RESUMO
GABA is synthesized from glutamate by glutamate decarboxylase (GAD), which exists in two isoforms, that is, GAD65 and GAD67. In line with GAD65 being located in the GABAergic synapse, several studies have demonstrated that this isoform is important during sustained synaptic transmission. In contrast, the functional significance of GAD65 in the maintenance of GABA destined for extrasynaptic tonic inhibition is less well studied. Using GAD65-/- and wild type GAD65+/+ mice, this was examined employing the cortical wedge preparation, a model suitable for investigating extrasynaptic GABA(A) receptor activity. An impaired tonic inhibition in GAD65-/- mice was revealed demonstrating a significant role of GAD65 in the synthesis of GABA acting extrasynaptically. The correlation between an altered tonic inhibition and metabolic events as well as the functional and metabolic role of GABA synthesized by GAD65 was further investigated in vivo. Tonic inhibition and the demand for biosynthesis of GABA were augmented by injection of kainate into GAD65-/- and GAD65+/+ mice. Moreover, [1-(13) C]glucose and [1,2-(13) C]acetate were administered to study neuronal and astrocytic metabolism concomitantly. Subsequently, cortical and hippocampal extracts were analyzed by NMR spectroscopy and mass spectrometry, respectively. Although seizure activity was induced by kainate, neuronal hypometabolism was observed in GAD65+/+ mice. In contrast, kainate evoked hypermetabolism in GAD65-/- mice exhibiting deficiencies in tonic inhibition. These findings underline the importance of GAD65 for synthesis of GABA destined for extrasynaptic tonic inhibition, regulating epileptiform activity.
Assuntos
Epilepsia/metabolismo , Glutamato Descarboxilase/fisiologia , Inibição Neural/fisiologia , Ácido gama-Aminobutírico/biossíntese , Animais , Córtex Cerebral/enzimologia , Córtex Cerebral/metabolismo , Corpo Caloso/enzimologia , Corpo Caloso/metabolismo , Epilepsia/enzimologia , Epilepsia/patologia , Glutamato Descarboxilase/deficiência , Isoenzimas/deficiência , Isoenzimas/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Vesículas Sinápticas/enzimologia , Vesículas Sinápticas/metabolismo , Ácido gama-Aminobutírico/fisiologiaRESUMO
THIP (4,5,6,7-tetrahydroisoxazolo[5,4-c]pyridin-3-ol) is a GABAA receptor agonist with varying potencies and efficacies at γ-subunit-containing receptors. More importantly, THIP acts as a selective superagonist at δ-subunit-containing receptors (δ-GABAA Rs) at clinically relevant concentrations. Evaluation of THIP as a potential anticonvulsant has given contradictory results in different animal models and for this reason, we reevaluated the anticonvulsive properties of THIP in the murine pentylenetetrazole (PTZ) kindling model. As loss of δ-GABAA R in the dentate gyrus has been associated with several animal models of epilepsy, we first investigated the presence of functional δ-GABAA receptors. Both immunohistochemistry and Western blot data demonstrated that δ-GABAA R expression is not only present in the dentate gyrus, but also the expression level was enhanced in the early phase after PTZ kindling. Whole-cell patch-clamp studies in acute hippocampal brain slices revealed that THIP was indeed able to induce a tonic inhibition in dentate gyrus granule cells. However, THIP induced a tonic current of similar magnitude in the PTZ-kindled mice compared to saline-treated animals despite the observed upregulation of δ-GABAA Rs. Even in the demonstrated presence of functional δ-GABAA Rs, THIP (0.5-4 mg/kg) showed no anticonvulsive effect in the PTZ kindling model using a comprehensive in vivo evaluation of the anticonvulsive properties.
RESUMO
Three 4-substituted 1,2,5-oxadiazol-3-ols containing aminoalkyl substituents (analogues and homologues of gamma-aminobutyric acid (GABA)) were synthesized to investigate the hydroxy-1,2,5-oxadiazolyl moiety as a bioisoster for a carboxyl group at GABA receptors. The pK(a) values of the target compounds were close to those of GABA. At GABA(A) receptors of cultured cerebral cortical neurons, weak agonist and partial agonist profiles were identified, demonstrating the 4-hydroxy-1,2,5-oxadiazol-3-yl unit to be a nonclassical carboxyl group bioisoster.
