RESUMO
Measurement of neurotransmitters during normal or altered function in cerebral slices could be an important tool to better understand the relationship between biochemical changes and electrophysiological activity. Some attempts of this analysis have been made; however, the current techniques do not have the appropriate time resolution to establish this relationship. The use of electrochemical biosensors has allowed for good time resolution, but problems related to the reduction of signal noise and biofouling of the electrode surface could be an important issue. In this work, we propose a new alternative to simultaneously measure glutamate and electrical activity with a high temporal resolution in brain slices. This approach is based on the use of enzymatic reactors that generate a fluorescent derivative from glutamate that can be measured at high temporal resolution. The results presented here show a reliable measurement of this neurotransmitter in brain slices obtained from intact animals under the effect of a glutamate transporter blocker DL-threo-beta-benzyloxyaspartate as well as the potassium channel blocker 4-aminopyridine. Differences in the levels of glutamate and high frequency and amplitude discharges as an effect of drug administration were found in brain slices obtained from epileptic rats (p<0.05). In conclusion, this method could be used to measure neurotransmitter concentration online at a near physiological temporal resolution, which can then be correlated to the electrical activity that is simultaneously recorded.
Assuntos
Encéfalo/citologia , Encéfalo/fisiologia , Eletrofisiologia/métodos , Espaço Extracelular/metabolismo , Ácido Glutâmico/metabolismo , 4-Aminopiridina/farmacologia , Sistema X-AG de Transporte de Aminoácidos/antagonistas & inibidores , Animais , Ácido Aspártico/farmacologia , Encéfalo/efeitos dos fármacos , Eletrofisiologia/instrumentação , Espaço Extracelular/efeitos dos fármacos , Corantes Fluorescentes/química , Ácido Glutâmico/química , Hipocampo/citologia , Hipocampo/efeitos dos fármacos , Hipocampo/fisiologia , Masculino , Ratos , Ratos WistarRESUMO
Fast ripples (FRs) are pathological high frequency oscillations that occur in patients with temporal lobe epilepsy (TLE), as well as in animal models of epilepsy in which seizures are induced with kainic acid or pilocarpine. These oscillations have been considered potential biomarkers of epileptogenesis in the hippocampus. Indeed, experimental evidence suggests an important role of serotonin in epilepsy and an increased frequency of FRs have been demonstrated in slow wave sleep, a period during which serotonin levels decrease. Accordingly, we investigated the role of serotonin in FRs modulation by evaluating the effects of citalopram, a blocker of serotonin uptake, on the occurrence of spontaneous FRs measured through intracranial bilateral EEG recording of the hippocampus of rats with spontaneous recurrent seizures. In addition, we recorded the mean number of oscillation cycles per FRs event and the average frequency (Hz) before, during and after citalopram administration in order to determine whether increases in extra-synaptic serotonin levels modulate FRs. The elevation of serotonin levels induced by citalopram (4.78 ± 1.69 nM) reduced the occurrence of spontaneous FRs (57%), the mean number of oscillation cycles per FRs event (34%) and the average frequency of FRs (33%). These findings suggest an important modulatory effect of serotonin on FRs.
Assuntos
Citalopram/farmacologia , Hipocampo/efeitos dos fármacos , Convulsões/tratamento farmacológico , Inibidores Seletivos de Recaptação de Serotonina/farmacologia , Serotonina/metabolismo , Animais , Cateteres de Demora , Modelos Animais de Doenças , Eletrodos Implantados , Eletroencefalografia , Epilepsia/tratamento farmacológico , Epilepsia/fisiopatologia , Espaço Extracelular/metabolismo , Hipocampo/fisiopatologia , Masculino , Microdiálise , Ratos Wistar , Convulsões/fisiopatologiaRESUMO
Hypoxia at birth is a major source of brain damage and it is associated with serious neurological sequelae in survivors. Alterations in the extracellular turnover of glutamate (Glu) and acetylcholine (ACh), two neurotransmitters that are essential for normal hippocampal function and learning and memory processes, may contribute to some of the neurological effects of perinatal hypoxia. We set out to determine the immediate and long-lasting effects of hypoxia on the turnover of these neurotransmitters by using microdialysis to measure the extracellular concentration of Glu and ACh in hippocampus, when hypoxia was induced in rats at postnatal day (PD) 7, and again at PD30. In PD7 rats, hypoxia induced an increase in extracellular Glu concentrations that lasted for up to 2.5 h and a decrease in extracellular ACh concentrations over this period. By contrast, perinatal hypoxia attenuated Glu release in asphyxiated rats, inducing a decrease in basal Glu levels when these animals reached PD30. Unlike Glu, the basal ACh levels in these animals were greater than in controls at PD30, although ACh release was stimulated less strongly than in control animals. These results provide the first evidence of the initial and long term consequences of the hypoxia on Glu and ACh turnover in the brain, demonstrating that hypoxia produces significant alterations in hippocampal neurochemistry and physiology.
