RESUMEN
We demonstrate the use of magnetic resonance imaging (MRI) for detection of neurotransmitter stimulation using the dopamine transporter ligands amphetamine and CFT (2beta-carbomethoxy-3beta-(4-fluorophenyl)tropane) as pharmacological challenges. We demonstrate that the unilateral loss of a hemodynamic response to either amphetamine or CFT challenge by unilateral 6-hydroxydopamine lesioning is restored by transplantation of fetal dopamine neurons in the striatum. The time course for the hemodynamic changes parallels the time courses for dopamine release, measured by prior microdialysis studies, and also for the rotational behavior in the unilaterally lesioned animals. Transplantation of the fetal cells results in hemodynamic time courses after CFT or amphetamine challenges at the graft site that are identical to those induced both before transplantation and on the intact contralateral side. The transplantation also results in complete behavioral recovery. The spatial extent of the dopaminergic recovery in the lesioned striatum is the same when measured using either PET of tracer levels of [11C]CFT binding or MRI. These results show great promise for the application of pharmacological MRI for application to studies of dopamine cell loss and potential recovery in Parkinson's disease.
Asunto(s)
Conducta Animal/fisiología , Trasplante de Células/fisiología , Dopamina/fisiología , Neuronas/fisiología , Anfetamina , Animales , Cocaína/análogos & derivados , Inhibidores de Captación de Dopamina , Femenino , Imagen por Resonancia Magnética , Microdiálisis , Ratas , Ratas Sprague-Dawley , Degeneración Estriatonigral , Simpatectomía Química , Tomografía Computarizada de EmisiónRESUMEN
Positron emission tomography (PET) and carbon-11-labeled 2B-carbomethoxy-3B-(4-fluorophenyl)tropane (11C-CFT or 11-WIN 35,428) were used as molecular markers for striatal presynaptic dopamine (DA) transporters in a unilateral Parkinson's disease rat neurotransplantation model. In the lesioned striatum, the binding ratio measured by the DA presynaptic marker was reduced to 15% to 35% of the intact side (or unoperated control). After grafting with non-DA cells (from dorsal mesencephalon), the DA binding ratio remained reduced to levels observed before transplantation and rats showed no behavioral recovery. In contrast, after DA neuronal transplantation, behavioral recovery occurred only after the 11C-CFT binding ratio had increased to 75% to 85% of the intact side. This study provides direct in vivo evidence for the dopaminergic molecular basis of functional recovery in the lesioned nigrostriatal system after neural transplantation.
Asunto(s)
Dopamina/metabolismo , Trasplante de Tejido Fetal , Mesencéfalo/embriología , Terminaciones Nerviosas/metabolismo , Enfermedad de Parkinson Secundaria/cirugía , Tomografía Computarizada de Emisión , Animales , Cuerpo Estriado/diagnóstico por imagen , Cuerpo Estriado/metabolismo , Cuerpo Estriado/cirugía , Femenino , Terminaciones Nerviosas/diagnóstico por imagen , Enfermedad de Parkinson Secundaria/inducido químicamente , Enfermedad de Parkinson Secundaria/diagnóstico por imagen , Ratas , Ratas Sprague-DawleyRESUMEN
The metabolic activation resulting from direct dopaminergic stimulation can be detected using auto-radiography, positron emission tomography (PET) or, potentially, fMRI techniques. To establish the validity of the latter possibility, we have performed a number of experiments. We measured the regional selectivity of two different dopaminergic ligands: the dopamine release compound D-amphetamine and the dopamine transporter antagonist 2 beta-carbomethoxy-3 beta-(4-fluoropheny) tropane (CFT). Both compounds led to increased signal intensity in gradient echo images in regions of the brain with high dopamine receptor density (frontal cortex, striatum, cingulate cortex > > parietal cortex). Lesioning the animals with unilaterally administered 6-hydroxydopamine (6-OHDA) led to ablation of the phMRI response on the ipsilateral side; control measurements of rCBV and rCBF using bolus injections of Gd-DTPA showed that the baseline rCBV and rCBF values were intact on the lesioned side. The time course of the BOLD signal changes paralleled the changes observed by microdialysis measurements of dopamine release in the striatum for both amphetamine and CFT; peaking at 20-40 min after injection and returning to baseline at about 70-90 min. Signal changes were not correlated with either heart rate, blood pressure or pCO2. Measurement of PET binding in the same animals showed an excellent correlation with the phMRI data when compared by either measurements of the number of pixels activated or percent signal change in a given region. The time course for the behavioral measurements of rotation in the 6-OHDA lesioned animals correlated with the phMRI. These experiments demonstrate that phMRI will become a valuable, noninvasive tool for investigation of neurotransmitter activity in vivo.