Assuntos
Agonistas de Receptores de GABA-A , Agonistas dos Receptores de GABA-B , Oxidiazóis/síntese química , Ácido gama-Aminobutírico/análogos & derivados , Animais , Astrócitos/efeitos dos fármacos , Astrócitos/metabolismo , Ligação Competitiva , Linhagem Celular , Células Cultivadas , Córtex Cerebral/citologia , Proteínas da Membrana Plasmática de Transporte de GABA/metabolismo , Antagonistas de Receptores de GABA-A , Antagonistas de Receptores de GABA-B , Humanos , Técnicas In Vitro , Camundongos , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Oxidiazóis/química , Oxidiazóis/farmacologia , Técnicas de Patch-Clamp , Ratos , Relação Estrutura-Atividade , Ácido gama-Aminobutírico/síntese química , Ácido gama-Aminobutírico/química , Ácido gama-Aminobutírico/farmacologiaRESUMO
Although animal models based on pentylenetetrazole (PTZ) are widely used, the mechanism by which PTZ elicits its action is not very well understood. At the molecular level, a generally accepted mechanism of PTZ is noncompetitive antagonism of the gamma-aminobutyric acid (GABA)(A) receptor complex. By a systematic pharmacological investigation of various GABA(A) receptor ligands, our aim was to gain a better understanding of the GABAergic mechanisms involved in different PTZ-induced seizures. We investigated anticonvulsant effects of various specific GABA(A) receptor ligands, which are believed to bind to different binding sites on the GABA(A) receptor complex, on PTZ-induced clonic seizures in drug naive and PTZ-kindled mice as well as their effects on the development of PTZ kindling. Diazepam and alphaxalone produced potent anticonvulsant effects and completely suppressed the development of kindling. In contrast, the antagonists bicuculline and dehydroepiandrosterone sulfate (DHEAS) displayed neither anticonvulsant nor antiepileptogenic effects. Flumazenil, often used as a reference antagonist at the GABA(A) receptor benzodiazepine (BZ) binding site, lacked anticonvulsant effects but surprisingly inhibited the development of PTZ-kindled seizures. The agonist 4,5,6,7-tetrahydroisoxazolo-(5,4-c)pyridin-3-ol (THIP) was devoid of both anticonvulsant and antiepileptogenic effects. Marked differences in drug sensitivity were observed between models based on single and chronic administration of PTZ showing that the two sets of models are fundamentally different. These results describe the pharmacology of a set of ligands believed to bind to different sites at the GABA(A) receptor complex in animal models based on PTZ and demonstrate that a drug's action in these models cannot be readily explained by agonistic or antagonistic properties at the receptor level.
Assuntos
Anticonvulsivantes/uso terapêutico , Convulsivantes , Epilepsia/prevenção & controle , Excitação Neurológica/efeitos dos fármacos , Pentilenotetrazol , Receptores de GABA-A/efeitos dos fármacos , Animais , Bicuculina/farmacologia , Sulfato de Desidroepiandrosterona/farmacologia , Diazepam/farmacologia , Relação Dose-Resposta a Droga , Epilepsia/induzido quimicamente , Flumazenil/farmacologia , Agonistas GABAérgicos/farmacologia , Antagonistas GABAérgicos/farmacologia , Moduladores GABAérgicos/farmacologia , Isoxazóis/farmacologia , Ligantes , Camundongos , Pregnanodionas/farmacologiaRESUMO
The N-acylethanolamine phospholipids (NAPE) are precursors for N-acylethanolamines (NAE), including anandamide (20:4-NAE), which is a ligand for the cannabinoid receptors. Previously, NAPE were believed to be found only in injured tissue, e.g., after neurodegenerative insults. Neuronal injury may occur in response to seizure activity. Therefore, we investigated the effect of pentylenetetrazol (PTZ)-induced seizures in PTZ-kindled mice on the level of NAPE in the brain. Male NMRI mice were kindled with PTZ injections 3 times/wk, thereby developing clonic seizures in response to PTZ. Mice were killed within 30 min after the clonic seizure on the test day (12th injection) and the brains were collected. Eight species of NAPE were analyzed as the glycerophospho-N-acylethanolamines by high-performance liquid chromatography-coupled electrospray ionization mass spectrometry. No effect of the PTZ kindling on the NAPE levels in murine brains was observed. Total NAPE in control mice cortex (n = 4) was 16.4 +/- 3.0 micromol/g wet weight of which 20:4-NAPE accounted for 3.6 mol%, and the major species was 16:0-NAPE, accounting for 52.1 mol%. Determination of the activity of NAPE-hydrolyzing phospholipase D and of N-acyltransferase in brain membrane preparations from adult and 3-d-old mice revealed an enzyme pattern in the adult mice that was favorable for NAE accumulation as opposed to NAPE accumulation. Thus, there was no difference in NAPE levels; at present, however, this does not exclude that NAE may accumulate during seizure.