Assuntos
Acetilcolina/metabolismo , Espaço Extracelular/metabolismo , Glutamatos/metabolismo , Hipóxia Encefálica/metabolismo , 4-Aminopiridina/farmacologia , Animais , Animais Recém-Nascidos , Química Encefálica/efeitos dos fármacos , Espaço Extracelular/efeitos dos fármacos , Feminino , Hipocampo/metabolismo , Bloqueadores dos Canais de Potássio/farmacologia , Gravidez , Ratos , Convulsões/metabolismoRESUMO
AIMS: The article highlights the general structural characteristics, functional properties and distribution of glutamate transporters, as well as the role they play in epilepsy and oxidative stress. DEVELOPMENT: Transporters of amino acids such as glutamate are considered to be proteins that are extremely important in the central nervous system because they participate in the capture of the neurotransmitter following its release in the synaptic cleft, thus putting an end to its effect and limiting glutamate-mediated excitability. These proteins belong to the family of Na+/K+ dependent transporters. A growing body of evidence has been gathered to show that these transporters are involved in several neuronal disorders, such as epilepsy and cerebral ischaemia. In this regard, it is considered that some defect in the structure of the transporters could affect their functioning and, therefore, favour the hyperexcitability produced by glutamate; this in turn would lead to the pathological disorders that are found in epilepsy. CONCLUSIONS: A detailed study of the structure and functioning of these transporters, as well as the role they play in the more common neurological diseases, such as epilepsy, would afford us a clearer view of new therapeutic alternatives with which to fight this kind of neuronal disorder in the future.
Assuntos
Sistema X-AG de Transporte de Aminoácidos/química , Sistema X-AG de Transporte de Aminoácidos/metabolismo , Epilepsia/metabolismo , Ácido Glutâmico/metabolismo , Estresse Oxidativo , Sistema X-AG de Transporte de Aminoácidos/genética , Animais , Transporte Biológico/fisiologia , Epilepsia/genética , Humanos , Modelos Moleculares , Estrutura MolecularRESUMO
In order to study the role of amino acids in the hippocampus and the entorhinal cortex during the convulsive process induced by 4-aminopyridine (4-AP), we have used a device allowing the simultaneous microdialysis and the recording of their electrical activity of both regions in freely moving rats. We found that infusion of 4-AP into the entorhinal cortex resulted in a large increase in extracellular glutamate and glutamine and small increases in glycine and taurine levels. Likewise, infusion of 4-AP into the hippocampus resulted in a major increase in glutamate, as well as slight increases in taurine and glycine. In both infused regions the peak concentration of extracellular glutamate was observed 15 min after 4-AP administration. No significant changes were found in the non-infused hippocampus or entorhinal cortex of the same rats. Simultaneous electroencephalographic recordings showed intense epileptiform activity starting during 4-AP infusion and lasting for the rest of the experiment (1 h) in both the entorhinal cortex and the hippocampus. The discharges were characterized by poly-spikes and spike-wave complexes that propagated almost immediately to the other region studied. These findings suggest that increased glutamatergic synaptic function in the circuit that connects both regions is involved in the epileptic seizures induced by 4-AP.
Assuntos
4-Aminopiridina/farmacologia , Aminoácidos/efeitos dos fármacos , Córtex Entorrinal/efeitos dos fármacos , Hipocampo/efeitos dos fármacos , Aminoácidos/metabolismo , Animais , Comportamento Animal/efeitos dos fármacos , Eletroencefalografia , Córtex Entorrinal/metabolismo , Espaço Extracelular , Glutamatos/efeitos dos fármacos , Glutamatos/metabolismo , Glutamina/efeitos dos fármacos , Glutamina/metabolismo , Glicina/efeitos dos fármacos , Glicina/metabolismo , Hipocampo/metabolismo , Masculino , Microdiálise , Potássio/farmacologia , Bloqueadores dos Canais de Potássio , Ratos , Ratos Wistar , Taurina/efeitos dos fármacos , Taurina/metabolismo , VigíliaRESUMO
We describe a rotatory electrical device that permits the simultaneous microdialysis and electroencephalographic (EEG) recording, by means of bipolar electrodes attached to the microdialysis probe, in two brain regions of awake rats. Using this device, we have found that the microdialysis infusion of 4-aminopyridine (4-AP) in the motor cerebral cortex produces intense behavioral convulsions and EEG seizures in both the infused and the contralateral cortex. This convulsant action is accompanied by a remarkable increase of extracellular dopamine (about 15-fold), norepinephrine (2.4-fold) and vanillylmandelic acid (1.8-fold) concentration in the infused cortex. Delayed increases of these amines were observed also in the contralateral cortex. The results suggest that 4-AP induces the release of catecholamines either through a direct effect on nerve endings or as a consequence of seizures.
