RESUMEN
5-HT(1A) autoreceptors regulate the firing of 5-HT neurons and their release of 5-HT. In previous immuno-electron microscopic studies, we have demonstrated an internalization of 5-HT(1A) autoreceptors in the nucleus raphe dorsalis (NRD) of rats, after the acute administration of a single dose of the specific agonist 8-hydroxy-2-(di-n-propylamine)tetralin (8-OH-DPAT) or of the selective 5-HT reuptake inhibitor, fluoxetine. Twenty-four hours after either treatment, the receptors were back in normal density on the plasma membrane of NRD neurons. Here, we examined the subcellular localization of these receptors and the in vivo binding of the 5-HT(1A) radioligand 4,2-(methoxyphenyl)-1-[2-(N-2-pyridinyl)-p-fluorobenzamido]ethylpiperazine labeled with [(18)F]fluorine ([(18)F]MPPF) after chronic fluoxetine treatment (10 mg/kg daily for 3 weeks, by minipump). Unexpectedly, after such a treatment, there were no more differences between treated and control rats in either the density of plasma membrane labeling of NRD dendrites, or in the in vivo binding of [(18)F]MPPF, as measured with beta-microprobes. This was in keeping with earlier reports of an unchanged density of 5-HT(1A) receptor binding sites after chronic fluoxetine treatment, but quite unexpected from the strong electrophysiological and biochemical evidence for a desensitization of 5-HT(1A) autoreceptors under such conditions. Indeed, when the fluoxetine-treated rats were challenged with a single dose of 8-OH-DPAT, there was no internalization of the 5-HT(1A) autoreceptors, at variance with the controls. Interestingly, several laboratories have reported an uncoupling of 5-HT(1A) autoreceptors from their G protein in the NRD of rats chronically treated with fluoxetine. Therefore, the best explanation for our results is that, after repeated internalization and retargeting, functional 5-HT(1A) autoreceptors are replaced by receptors uncoupled from their G protein on the plasma membrane of NRD 5-HT neurons. Thus, the regulatory function of these autoreceptors may depend on a dynamic balance among their production, activation, internalization and recycling to the plasma membrane in inactivated (desensitized) form.
Asunto(s)
Membrana Celular/efectos de los fármacos , Fluoxetina/farmacología , Neuronas/diagnóstico por imagen , Núcleos del Rafe/citología , Receptor de Serotonina 5-HT1A/metabolismo , Inhibidores Selectivos de la Recaptación de Serotonina/farmacología , 8-Hidroxi-2-(di-n-propilamino)tetralin/farmacología , Animales , Interacciones Farmacológicas , Fluoxetina/farmacocinética , Hipocampo/ultraestructura , Masculino , Microscopía Inmunoelectrónica , Modelos Neurológicos , Neuronas/efectos de los fármacos , Unión Proteica/efectos de los fármacos , Ratas , Ratas Sprague-Dawley , UltrasonografíaRESUMEN
Alzheimer's disease (AD) is characterized by increases in amyloid-beta (Abeta) peptides, neurofibrillary tangles, oxidative stress and cholinergic deficits. However, the selectivity of these deficits and their relation with the Abeta pathology or oxidative stress remain unclear. We therefore investigated amyloidosis-related changes in acetylcholine (ACh) and serotonin (5-HT) innervations of hippocampus and parietal cortex by quantitative choline acetyltransferase (ChAT) and 5-HT immunocytochemistry, in 6, 12/14 and 18 month-old transgenic mice carrying familial AD-linked mutations (hAPP(Sw,Ind)). Further, using manganese superoxide dismutase (MnSOD) and nitrotyrosine immunoreactivity as markers, we evaluated the relationship between oxidative stress and the ACh deficit in 18 month-old mice. Thioflavin-positive Abeta plaques were seen in both regions at all ages; they were more numerous in hippocampus and increased in number (>15-fold) and size as a function of age. A majority of plaques exhibited or were surrounded by increased MnSOD immunoreactivity, and dystrophic ACh or 5-HT axons were seen in their immediate vicinity. Counts of immunoreactive axon varicosities revealed significant decreases in ACh innervation, with a sparing of the 5-HT, even in aged mice. First apparent in hippocampus, the loss of ACh terminals was in the order of 20% at 12/14 months, and not significantly greater (26%) at 18 months. In parietal cortex, the ACh denervation was significant at 18 months only, averaging 24% across the different layers. Despite increased perivascular MnSOD immunoreactivity, there was no evidence of dystrophic ACh varicosities or their accentuated loss in the perivascular area. Moreover, there was virtually no sign of tyrosine nitration in ChAT nerve terminals or neuronal cell bodies. These data suggest that aggregated Abeta exerts an early, non-selective and focal neurotoxic effect on both ACh and 5-HT axons, but that a selective, plaque- and oxidative stress-independent diffuse cholinotoxicity, most likely caused by soluble Abeta assemblies, is responsible for the hippocampal and cortical ACh denervation.
Asunto(s)
Vías Aferentes/fisiopatología , Enfermedad de Alzheimer/metabolismo , Péptidos beta-Amiloides/metabolismo , Corteza Cerebral/fisiopatología , Fibras Colinérgicas/patología , Tirosina/análogos & derivados , Degeneración Walleriana/fisiopatología , Acetilcolina/metabolismo , Vías Aferentes/metabolismo , Vías Aferentes/patología , Enfermedad de Alzheimer/fisiopatología , Precursor de Proteína beta-Amiloide/genética , Animales , Axones/metabolismo , Axones/patología , Corteza Cerebral/metabolismo , Corteza Cerebral/patología , Colina O-Acetiltransferasa/metabolismo , Fibras Colinérgicas/metabolismo , Desnervación , Modelos Animales de Enfermedad , Femenino , Hipocampo/metabolismo , Hipocampo/patología , Hipocampo/fisiopatología , Humanos , Inmunohistoquímica , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Estrés Oxidativo/fisiología , Terminales Presinápticos/metabolismo , Serotonina/metabolismo , Superóxido Dismutasa/metabolismo , Tirosina/metabolismo , Degeneración Walleriana/metabolismo , Degeneración Walleriana/patologíaRESUMEN
To follow on prior studies of the cerebral cortex, we examined the acetylcholine innervation in the developing hippocampus of rat, by means of light and electron microscopic immunocytochemistry with a highly sensitive antibody against choline acetyltransferease. As in neocortex, the growth of this innervation mostly occurred within the first two weeks after birth. A preliminary ultrastructural survey indicated that a vast majority of these ChAT-immunostained axon varicosities were asynaptic during development as in the adult. In parallel, we quantified the cholinergic innervations of cerebral cortex and hippocampus in transgenic mice overexpressing human beta-amyloid peptide (hAPP(SW,IND)). A selective, widespread, plaque independent cholinergic denervation was thus demonstrated, first in hippocampus and then neocortex, in addition to a non-selective, plaque-dependent, local neurotoxic effect of aggregated beta-amyloid on ACh and 5-HT axons.