Assuntos
Córtex Cerebral/metabolismo , Fosfatidiletanolaminas/metabolismo , Convulsões/metabolismo , Fatores Etários , Animais , Arilamina N-Acetiltransferase/metabolismo , Radioisótopos de Carbono , Córtex Cerebral/efeitos dos fármacos , Convulsivantes , Excitação Neurológica/efeitos dos fármacos , Excitação Neurológica/metabolismo , Masculino , Camundongos , Pentilenotetrazol , Fosfolipase D/metabolismo , Convulsões/induzido quimicamenteRESUMO
The cortical wedge is a commonly applied model for in vitro screening of new antiepileptic drugs (AEDs) and has been extensively used in characterization of well-known AEDs. However, the predictive validity of this model as a screening model has been questioned as, e.g., carbamazepine has been reported to lack effect in this model. The neuroplastic changes induced in acute and chronic animal models of epilepsy are known to affect the pharmacological profile of AEDs in vivo. Hence, we investigated whether brain slices from pentylenetetrazole (PTZ)-kindled animals could provide a more predictive screening model for AEDs. To this end, we compared the in vitro and in vivo pharmacological profile of several selected AEDs (phenobarbital, phenytoin, tiagabine, fosphenytoin, valproate, and carbamazepine) along with citalopram using the PTZ-kindled model and brain slices from naïve, saline-injected and PTZ-kindled mice. Our data suggest that the use of slices from PTZ-kindled mice in the cortical wedge does not increase the predictive validity of the model as an in vitro screening model for AEDs. Traditionally, the incidence of certain seizure types is widely used as a measure to characterize drug action in animal models of epilepsy. In our study, the anticonvulsant effect of the AEDs was investigated in vivo using several observational parameters (i.e., incidence and duration of convulsions, latency to clonic convulsions, and severity of convulsions). We found that including the observational parameter "severity" offered important additional information about the drug profile that would otherwise be lost if only a single parameter as "incidence" was used.
Assuntos
Anticonvulsivantes/farmacologia , Encéfalo/efeitos dos fármacos , Avaliação Pré-Clínica de Medicamentos/métodos , Pentilenotetrazol/farmacologia , Animais , Encéfalo/citologia , Córtex Cerebral/efeitos dos fármacos , Masculino , CamundongosRESUMO
RATIONALE: Schizophrenia is a complex psychiatric disorder comprised of three main classes of symptoms: positive, negative and cognitive symptoms. Currently, no approved treatment exists for the cognitive symptoms. There is thus a great need for research aiming at identifying novel targets for treatment of this indication. Several neurotransmitter systems are affected in schizophrenia patients, including the γ-amino butyric acid (GABAergic) system, demonstrated by reduced parvalbumin-containing interneurons, glutamate decarboxylase (GAD) and the GABA transporter GAT-1. Furthermore, gene expression of several GABA(A) receptor sub-units, such as α1, α4 and δ is reduced in the dorsolateral prefrontal cortex of schizophrenia patients. OBJECTIVES: The psychotomimetic NMDA receptor antagonist phencyclidine (PCP) is frequently employed to model schizophrenia in animal disease models. Sub-chronic PCP treatment of female hooded Lister rats has repeatedly been shown to induce impairments in object recognition memory, and this model was therefore chosen for the examination of the potential of positive modulation of extrasynaptic GABA(A) receptors in alleviating the PCP-induced deficit. RESULTS: Rats treated sub-chronically with PCP showed significant impairments in recognition memory. This deficit was reversed by positive modulation of extrasynaptic GABA(A) receptors. CONCLUSION: The present study shows that extrasynaptic GABA(A) receptors may present a novel target for the development of therapeutics aimed at improving cognitive deficits in schizophrenia.