Assuntos
4-Aminopiridina/farmacologia , Catecolaminas/metabolismo , Córtex Cerebral/efeitos dos fármacos , Canais de Potássio/efeitos dos fármacos , Convulsões/induzido quimicamente , Animais , Córtex Cerebral/metabolismo , Diálise , Eletroencefalografia , Masculino , Microeletrodos , Ratos , Ratos Wistar , Convulsões/metabolismo , Convulsões/patologiaRESUMO
Diversos estudios han demostrado que la norepinefrina (NE) y la dopamina (DA) participan en la regulación del umbral convulsivo en diferentes modelos de epilepsia experimental a través de una estrecha relación entre la concentración y la predisposición para desarrollar crisis convulsivas. También, se ha demostrado que el mecanismo de acción de algunos anticonvulsionantes conocidos, se realiza bajo una regulación de la neurotransmisión a nivel sináptico. Por otro lado, los derivados de las butiramidas presentan una importante actividad anticonvulsionante en algunos animales de experimentación. Por lo que en el presente trabajo se investigó el efecto de un derivado de las butiramidas, la 4-hidroxi, 4-etil, 4-fenil butiramida (HEPB) sobre la concentración de NE y DA en diferentes regiones cerebrales como son: la corteza cerebral, el núcleo caudado y el hipocampo. Se utilizaron ratones adultos Balb/c tratados con solución salina fisiológica (testigo), propilenglicol al 10 por ciento como vehículo (testigo) y HEPB (experimental). Las regiones cerebrales se ontuvieron previa decapacitación de los animales y la concentración de NE y DA se realizó por cromatografía de líquidos de alta resolución con detección electroquímica. Los resultados muestran que el HEPB induce un incremento de NE (70 por ciento) en el núcleo caudado a la dosis de 20 mg/kg de peso corporal. Mientras que la DA sólo se incremento en 38 por ciento en esta misma región. En la corteza cerebral y el hipocampo no se encontró diferencia significativa respecto a los grupos testigo. Con estos resultados es difícil explicar el efecto del HEPB sobre el sistema catecolaminérgico en el núcleo caudado, no obstante, es necesario continuar con estudios complementarios que permitan precisar el posible mecanismo de acción del HEPB como anticonvulsionante y en particular su efecto sobre el transporte de DA en el núcleo caudado
Assuntos
Camundongos , Animais , Anticonvulsivantes/farmacologia , Butiratos/farmacocinética , Catecolaminas/análise , Sistema Nervoso Central/efeitos dos fármacos , Dopamina/farmacologia , Epilepsia/fisiopatologia , Neurotransmissores/fisiologia , Norepinefrina/farmacologiaRESUMO
The effects of NMDA receptor antagonists on the convulsant action of the administration of 4-aminopyridine in the rat lateral cerebral ventricle (i.c.v. injection) and motor cerebral cortex (i.cx. injection) were studied. 4-Aminopyridine administration in both regions induced various preconvulsive symptoms, such as salivation, tremors, chewing and rearing, followed by continuous clonic convulsions and, only after i.c.v. injection, running fits and generalized tonic convulsions. This behavioral pattern appeared 5-9 min after administration of 4-aminopyridine and persisted for 100-150 min. 4-Aminopyridine also generated epileptiform electroencephalographic (EEG) discharges characterized by isolated spikes, poly-spikes and spike-wave complexes, which began some seconds after administration of the drug and were present for more than 2 h. The NMDA receptor antagonists (+/-)-3-(2-carboxy-piperazin-4-yl)-propyl-1-phosphonic acid (CPP), (+/-)-2-amino-7-phosphono-heptanoic acid (AP7) and (+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine hydrogen maleate (MK-801) clearly protected against some of the behavioral alterations induced by i.c.v. 4-aminopyridine, particularly the tonic convulsions, but were less effective against those produced by i.cx. 4-aminopyridine. These antagonists also delayed the appearance of EEG epileptiform discharges, reduced its amplitude, frequency and duration, and blocked their propagation to other cortical regions after i.cx. 4-aminopyridine. These results, together with previous data showing that 4-aminopyridine stimulates the release of glutamate in vivo, suggest that an excessive glutamatergic neurotransmission involving NMDA receptors is implicated in 4-amino-pyridine-induced seizures.