Asunto(s)
Conducta Animal/fisiología , Encéfalo/metabolismo , Dopamina/metabolismo , Imagen por Resonancia Magnética/métodos , Microdiálisis/métodos , Receptores Dopaminérgicos/metabolismo , Tomografía Computarizada de Emisión/métodos , Adrenérgicos/farmacología , Animales , Encéfalo/diagnóstico por imagen , Encéfalo/efectos de los fármacos , Cocaína/análogos & derivados , Cocaína/farmacología , Dextroanfetamina/farmacología , Inhibidores de Captación de Dopamina/farmacología , Oxidopamina/farmacología , Ratas , Ratas Sprague-Dawley , Sensibilidad y EspecificidadRESUMEN
Nerve growth factor (NGF)-secreting fibroblasts are able to protect against the Huntington-like striatal neurodegeneration induced by the mitochondrial toxin 3-nitropropionic acid (3-NP). In the present study, we investigated whether the neuroprotective effects of NGF are mediated through antioxidative mechanisms. Rats were grafted in the corpus callosum with NGF[+] or NGF[-] fibroblasts 7 days before administration of 3-NP. The generation of peroxynitrite was evaluated by measuring the striatal levels of 3-nitrotyrosine. NGF significantly decreased the 3-NP induced generation of 3-nitrotyrosine, presumably by decreasing peroxynitrite formation. These findings suggest that NGF might protect against neuronal death by inhibiting the production of nitric oxide or decreasing the levels of superoxide radicals, thereby decreasing the generation of oxidative agents such as peroxynitrite.
Asunto(s)
Cuerpo Estriado/efectos de los fármacos , Enfermedad de Huntington/tratamiento farmacológico , Factores de Crecimiento Nervioso/farmacología , Propionatos/farmacología , Tirosina/análogos & derivados , Animales , Modelos Animales de Enfermedad , Masculino , Nitrocompuestos , Ratas , Ratas Sprague-Dawley , Tirosina/metabolismoRESUMEN
Neurotransplantation of human fetal dopamine (DA) neurons is currently being investigated as a therapeutic modality for Parkinson's disease (PD). However, the practical limitations of human fetal transplantation indicate a need for alternative methodologies. Using the 6-hydroxydopamine rat model of PD, we transplanted dopaminergic neurons derived from Embryonic Day 27 porcine fetuses into the denervated striatum of cyclosporine-A (CyA)-treated or non-CyA-treated rats. Functional recovery was assessed by amphetamine-induced rotation, and graft survival and morphology were analyzed using neuronal and glial immunostaining as well as in situ hybridization with a porcine repeat element DNA probe. A significant, sustained reduction in amphetamine-induced rotational asymmetry was present in the CyA-treated rats whereas the non-CyA-treated rats showed a transient behavioral recovery. The degree of rotational recovery was highly correlated to the number of surviving transplanted porcine dopaminergic neurons. TH+ neuronal survival and graft volume were significantly greater in the CyA-treated group as compared to the non-CyA group. By donor-specific neuronal and glial immunostaining as well as donor-specific DNA labeling, we demonstrate that porcine fetal neuroblasts are able to survive in the adult brain of immunosuppressed rats, mediate functional recovery, and extensively reinnervate the host striatum. These findings suggest that porcine DA neurons may be a suitable alternative to the use of human fetal tissue in neurotransplantation for PD.
