Artigo em Inglês | MEDLINE | ID: mdl-33318667

RESUMO

Microglial surveillance is a key feature of brain physiology and disease. Here, we found that Gi-dependent microglial dynamics prevent neuronal network hyperexcitability. By generating MgPTX mice to genetically inhibit Gi in microglia, we show that sustained reduction of microglia brain surveillance and directed process motility induced spontaneous seizures and increased hypersynchrony after physiologically evoked neuronal activity in awake adult mice. Thus, Gi-dependent microglia dynamics may prevent hyperexcitability in neurological diseases.


Assuntos
Receptor Quinase 1 Acoplada a Proteína G/fisiologia , Microglia/fisiologia , Rede Nervosa/fisiologia , Animais , Sinalização do Cálcio , Movimento Celular , Convulsivantes , Eletroencefalografia , Vigilância Imunológica , Camundongos , Microglia/enzimologia , Microglia/ultraestrutura , Doenças do Sistema Nervoso/fisiopatologia , Fenômenos Fisiológicos do Sistema Nervoso , Pilocarpina , Convulsões/fisiopatologia , Transdução de Sinais , Proteínas rho de Ligação ao GTP/metabolismo
20.
Int J Mol Sci ; 21(23)2020 Dec 04.
Artigo em Inglês | MEDLINE | ID: mdl-33291789

RESUMO

BACKGROUND: Epilepsy is a chronic neurological disorder characterized by the recurrence of seizures. One-third of patients with epilepsy may not respond to antiseizure drugs. PURPOSE: We aimed to examine whether D-limonene, a cyclic monoterpene, exhibited any antiseizure activity in the pentylenetetrazole (PTZ)-induced kindling mouse model and in vitro. METHODS: PTZ kindling mouse model was established by administering PTZ (30 mg/kg) intraperitoneally to mice once every 48 h. We performed immunoblot blots, immunohistochemistry (IHC), and high-performance liquid chromatography (HPLC) analysis after the behavioral study. RESULTS: An acute injection of PTZ (60 mg/kg) induced seizure in mice, while pretreatment with D-limonene inhibited PTZ-induced seizure. Repeated administration of PTZ (30 mg/kg) increased the seizure score gradually in mice, which was reduced in D-limonene (10 mg/kg)-pretreated group. In addition, D-limonene treatment increased glutamate decarboxylase-67 (GAD-67) expression in the hippocampus. Axonal sprouting of hippocampal neurons after kindling was inhibited by D-limonene pretreatment. Moreover, D-limonene reduced the expression levels of Neuronal PAS Domain Protein 4 (Npas4)-induced by PTZ. Furthermore, the adenosine A2A antagonist SCH58261 and ZM241385 inhibited anticonvulsant activity and gamma-aminobutyric acid (GABA)ergic neurotransmission-induced by D-limonene. CONCLUSION: These results suggest that D-limonene exhibits anticonvulsant activity through modulation of adenosine A2A receptors on GABAergic neuronal function.


Assuntos
Neurônios GABAérgicos/metabolismo , Limoneno/farmacologia , Pentilenotetrazol/efeitos adversos , Receptor A2A de Adenosina/metabolismo , Convulsões/etiologia , Convulsões/metabolismo , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Convulsivantes/administração & dosagem , Convulsivantes/efeitos adversos , Modelos Animais de Doenças , Regulação da Expressão Gênica/efeitos dos fármacos , Glutamato Descarboxilase/genética , Glutamato Descarboxilase/metabolismo , Hipocampo/citologia , Hipocampo/efeitos dos fármacos , Hipocampo/metabolismo , Camundongos , Plasticidade Neuronal/efeitos dos fármacos , Pentilenotetrazol/administração & dosagem , Fosforilação , Ratos , Convulsões/fisiopatologia
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