RESUMEN
Estrogens are known to have broad effects on neuronal plasticity, but their specific role in neuronal cell death has not been determined. In the present study, we investigated the effects of beta-estradiol on an experimental model of apoptosis of granule cells of the dentate gyrus, i.e., apoptosis induced by intraventricular injection of the microtubule polymerization inhibitor colchicine. Cell death was characterized with multiple methods, including TUNEL and DNA electrophoresis. Nonrandom digestion of DNA was observed within 8-10 hours after colchicine injection, followed by condensation and fragmentation of granule cell nuclei and extensive anterograde degeneration of mossy fibers/terminals in 2 days. We compared the outcomes of the above-described manipulation in ovariectomized or sham-operated rats and animals treated daily with beta-estradiol or vehicle. Animals were lesioned with colchicine or vehicle 2 weeks after ovariectomy or sham operation. Beta-estradiol or vehicle was administered for 1 week prior to lesion and was continued for a further 2 weeks. Total numbers and densities of granule cells in different animal groups were counted by stereology in various anteroposterior levels of the hippocampus. Our results show that ovariectomy intensifies colchicine-induced granule cell apoptosis, which is ameliorated by exogenous beta-estradiol. In doses that ameliorate the adverse effect of ovariectomy, exogenous beta-estradiol appears to have no effect of preventing granule cell death in animals with intact ovaries; i.e., an estrogen excess is not more neuroprotective than physiological levels of these hormones. Taken together, our results indicate that estrogen deprivation increases the vulnerability of hippocampal neurons to injury and may predispose to neurological diseases occurring after menopause.
Asunto(s)
Apoptosis/efectos de los fármacos , Giro Dentado/citología , Estradiol/farmacología , Fármacos Neuroprotectores/farmacología , Ratas Sprague-Dawley/fisiología , Enfermedad de Alzheimer/patología , Animales , Recuento de Células , Colchicina , Femenino , Etiquetado Corte-Fin in Situ , Menopausia , Degeneración Nerviosa/patología , Ovariectomía , RatasRESUMEN
BACKGROUND: The delta(delta)-opioid receptors belong to the G protein-coupled receptors and in vitro studies have shown that delta-opioid receptors undergo an internalization process in response to agonist stimulation. The immediate consequence is the disappearance of receptors from the plasma membrane. This adaptation process reveals the cell's capacity to desensitize after a strong agonist stimulus. This process, if it occurs in vivo, could contribute to the tolerance phenomenon observed after opiate treatment. To study the mechanisms underlying regulation of the delta-opioid receptors in vivo, the effects of an application of the drug dermenkephalin, a potent and selective agonist of the delta-opioid receptor, were analysed in the rat spinal cord. RESULTS: Using immunocytochemistry and electron microscopy, we observed in control rats that membrane labelling was strictly localized at the interface between two neurites. Fifteen minutes after dermenkephalin stimulation, the plasma membrane labelling was associated with invaginated areas. Thirty minutes after stimulation, labelled vesicles were found in the cytoplasm confirming the internalization process. CONCLUSIONS: The present findings support the view that delta-opioid receptors are internalized in response to prolonged exposure to dermenkephalin in vivo and confirm the presynaptic localization of delta-opioid receptors in the dorsal horn of the rat spinal cord.
Asunto(s)
Endocitosis/fisiología , Oligopéptidos/farmacología , Receptores Opioides delta/metabolismo , Médula Espinal/efectos de los fármacos , Médula Espinal/metabolismo , Animales , Axones/ultraestructura , Dendritas/ultraestructura , Endocitosis/efectos de los fármacos , Inmunohistoquímica , Masculino , Microscopía Electrónica , Neuritas/ultraestructura , Células del Asta Posterior/metabolismo , Células del Asta Posterior/ultraestructura , Ratas , Ratas Wistar , Receptores Opioides delta/agonistas , Médula Espinal/ultraestructuraRESUMEN
Using a specific rat monoclonal anti-idiotypic antibody we have examined the subcellular distribution of -opioid receptors in various neuronal subtypes of the rat spinal cord. The immunofluorescence was detected with a confocal microscope and in some cases serial images were processed for a three-dimensional (3-D) reconstruction of the neurons. Immunolabelling was found to be distributed throughout the spinal cord grey matter specially in the most superficial layers of the dorsal horn, around the central canal and in the region of motoneurons of the ventral horn. The 3-D reconstruction made on large neurons of lamina IX in the ventral horn and on neurons of lamina X around the central canal allowed the visualization of 5 -opioid receptors in the cytoplasm of the soma and proximal neurites of immunofluorescent neurons. Some immunolabelled receptors were also detected at the level of the plasma membrane of the cell bodies and in the nuclear matrix. Interestingly, a particular arrangement of delta-opioid receptors organized along parallel alignments was observed on the plasma membrane of some neurons. This study emphasizes the potential usefulness of a 3-D reconstruction in the study of the spatial arrangement of cellular components.