Asunto(s)
Trasplante de Tejido Encefálico , Mesencéfalo/trasplante , Trasplante Heterólogo , Animales , Ciclosporina/farmacología , Modelos Animales de Enfermedad , Femenino , Enfermedad de Parkinson/terapia , Ratas , Ratas Sprague-Dawley , Porcinos , Factores de TiempoRESUMEN
The controlled differentiation of mouse embryonic stem (ES) cells into near homogeneous populations of both neurons and skeletal muscle cells that can survive and function in vivo after transplantation is reported. We show that treatment of pluripotent ES cells with retinoic acid (RA) and dimethylsulfoxide (DMSO) induce differentiation of these cells into highly enriched populations of gamma-aminobutyric acid (GABA) expressing neurons and skeletal myoblasts, respectively. For neuronal differentiation, RA alone is sufficient to induce ES cells to differentiate into neuronal cells that show properties of postmitotic neurons both in vitro and in vivo. In vivo function of RA-induced neuronal cells was demonstrated by transplantation into the quinolinic acid lesioned striatum of rats (a rat model for Huntington's disease), where cells integrated and survived for up to 6 wk. The response of embryonic stem cells to DMSO to form muscle was less dramatic than that observed for RA. DMSO-induced ES cells formed mixed populations of muscle cells composed of cardiac, smooth, and skeletal muscle instead of homogeneous populations of a single muscle cell type. To determine whether the response of ES cells to DMSO induction could be further controlled, ES cells were stably transfected with a gene coding for the muscle-specific regulatory factor, MyoD. When induced with DMSO, ES cells constitutively expressing high levels of MyoD differentiated exclusively into skeletal myoblasts (no cardiac or smooth muscle cells) that fused to form myotubes capable of spontaneous contraction. Thus, the specific muscle cell type formed was controlled by the expression of MyoD. These results provided evidence that the specific cell type formed (whether it be muscle, neuronal, or other cell types) can be controlled in vitro. Further, these results demonstrated that ES cells can provide a source of multiple differentiated cell types that can be used for transplantation.
Asunto(s)
Interleucina-6 , Trasplante de Células Madre , Animales , Northern Blotting , Diferenciación Celular/efectos de los fármacos , Diferenciación Celular/fisiología , Línea Celular , Dimetilsulfóxido/farmacología , Trasplante de Tejido Fetal , Feto/citología , Técnica del Anticuerpo Fluorescente , Regulación del Desarrollo de la Expresión Génica/efectos de los fármacos , Inhibidores de Crecimiento/farmacología , Factor Inhibidor de Leucemia , Linfocinas/farmacología , Ratones , Músculo Esquelético/citología , Músculo Esquelético/embriología , Proteína MioD/genética , Neuronas/citología , Neuronas/trasplante , Regiones Promotoras Genéticas/efectos de los fármacos , Regiones Promotoras Genéticas/fisiología , Ratas , Ratas Sprague-Dawley , Células Madre/química , Células Madre/citología , Transfección , Tretinoina/farmacología , Ácido gamma-Aminobutírico/análisis , Ácido gamma-Aminobutírico/fisiologíaRESUMEN
Brain-derived neurotrophic factor (BDNF) promotes the survival of fetal mesencephalic dopaminergic cells and protects dopaminergic neurons against the toxicity of MPP+ in vitro. Supranigral implantation of fibroblasts genetically engineered to secrete BDNF attenuates the loss of substantia nigra pars compacta (SNc) dopaminergic neurons associated with striatal infusion of MPP+ in the adult rat. Using this MPP+ rat model of nigral degeneration, we evaluated the neurochemical effects of supranigral, cell-mediated delivery of BDNF on substantia nigra (SN) dopamine (DA) content and turnover. Genetically engineered BDNF-secreting fibroblasts (approximately 12 ng BDNF/24 h) were implanted dorsal to the SN 7 days prior to striatal MPP+ administration. The present results demonstrate that BDNF-secreting fibroblasts, as compared to control fibroblasts, enhance SN DA levels ipsilateral as well as contralateral to the graft without altering DA turnover. This augmentation of DA levels suggests that local neurotrophic factor delivery by genetically engineered cells may provide a therapeutic strategy for preventing neuronal death or enhancing neuronal function in neurodegenerative diseases characterized by dopaminergic neuronal dysfunction, such as Parkinson's disease.