Assuntos
Comportamento Animal/efeitos dos fármacos , Agonistas de Receptores de GABA-A/farmacologia , Isoxazóis/farmacologia , Receptores de GABA-A/efeitos dos fármacos , Reconhecimento Psicológico/efeitos dos fármacos , Esquizofrenia/tratamento farmacológico , Psicologia do Esquizofrênico , Análise de Variância , Animais , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Ciclo Estral , Comportamento Exploratório/efeitos dos fármacos , Feminino , Atividade Motora/efeitos dos fármacos , Fenciclidina , Ratos , Receptores de GABA-A/metabolismo , Esquizofrenia/induzido quimicamente , Esquizofrenia/metabolismo , Fatores de TempoRESUMO
Cognitive deficits are a major clinical unmet need in schizophrenia. The psychotomimetic drug phencyclidine (PCP) is widely applied in rodents to mimic symptoms of schizophrenia, including cognitive deficits. Previous studies have shown that sub-chronic PCP induces an enduring episodic memory deficit in female Lister Hooded rats in the novel object recognition (NOR) task. Here we show that positive modulation of AMPA receptor (AMPAR) mediated glutamate transmission alleviates cognitive deficits induced by sub-chronic PCP treatment. Female Lister hooded rats were treated sub-chronically with either vehicle (0.9% saline) or PCP (2mg/kg two doses per day for 7 days), followed by a 7 days washout period. 30 min prior to the acquisition trial of the NOR task animals were dosed with either vehicle, CX546 (10, 40 or 80 mg/kg) or CX516 (0.5, 2.5, 10, 40 or 80 mg/kg). Our results show that sub-chronic PCP treatment induced a significant decrease in the discrimination index (DI) and both ampakines CX546 and CX516 were able to reverse this disruption of object memory in rats in the novel object recognition task. These data suggest that positive AMPAR modulation may represent a mechanism for treatment of cognitive deficits in schizophrenia.
Assuntos
Comportamento Exploratório/efeitos dos fármacos , Transtornos da Memória/tratamento farmacológico , Receptores de AMPA/metabolismo , Reconhecimento Psicológico/efeitos dos fármacos , Análise de Variância , Animais , Comportamento Animal/efeitos dos fármacos , Química Encefálica , Dioxóis/análise , Dioxóis/farmacologia , Relação Dose-Resposta a Droga , Feminino , Alucinógenos , Transtornos da Memória/induzido quimicamente , Atividade Motora/efeitos dos fármacos , Fenciclidina/toxicidade , Piperidinas/análise , Piperidinas/farmacologia , Ratos , Gravação em Vídeo , Percepção Visual/efeitos dos fármacosRESUMO
ETHNOPHARMACOLOGICAL RELEVANCE: Several medicinal plants are used in Mali to treat epilepsy and convulsions. So far, no studies have investigated the pharmacological effect of these plants. AIMS: The aim of this study was to investigate the in vitro and in vivo antiepileptic potential of the ethanolic extracts of 11 Malian medicinal plants. MATERIALS AND METHODS: The extracts were screened for antiepileptic properties in the mouse cortical wedge preparation and in the [3H]-flumazenil binding assay. Two of the plant extracts were investigated for anticonvulsive properties in the pentylenetetrazol (PTZ) kindling model in mice. Possible side effects on motor impairment were evaluated using the rota-rod test. RESULTS: Extracts of 10 of the 11 medicinal plants showed affinity to the benzodiazepine binding site on the GABAA receptor. Seven of the 11 extracts inhibited the spontaneous discharges (SEDs) in the mouse cortical wedge preparation, with the extracts of Flueggea virosa and Psorospermum senegalense being the most potent. However, when tested for in vivo anticonvulsive properties these two extracts failed to show any effect on PTZ-induced seizures in mice. CONCLUSIONS: The pharmacological screening of the ethanolic extracts of 11 Malian medicinal plants in vitro lead to the identification of several extracts with potential anticonvulsant properties.