Asunto(s)
Procesamiento de Imagen Asistido por Computador/métodos , Neuronas/metabolismo , Neuronas/ultraestructura , Receptores Opioides delta/metabolismo , Médula Espinal/metabolismo , Animales , Membrana Celular/metabolismo , Membrana Celular/ultraestructura , Núcleo Celular/metabolismo , Núcleo Celular/ultraestructura , Leucina Encefalina-2-Alanina/inmunología , Leucina Encefalina-2-Alanina/farmacocinética , Inmunohistoquímica , Microscopía Confocal , Neuronas Motoras/metabolismo , Neuronas Motoras/ultraestructura , Ratas , Ratas Sprague-Dawley , Médula Espinal/citología , Médula Espinal/ultraestructura , Fracciones Subcelulares/metabolismo , Fracciones Subcelulares/ultraestructuraRESUMEN
Calcitonin gene-related peptide (CGRP)-like immunoreactivity of motor nerve terminals was investigated at different times after local in vivo injection of botulinum type-A toxin (BoNT/A) close to the mouse levator auris longus muscle. CGRP expression in most of control nerve terminals was undetectable, but markedly increased during muscle paralysis and synaptic remodelling and, declined once functional recovery occurred.
Asunto(s)
Toxinas Botulínicas Tipo A/toxicidad , Péptido Relacionado con Gen de Calcitonina/biosíntesis , Neuronas Motoras/fisiología , Unión Neuromuscular/fisiología , Regulación hacia Arriba/efectos de los fármacos , Animales , Electrofisiología , Inmunohistoquímica , Rayos Láser , Ratones , Microscopía Confocal , Neuronas Motoras/efectos de los fármacos , Músculo Esquelético/inervación , Músculo Esquelético/fisiología , Unión Neuromuscular/efectos de los fármacosRESUMEN
The ultrastructural localization of delta-opioid receptors was studied using monoclonal anti-idiotypic antibody prepared with an anti-D-Ala2-D-Leu5-enkephalin. Immunocytochemical techniques were used on vibratome sections from rats perfused with paraformaldehyde. A high density of immunoreactivity was observed in the dorsal horn of the spinal cord, particularly the two superficial layers, the dorsolateral funiculus and the area surrounding the central canal. The labelling was absent when the antibody was preincubated with the immunogen. Competition between the anti-idiotypic antibody and different ligands, delta or mu, was controlled by preincubation of tissue sections with the ligand in the presence of peptidase inhibitors for 3-4 h before addition of the anti-idiotypic antibody. Enkephalin, dermenkephalin and naltrindole induced disappearance of the labelling at 10(-9) M while dermorphin or dermorphin Lys7 were ineffective at the same concentration. Lamina II of the dorsal horn was studied by electron microscopy. The immunolabelling was mainly localized on cell membranes at appositions between the two neurons. About one third were localized between an axon terminal and a dendrite, the same proportion of labellings were between two axon terminals. Labelling was occasionally observed at appositions between a glomerular terminal and a dendrite or a terminal or at axoglial appositions. Axosomatic localizations were rare. The presynaptic localization of the labelling is in favor of a presynaptic mechanism of action for delta-opioids in the spinal cord, providing that these receptors are functional. delta-Opioid peptides probably act non-synaptically since receptors were never localized on synaptic differentiations.
Asunto(s)
Anticuerpos Antiidiotipos , Receptores Opioides delta/análisis , Médula Espinal/química , Animales , Anticuerpos Monoclonales , Especificidad de Anticuerpos , Inmunohistoquímica , Microscopía Electrónica , Ratas , Ratas Wistar , Receptores Opioides delta/ultraestructuraRESUMEN
The responses to the glutamate agonist N-methyl-D-aspartate (NMDA) were studied in the sensori-motor cortex of rats with petit mal-like seizures. In a first study, the changes in extracellular concentration of calcium elicited through ionophoretic application of NMDA at various depths in the cortex were measured in vivo. The results show that in the cortex of epileptic rats the NMDA responses are much more widely distributed than in the cortex of control rats. In a second study, a current-source density analysis of the responses elicited through electrical stimulation of the white matter was performed in slices of neocortex in vitro. These findings show that the NMDA-dependent component of the synaptic responses are more widely distributed and of longer duration in the cortex of epileptic rats than in that of control rats. Taken together, these results suggest that in this model of absence epilepsy NMDA-dependent mechanisms are important in the triggering and maintenance of epileptic activity.