Asunto(s)
1-Metil-4-fenilpiridinio/farmacología , Dopamina/metabolismo , Fibroblastos/trasplante , Proteínas del Tejido Nervioso/metabolismo , Sustancia Negra/fisiología , Animales , Northern Blotting , Factor Neurotrófico Derivado del Encéfalo , Fibroblastos/metabolismo , Fibroblastos/fisiología , Ingeniería Genética , Masculino , Microinyecciones , Neostriado/citología , Neostriado/efectos de los fármacos , Degeneración Nerviosa/efectos de los fármacos , Degeneración Nerviosa/fisiología , Factores de Crecimiento Nervioso/metabolismo , Proteínas del Tejido Nervioso/genética , Fármacos Neuroprotectores/farmacología , ARN Mensajero/metabolismo , Ratas , Ratas Sprague-Dawley , Sustancia Negra/citología , Sustancia Negra/metabolismoRESUMEN
Clinical trials are under way using fetal cells to repair damaged neuronal circuitry. However, little is known about how transplanted immature neurons can grow anatomically correct connections in the adult central nervous system (CNS). We transplanted embryonic porcine neural cells in vivo into adult rat brains with neuronal and axonal loss typical of Parkinson's or Huntington's disease. Using complementary species-specific cellular markers, we found donor axons and CD44+ astroglial fibres in host white matter tracts up to 8 mm from CNS transplant sites, although only donor axons were capable of reaching correct gray matter target regions. This work demonstrates that adult host brain can orient growth of transplanted neurons and that there are differences in transplant donor glial and axonal growth patterns in cellular repair of the mature CNS.
Asunto(s)
Axones/ultraestructura , Enfermedades de los Ganglios Basales/cirugía , Encéfalo/citología , Neuroglía/citología , Neuronas/trasplante , Trasplante Heterólogo , Animales , Biomarcadores/análisis , Encéfalo/patología , Encéfalo/cirugía , Células Cultivadas , Modelos Animales de Enfermedad , Enfermedad de Huntington/cirugía , Inmunohistoquímica , Masculino , Enfermedad de Parkinson/cirugía , Ratas , Ratas Sprague-Dawley , PorcinosRESUMEN
The relationship between the specific neuronal loss observed in Huntington's disease and the mutation in the IT15 gene responsible for this disease remains obscure. Using an antipeptide antibody against amino acids 3114-3141 of the human huntington protein, we demonstrate that striatal injection of quinolinic acid in mice induces increased immunoreactivity for huntington in some remaining neurons but not in glial cells. This increase is apparent in both neuronal cell bodies and in cell processes in the white matter six hours after excitotoxic challenge. This finding suggests that huntington may be involved in the response to excitotoxic stress in these neurons.
Asunto(s)
Cuerpo Estriado/efectos de los fármacos , Enfermedad de Huntington/inmunología , Proteínas del Tejido Nervioso/inmunología , Neurotoxinas/farmacología , Proteínas Nucleares/inmunología , Ácido Quinolínico/farmacología , Animales , Anticuerpos/inmunología , Muerte Celular , Aminoácidos Excitadores/farmacología , Expresión Génica , Proteína Huntingtina , Inmunohistoquímica , Masculino , Ratones , Ratones Endogámicos , Mutación , Neuronas/efectos de los fármacos , Putamen/efectos de los fármacosRESUMEN
The trophism of brain-derived neurotrophic factor (BDNF) for dopaminergic cells in culture has led to significant interest in the role of BDNF in the etiology and potential treatment of Parkinson disease. Previous in vivo investigation of BDNF delivery to axotomized substantia nigra dopaminergic neurons in the adult rat has shown no protective effect. In this study, we produced nigral degeneration by infusing 1-methyl-4-phenylpyridinium (MPP+), a mitochondrial complex I inhibitor and the active metabolite of 1-methyl-4-phenyl-1,2,3,6- tetrahydropyridine (MPTP), into the rat striatum. The subsequent loss of nigral neurons was presumably due to mitochondrial toxicity after MPP+ uptake and retrograde transport to the substantia nigra. We engineered immortalized rat fibroblasts to secrete human BDNF and implanted these cells near the substantia nigra 7 days before striatal MPP+ infusion. We found that BDNF-secreting fibroblasts markedly increased nigral dopaminergic neuronal survival when compared to control fibroblast implants. The observation that BDNF prevents MPTP-induced dopaminergic neuronal degeneration in the adult brain has significance for the treatment of neurodegenerative disorders, which may involve mitochondrial dysfunction, such as Parkinson disease.