Assuntos
Anticonvulsivantes/uso terapêutico , Epilepsia/tratamento farmacológico , Agonistas de Receptores de GABA-A , Fitoterapia , Extratos Vegetais/uso terapêutico , Plantas Medicinais , Convulsões/tratamento farmacológico , Animais , Anticonvulsivantes/farmacologia , Epilepsia/induzido quimicamente , Técnicas In Vitro , Masculino , Mali , Camundongos , Pentilenotetrazol , Extratos Vegetais/farmacologia , Ratos , Convulsões/induzido quimicamenteRESUMO
The ketogenic diet (KD) is used for the treatment of refractory epilepsy in children, however, the mechanism(s) remains largely unknown. Also, the antiepileptogenic potential in animal models of epilepsy has been poorly addressed. Activation of cannabinoid type-1 receptor (CB(1)-R) upon seizure activity may mediate neuroprotection as may several N-acylethanolamines. It is unknown how the KD interfere with the endocannabinoid system. We investigated the antiepileptogenic potential of the KD in the pentylenetetrazole kindling model in young mice and measured the hippocampal levels of CB(1)-R by Western blot and of endocannabinoids and N-acylethanolamines by mass spectrometry. The KD significantly decreased incidence and severity of seizures, and significantly increased the latency to clonic convulsions. There were no changes in levels of endocannabinoids or CB(1)-R expression by either seizure activity or type of diet. The level of oleoylethanolamide as well as the sum of N-acylethanolamines were significantly decreased by the KD, but were unaffected by seizure activity. The study shows that the KD had clear antiepileptogenic properties in the pentylenetetrazole kindling model and does not support a role of endocannabinoids in this model. The significance of the decreased hippocampal level of oleoylethanolamide awaits further studies.
Assuntos
Dieta Cetogênica/métodos , Epilepsia/dietoterapia , Etanolaminas/metabolismo , Hipocampo/metabolismo , Receptor CB1 de Canabinoide/metabolismo , Animais , Moduladores de Receptores de Canabinoides/metabolismo , Convulsivantes/farmacologia , Modelos Animais de Doenças , Regulação para Baixo/fisiologia , Endocanabinoides , Epilepsia/metabolismo , Epilepsia/fisiopatologia , Hipocampo/fisiopatologia , Excitação Neurológica/efeitos dos fármacos , Excitação Neurológica/metabolismo , Masculino , Camundongos , Ácidos Oleicos/metabolismo , Pentilenotetrazol/farmacologiaRESUMO
In a recent study, EF1502 [N-[4,4-bis(3-methyl-2-thienyl)-3-butenyl]-3-hydroxy-4-(methylamino)-4,5,6,7-tetrahydrobenzo [d]isoxazol-3-ol], which is an N-substituted analog of the GAT1-selective GABA uptake inhibitor exo-THPO (4-amino-4,5,6,7-tetrahydrobenzo[d]isoxazol-3-ol), was found to inhibit GABA transport mediated by both GAT1 and GAT2 in human embryonic kidney (HEK) cells expressing the mouse GABA transporters GAT1 to 4 (mGAT1-4). In the present study, EF1502 was found to possess a broad-spectrum anticonvulsant profile in animal models of generalized and partial epilepsy. When EF1502 was tested in combination with the clinically effective GAT1-selective inhibitor tiagabine [(R)-N-[4,4-bis(3-methyl-2-thienyl)-3-butenyl]nipecotic acid] or LU-32-176B [N-[4,4-bis(4-fluorophenyl)-butyl]-3-hydroxy-4-amino-4,5,6,7-tetrahydrobenzo[d]isoxazol-3-ol], another GAT1-selective N-substituted analog of exo-THPO, a synergistic rather than additive anticonvulsant interaction was observed in the Frings audiogenic seizure-susceptible mouse and the pentylenetetrazol seizure threshold test. In contrast, combination of the two mGAT1-selective