Assuntos
4-Aminopiridina/efeitos adversos , Convulsivantes/efeitos adversos , Receptores de N-Metil-D-Aspartato/antagonistas & inibidores , Convulsões/induzido quimicamente , 2-Amino-5-fosfonovalerato/análogos & derivados , 2-Amino-5-fosfonovalerato/farmacologia , 4-Aminopiridina/administração & dosagem , Animais , Anticonvulsivantes/farmacologia , Maleato de Dizocilpina/farmacologia , Eletroencefalografia , Injeções Intraventriculares , Masculino , Córtex Motor/fisiologia , Fármacos Neuroprotetores/farmacologia , Piperazinas/farmacologia , Ratos , Ratos Wistar , Convulsões/prevenção & controle , Técnicas EstereotáxicasRESUMO
L-Glutamate has an excitatory and cytotoxic effect on the central nervous system. It was shown previously that norepinephrine and dopamine uptake and release were affected by in vivo administration of glutamate to adult rats. The kinetic parameters, Km and Vmax of [14C]DA uptake and release were measured on synaptosomal and slices from caudate nucleus under in vitro conditions at different glutamate concentrations. Results showed an important increase in [14C]DA uptake on synaptosomal (> 100%) and slices by lower glutamate concentrations, the affinity for transport system was increased (100%) and its release of high potassium evoked was also increased at 0.5 microM of glutamate. The results suggest the possibility that glutamate may modify DA uptake and release interacting with the DA transporter complex at the synaptic level.
Assuntos
Núcleo Caudado/efeitos dos fármacos , Dopamina/metabolismo , Glutamato de Sódio/farmacologia , Animais , Núcleo Caudado/metabolismo , Técnicas In Vitro , Cinética , Masculino , Ratos , Ratos Wistar , Sinaptossomos/efeitos dos fármacos , Sinaptossomos/metabolismoRESUMO
Effect of intraperitoneal (i.p.) injection of ruthenium red (RuR) to adults rats on catecholamine (CA) uptake and dopamine (DA) release was evaluated in brain synaptosomal fractions. No effect on CA uptake by synaptosomal fractions was seen when RuR was added in vitro. There was no effect of RuR injection i.p. to rats, on CA uptake by their brain synaptosomal fractions. A blocking effect on depolarizing induced released of DA from brain synaptosomal fraction obtained from rats previously injected i.p. with RuR was seen. A similar effect was observed on the basal DA efflux from synaptosomes loaded with (14C)DA obtained from rats i.p. injected with RuR. It is concluded that RuR, a non-liposoluble substance, when injected i.p. reaches the brain parenchyma through areas devoid of blood-brain barrier and interferes with Ca(++)-dependent release of neurotransmitters, inducing cerebral hyperexcitability and convulsions.
Assuntos
Química Encefálica/efeitos dos fármacos , Bloqueadores dos Canais de Cálcio/farmacologia , Convulsivantes/farmacologia , Neurotransmissores/metabolismo , Rutênio Vermelho/farmacologia , Sinaptossomos/efeitos dos fármacos , Animais , Barreira Hematoencefálica , Cálcio/metabolismo , Bloqueadores dos Canais de Cálcio/administração & dosagem , Bloqueadores dos Canais de Cálcio/toxicidade , Convulsivantes/administração & dosagem , Convulsivantes/toxicidade , Injeções Intraperitoneais , Ratos , Ratos Endogâmicos , Rutênio Vermelho/administração & dosagem , Rutênio Vermelho/toxicidade , Convulsões/induzido quimicamente , Convulsões/fisiopatologia , Sinaptossomos/metabolismoRESUMO
Adult rats (60 days old) were injected intraperitoneally with 5 mg/g monosodium L-glutamate (MSG). During the convulsive period (1 h after injection), uptake and release of [3H]norepinephrine (3H-NE) and [14C]dopamine (14C-DA) were measured in a crude synaptosomal fraction and in slices of cerebral cortex and caudate nucleus, respectively. A significant reduction of 3H-NE uptake was detected in cortical slices (by 42%) and in synaptosomal fraction (by 33%) of rats treated with MSG, whereas K+- stimulated 3H-NE release was decreased by 32% and 39% in brain slices and in a synaptosomal fraction of cerebral cortex, respectively, in comparison with animals injected with 0.9% NaCl aqueous solution (PSS). In the caudate nucleus, 14C-DA uptake was increased by 100% in brain slices and by 36% in the synaptosomal fraction following MSG administration, whereas K+- stimulated 14C-DA release was enhanced by 80% in slices and by 25% in synaptosomes as compared to PSS-injected rats. Data suggest that catecholaminergic neurotransmission may play an important role in the etiopathology of convulsions in the experimental model using MSG.