Asunto(s)
1-Metil-4-fenilpiridinio/toxicidad , Fibroblastos/trasplante , Factores de Crecimiento Nervioso/genética , Proteínas del Tejido Nervioso/genética , Neuronas/efectos de los fármacos , 1-Metil-4-fenilpiridinio/antagonistas & inhibidores , Animales , Factor Neurotrófico Derivado del Encéfalo , Línea Celular , Supervivencia Celular , Células Cultivadas , Fibroblastos/metabolismo , Ingeniería Genética , Masculino , Degeneración Nerviosa , Factores de Crecimiento Nervioso/farmacología , Proteínas del Tejido Nervioso/farmacología , Neuronas/metabolismo , Neuronas/patología , Ratas , Ratas Sprague-Dawley , Receptores Dopaminérgicos/efectos de los fármacos , Receptores Dopaminérgicos/metabolismo , Sustancia Negra/efectos de los fármacos , Sustancia Negra/metabolismoRESUMEN
Benzodiazepine (BDZ) hypnotics bind at a specific receptor located on postsynaptic neurons. Some data support specificity of binding for several hypnotics to receptor subtypes. We evaluated BDZ receptor binding in cerebral cortical membranes using agonist, antagonist, and subtype-specific ligands for commonly used hypnotics and their metabolites. All hypnotics competed similarly at BDZ1 and BDZ2 receptor subtypes except quazepam and its metabolite 2-oxo-quazepam and to a lesser extent hydroxyethyl flurazepam (EtOH) flurazepam. These compounds had relative specificity for the BDZ1 site. Triazolam, estazolam, and flurazepam bound equally to sites labeled by agonists and antagonists but desalkylflurazepam, EtOH flurazepam, temazepam, quazepam, and 2-oxo-quazepam did not; in addition, these four compounds did not bind to the "peripheral" BDZ site labeled by Ro 5-4864. BDZ hypnotics differ in their receptor subtype and ligand binding characteristics.
Asunto(s)
Ansiolíticos/farmacocinética , Hipnóticos y Sedantes/farmacocinética , Receptores de GABA-A/metabolismo , Animales , Ansiolíticos/farmacología , Benzodiazepinas , Unión Competitiva/efectos de los fármacos , Antagonistas de Receptores de GABA-A , Ligandos , Masculino , Ratones , Piridazinas/farmacología , Receptores de GABA-A/efectos de los fármacosRESUMEN
Chronic administration of benzodiazepine active at the tau-aminobutyric acidA receptor ("central" benzodiazepine sites) is associated with behavioral tolerance and receptor downregulation. Recent reports indicate possible interactions between central sites and benzodiazepines active at "peripheral-type" sites located primarily on non-neuronal cells. To evaluate these interactions during chronic administration, we treated mice with lorazepam for 1 to 14 days alone or in combination with the peripheral-type site ligand PK11195 [N-methyl-N-(methyl-1-propyl)chloro-2-phenyl-1-isoquinoline-3-carboxamid e]. Lorazepam was associated with tolerance at 7 days, but tolerance was not observed during concurrent administration of PK11195. Lorazepam was also associated with benzodiazepine receptor down-regulation in cortex and hippocampus at 7 days. With concurrent administration of PK11195, this effect remained in cortex but was absent in hippocampus. tau-Aminobutyric acid-dependent chloride uptake was reduced in both cortex and hippocampus with lorazepam, but not with concurrent lorazepam and PK11195. PK11195 administration alone did not affect behavior or neurochemical parameters, or did it alter brain lorazepam concentrations. These data indicate that concurrent PK11195 administration attenuates behavioral and neurochemical effects of chronic lorazepam administration.