inhibitors, tiagabine and LU-32-176B, resulted in only an additive anticonvulsant effect. Importantly, the combination of EF1502 and tiagabine did not result in a greater than additive effect in the rotarod behavioral impairment test. In subsequent in vitro studies conducted in HEK-293 cells expressing the cloned mouse GAT transporters mGAT1 and mGAT2, EF1502 was found to noncompetitively inhibit both mGAT1 and the betaine/GABA transporter mGAT2 (K(i) of 4 and 5 muM, respectively). Furthermore, in a GABA release study conducted in neocortical neurons, EF1502 did not act as a substrate for the GABA carrier. Collectively, these findings support a functional role for mGAT2 in the control of neuronal excitability and suggest a possible utility for mGAT2-selective inhibitors in the treatment of epilepsy.
Assuntos
Aciltransferases/antagonistas & inibidores , Aciltransferases/fisiologia , Anticonvulsivantes/farmacologia , Estimulação Acústica , Aciltransferases/genética , Tonsila do Cerebelo/fisiologia , Animais , Comportamento Animal/efeitos dos fármacos , Células Cultivadas , Clonagem Molecular , Convulsivantes , Sinergismo Farmacológico , Epilepsia/induzido quimicamente , Epilepsia/prevenção & controle , Epilepsia Reflexa/induzido quimicamente , Epilepsia Reflexa/prevenção & controle , Excitação Neurológica , Masculino , Camundongos , N-Acetilglucosaminiltransferases , Neurônios/efeitos dos fármacos , Ácidos Nipecóticos/farmacologia , Pentilenotetrazol , Pilocarpina , Transtornos Psicomotores/induzido quimicamente , Transtornos Psicomotores/prevenção & controle , Proteínas Recombinantes/antagonistas & inibidores , Proteínas Recombinantes/genética , Convulsões/induzido quimicamente , Convulsões/prevenção & controle , Tiagabina , Ácido gama-Aminobutírico/metabolismoRESUMO
A number of neurosteroids bind to GABAA receptors and alter their responsiveness to neurotransmitters. Considerable effort has been devoted to understanding how this form of receptor modulation alters inhibitory synaptic function. Neurosteroid-sensitive GABAA receptors have also been demonstrated in many endocrine cells, but little is known about how neurosteroids modulate the release of hormones. Here, the action of allopregnanolone, a neurosteroid that enhances GABAA receptor-mediated responses, was investigated in posterior pituitary nerve terminals and intermediate pituitary endocrine cells. Patch clamp recordings showed that GABA-evoked currents were enhanced to similar degrees and with similar concentration dependences in both locations. An organ bath preparation of the neurointermediate lobe was used to investigate drug effects on secretion of vasopressin and alpha-melanocyte stimulating hormone. GABA increased the basal release of vasopressin and alpha-melanocyte stimulating hormone from the posterior and intermediate pituitary lobe, respectively, an effect that could be blocked by picrotoxinin. Vasopressin release evoked by electrical stimulation was also examined, and a small statistically significant inhibition by 5 microM GABA was observed. Allopregnanolone increased the basal release of vasopressin, and this effect was blocked by the GABAA receptor antagonist picrotoxinin. Allopregnanolone had no effect in conjunction with GABA. In contrast to the posterior lobe, allopregnanolone had no effect on release from the intermediate lobe. Thus, allopregnanolone in physiological relevant concentrations modulates GABAA receptors in both the posterior and intermediate lobes, but only affects hormone release in the posterior lobe.