Asunto(s)
Encéfalo/efectos de los fármacos , Compuestos Bicíclicos Heterocíclicos con Puentes , Convulsivantes/farmacología , Isoquinolinas/farmacología , Lorazepam/antagonistas & inhibidores , Receptores de GABA-A/efectos de los fármacos , Animales , Encéfalo/metabolismo , Compuestos Bicíclicos con Puentes/metabolismo , Convulsivantes/metabolismo , Regulación hacia Abajo/efectos de los fármacos , Flumazenil/metabolismo , Isoquinolinas/metabolismo , Ligandos , Lorazepam/metabolismo , Lorazepam/toxicidad , Masculino , Ratones , Actividad Motora/efectos de los fármacos , Receptores de GABA-A/metabolismoRESUMEN
Clinical studies suggest that carbamazepine may attenuate effects of alprazolam discontinuation. Since discontinuation of chronic alprazolam in a mouse model is associated with behavioral alterations and upregulation at the gamma-aminobutyric acidA (GABAA) receptor, we studied the effects of carbamazepine administration after alprazolam (2 mg/kg/day) discontinuation. Open-field activity was increased in mice 4 days after alprazolam discontinuation, but this effect was reduced significantly by continuous infusion of carbamazepine, 25 or 100 mg/kg/day. Benzodiazepine receptor binding in vivo was increased in cortex at 2 and 4 days after alprazolam discontinuation, and in hypothalamus at 4 days; with carbamazepine, 100 mg/kg/day, binding in both regions at these time points was similar to control values. Similar results were observed in cortex with benzodiazepine receptor binding in vitro. GABA-dependent chloride uptake was also increased at 4 days alprazolam administration. Treatment with carbamazepine attenuated (P less than 0.10) this increase. Carbamazepine alone after vehicle did not alter benzodiazepine binding or GABA-dependent chloride uptake. These results indicate that carbamazepine administration after alprazolam discontinuation attenuates behavioral and neurochemical alterations associated with discontinuation.
Asunto(s)
Alprazolam/efectos adversos , Ansiedad/prevención & control , Carbamazepina/uso terapéutico , Receptores de GABA-A/efectos de los fármacos , Convulsiones/prevención & control , Síndrome de Abstinencia a Sustancias/tratamiento farmacológico , Animales , Carbamazepina/administración & dosificación , Corteza Cerebral/metabolismo , Modelos Animales de Enfermedad , Esquema de Medicación , Evaluación Preclínica de Medicamentos , Hipocampo/metabolismo , Hipotálamo/metabolismo , Masculino , Ratones , Actividad Motora/efectos de los fármacos , Receptores de GABA-A/metabolismo , Factores de TiempoRESUMEN
Prenatal benzodiazepine exposure has been associated with neurobehavioral alterations in humans and animals. To determine effects of prenatal benzodiazepine exposure on binding at the benzodiazepine and t-butylbicyclophosphorothionate (TBPS) sites on the GABAA receptor in mature offspring, we treated mice with lorazepam, 2 mg/kg/day, during days 13-20 of gestation. Binding was assessed at 6 weeks of age. There were no differences among controls, vehicle- or lorazepam-exposed mice in benzodiazepine receptor binding determined in vivo or in vitro. However, receptor density for [35S]TBPS binding sites was decreased in lorazepam-exposed offspring compared to the other groups. These data are consistent with prior neurochemical results indicating decreased TBPS binding and GABAA receptor function in several systems.
Asunto(s)
Compuestos Bicíclicos Heterocíclicos con Puentes , Compuestos Bicíclicos con Puentes/metabolismo , Lorazepam/toxicidad , Receptores de GABA-A/efectos de los fármacos , Animales , Peso Corporal/efectos de los fármacos , Femenino , Ratones , Embarazo , Efectos Tardíos de la Exposición Prenatal , Receptores de GABA-A/metabolismo , Reproducción/efectos de los fármacosRESUMEN
Treatment with benzodiazepine agonists is associated with the development of behavioral tolerance and receptor downregulation, whereas antagonist administration has been reported to lead to increased activity and receptor upregulation. To determine the effects of chronic inverse agonist administration, mice were treated with FG-7142 (20 mg/kg/day) by implanted s.c. osmotic pumps for 1 to 14 days. No seizures were observed in FG-7142-exposed animals. Open-field activity was unchanged as compared to controls at days 1 and 2, but was elevated significantly at days 4 and 7. Activity was reduced below control values at day 14. Benzodiazepine receptor binding determined in vivo was unchanged for days 1, 4 and 7 within the hippocampus but was elevated at day 14. Binding remained unchanged in the cortex, cerebellum, hypothalamus and pons-medulla for the duration of drug exposure. Cortical benzodiazepine binding assessed in vitro was unchanged at days 1 and 2 but increased at days 4, 7 and 14 vs. vehicle treated controls. Binding at the gamma-aminobutyric acid site was increased at day 7 whereas binding of t-[35S]butylbicyclophosphoro-thionate was increased at days 7 and 14 of FG-7142 exposure. Maximal muscimol-slimulated [36Cl-] uptake was elevated at days 4, 7 and 14 compared to day 1 of exposure. These results demonstrate that chronic continuous exposure to FG-7142 is associated with an absence of kindled seizures and with behavioral and neurochemical changes indicative of gamma-aminobutyric acidA receptor upregulation.
Asunto(s)
Depresores del Apetito/farmacología , Benzodiazepinas/metabolismo , Carbolinas/farmacología , Receptores de GABA-A/efectos de los fármacos , Receptores de GABA-A/metabolismo , Regulación hacia Arriba/efectos de los fármacos , Animales , Sitios de Unión , Flumazenil/farmacología , Inyecciones Intravenosas , Masculino , Ratones , Actividad Motora/efectos de los fármacosRESUMEN
The irreversible protein-modifying reagent N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline (EEDQ) was used to investigate binding site characteristics on the gamma-aminobutyric acidA (GABAA) receptor complex. In vitro, preincubation with EEDQ led to a concentration-dependent decrease in receptor number for benzodiazepine, t-butylbicyclophosphorothionate (TBPS), and GABA binding sites in cerebral cortex. The effect was maximal at the highest concentration of EEDQ used (10(-4) M) and was greatest for the benzodiazepine site. Pretreatment of membranes with the benzodiazepine antagonist Ro 15-1788, 1 or 10 microM, or the agonist lorazepam, 10 microM, largely prevented the effects of EEDQ. Scatchard analysis indicated no effect of EEDQ, 10(-4) M, on apparent affinity, but a decrease in receptor density for each site. Administration of EEDQ to mice, 12.5 mg/kg i.p., led to a substantial (55-65%) decrease in number of benzodiazepine binding sites in cortex after 4 h. Slightly smaller changes were observed for TBPS and GABA binding. No changes were observed in apparent affinity at any site. Prior administration of Ro 15-1788, 5 mg/kg, prevented the effect of EEDQ on benzodiazepine binding. Density of benzodiazepine binding sites gradually recovered over time, and receptor density returned to control values by 96 h after EEDQ injection. Number of binding sites in cortex for TBPS and GABA also increased over time after EEDQ. Benzodiazepine sites in cerebellum were decreased proportionally to cortex after EEDQ, and increased over a similar time course. Function of the GABAA receptor in chloride uptake in cortex was markedly reduced (65%) by EEDQ.(ABSTRACT TRUNCATED AT 250 WORDS)
Asunto(s)
Envejecimiento/metabolismo , Compuestos Bicíclicos Heterocíclicos con Puentes , Quinolinas/farmacología , Receptores de GABA-A/metabolismo , Animales , Benzodiazepinas/metabolismo , Compuestos Bicíclicos con Puentes/metabolismo , Cerebelo/metabolismo , Corteza Cerebral/metabolismo , Relación Dosis-Respuesta a Droga , Flumazenil/farmacología , Cinética , Lorazepam/farmacología , Masculino , Ratones , Ratones Endogámicos , Receptores de GABA-A/efectos de los fármacos , Ácido gamma-Aminobutírico/metabolismoRESUMEN
Interleukin-1 (IL-1), a cytokine involved in the acute phase reaction to injury and infection, has multiple effects in the central nervous system, including induction of fever and sleep and the release of several neuropeptides. We evaluated effects of IL-1 beta on inhibitory postsynaptic function at the gamma-aminobutyric acidA (GABAA) receptor. IL-1 (100 pg/ml to 10 ng/ml) augmented GABAA receptor function in cortical synaptic preparations. This effect of IL-1 was largely prevented by incubation with a specific IL-1 receptor antagonist. The related cytokines interleukin-6 and tumor necrosis factor did not augment GABA-dependent chloride transport. Similar enhancement of GABAA receptor function was observed in tissue prepared from mice previously injected intraperitoneally with IL-1 (1 microgram). Electrophysiological studies in cultured primary cortical neurons demonstrated that IL-1 enhanced the GABA-mediated increase in chloride permeability, whereas IL-1 alone produced no alterations in resting conductance. Behavioral studies indicated that IL-1 is similarly active in vivo; mice treated with IL-1 showed a decrease in open-field activity and an increase in the threshold for pentylenetetrazol-induced seizures. The interaction of IL-1 with GABAA receptors might account for the somnogenic and motor-depressant effects of this cytokine.
Asunto(s)
Encéfalo/ultraestructura , Interleucina-1/farmacología , Receptores de GABA-A/fisiología , Animales , Encéfalo/fisiología , Cloruros/farmacocinética , Interleucina-6/farmacología , Masculino , Ratones , Ratones Endogámicos , Muscimol/farmacología , Neuronas/metabolismo , Receptores de GABA-A/efectos de los fármacos , Especificidad por Sustrato , Sinapsis/metabolismo , Sinaptosomas/metabolismo , Factores de Tiempo , Factor de Necrosis Tumoral alfa/farmacologíaRESUMEN
Clonazepam administration may lead to tolerance and "withdrawal" syndromes in clinical use. To assess the effects of this drug in a mouse model, we administered clonazepam (1.5 mg/kg/day) for 1-14 days and evaluated open-field activity, cortical clonazepam concentrations, and binding and function at the GABAA receptor. We also evaluated the same parameters at 1, 2, 4 and 7 days after discontinuation of 7 days of clonazepam administration. During chronic treatment, tolerance developed to the effects of clonazepam on motor activity at 7 days and persisted to 14 days. Cortical clonazepam concentrations did not change significantly during this period. Benzodiazepine receptor binding in vivo was decreased in cortex at days 7 and 14 of clonazepam, but was unchanged in other regions. Binding determined in vitro was also decreased at these points. TBPS (t-butylbicyclophosphorothionate) binding in cortex was slightly, but not significantly, decreased. Muscimol-stimulated chloride uptake was also decreased at days 7 and 14. After clonazepam discontinuation, open-field activity returned to control values at 1 day but was increased above baseline at 4 days. Benzodiazepine binding in vivo and in vitro, as well as TBPS binding, were increased at 4 days. Muscimol-stimulated chloride uptake was also increased at this point. These results indicate that chronic clonazepam administration is associated with tolerance to motoric effects, with discontinuation effects, and with receptor alterations in a mouse model. Clonazepam is similar to other benzodiazepines in this regard.
Asunto(s)
Conducta Animal/efectos de los fármacos , Compuestos Bicíclicos Heterocíclicos con Puentes , Clonazepam/farmacología , Receptores de GABA-A/efectos de los fármacos , Síndrome de Abstinencia a Sustancias/psicología , Animales , Compuestos Bicíclicos con Puentes/farmacología , Corteza Cerebral/efectos de los fármacos , Corteza Cerebral/metabolismo , Cloruros/metabolismo , Convulsivantes/farmacología , Tolerancia a Medicamentos , Flumazenil/farmacología , Masculino , Ratones , Actividad Motora/efectos de los fármacos , Muscimol/metabolismoRESUMEN
Prenatal exposure to benzodiazepines may lead to developmental abnormalities in humans and animals. To assess the behavioral and neurochemical effects of such exposure, pregnant mice were treated with lorazepam, 2 mg/kg/day, from days 13-20 of gestation, and open-field activity was assessed in offspring at 3 and 6 weeks of age and the function of GABAA receptors at 6 weeks of age. Activity was increased in mice exposed to lorazepam, compared to untreated or vehicle-treated controls at 3 weeks, but was unchanged at 6 weeks. Muscimol-stimulated uptake of chloride was decreased in lorazepam-treated mice, compared to controls, with a decrease in maximum uptake but no change in the EC50 for muscimol. Concentrations of lorazepam in maternal plasma and brain showed a similar brain:plasma ratio as previously reported and concentrations in fetal brain were about 50% of maternal levels. Lorazepam persisted for 48 hours after birth in dams but not in the offspring. These results indicate persistent behavioral and neurochemical alterations after prenatal exposure to lorazepam. This model may be useful in assessing other effects of prenatal exposure to benzodiazepine.