RESUMEN
The neurotrophin survival dependence of peripheral neurons in vitro is regulated by the proapoptotic BCL-2 homolog BAX. To study peripheral neuron development in the absence of neurotrophin signaling, we have generated mice that are double null for BAX and nerve growth factor (NGF), and BAX and the NGF receptor TrkA. All dorsal root ganglion (DRG) neurons that normally die in the absence of NGF/TrkA signaling survive if BAX is also eliminated. These neurons extend axons through the dorsal roots and collateral branches into the dorsal horn. In contrast, superficial cutaneous innervation is absent. Furthermore, rescued sensory neurons fail to express biochemical markers characteristic of the nociceptive phenotype. These findings establish that NGF/TrkA signaling regulates peripheral target field innervation and is required for the full phenotypic differentiation of sensory neurons.
Asunto(s)
Factor de Crecimiento Nervioso/farmacología , Neuronas Aferentes/citología , Proteínas Proto-Oncogénicas c-bcl-2 , Proteínas Proto-Oncogénicas/genética , Receptor trkA/genética , Transducción de Señal/fisiología , Animales , Péptido Relacionado con Gen de Calcitonina/genética , Recuento de Células , Diferenciación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , Femenino , Ganglios Espinales/citología , Ganglios Espinales/embriología , Regulación del Desarrollo de la Expresión Génica , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Mutantes , Neuronas Aferentes/química , Neuronas Aferentes/fisiología , Fenotipo , Piel/inervación , Médula Espinal/citología , Sustancia P/genética , Proteína X Asociada a bcl-2RESUMEN
We describe the production and characterization of a specific anti-5-HT1A receptor antibody made against a fusion protein consisting of glutathione-S-transferase (GST) coupled to a 75-amino acid sequence from the middle portion of the third intracellular loop (5-HT1A-m3i, serine253-arginine327) of the rat 5-HT1A receptor protein. This region was chosen to avoid putative phosphorylation and glycosylation sites and regions of known homology with other 5-HT receptors. Western blot analysis indicated that the polyclonal anti-5-HT1A-m3i antibody accurately recognized the fusion protein expressed in bacteria and labeled a prominent 67 kDa protein band in the hippocampus, cortex, brainstem, cerebellum and kidney with a density profile corresponding to the relative abundance of the 5-HT1A receptor in these tissues. No protein was detected in liver or muscle tissue preparations, and no protein bands were labeled in any of the above tissues following preabsorption of the antibody with the 5-HT1A-m3i fusion protein. Immunohistochemistry revealed prominent labeling in limbic structures including the hippocampus, amygdala, entorhinal cortex, and septum as well as in raphe nuclei. In the hippocampus, 5-HT1A-m3i labeling revealed a characteristic laminar pattern that coincided with that seen by autoradiographic binding of the 5-HT1A agonist [3H]-8-OH-DPAT in all strata of the hippocampal formation. In the dorsal and medial raphe nuclei, anti-5-HT1A-m3i antibodies labeled the somatodendritic membranes of 5-HT neurons, consistent with its role as an autoreceptor. The detailed matching of the anti-5-HT1A-m3i antibody with [3H]-8-OH-DPAT binding suggests that the antibody recognizes a functionally active form of the 5-HT1A receptor protein capable of binding 5-HT1A agonist ligands. These anti-5-HT1A antibodies may therefore be useful tools in localizing functional 5-HT1A receptors in specific regions of the brain as well as in studying the plasticity and ontogeny of the 5-HT1A receptor at the cellular and subcellular level.
Asunto(s)
Receptores de Serotonina/inmunología , Secuencia de Aminoácidos , Animales , Formación de Anticuerpos , Glutatión Transferasa/inmunología , Inmunohistoquímica , Masculino , Datos de Secuencia Molecular , Ratas , Ratas Sprague-Dawley , Proteínas Recombinantes de Fusión/inmunología , Homología de Secuencia de AminoácidoRESUMEN
We found previously that alcohol-preferring (P) rats have fewer serotonin (5-HT) neurons and fibers in key brain regions than alcohol-nonpreferring (NP) rats. Because 5-HT uptake blockers increase synaptic 5-HT content and 5-HT1A receptor antagonists increase 5-HT release by disinhibiting 5-HT autoinnervation, in the present study, our intent was to determine whether increased synaptic 5-HT content and/or 5-HT release in P rats would effectively reduce alcohol consumption. In experiment 1, the 5-HT antagonist WAY 100635 (WAY) was tested on adult female P rats maintained on 24-hr free-choice access to ethanol (10% v/v) and water. Twice daily doses of WAY (0.05, 0.1, 0.5, and 1.0 mg/kg, subcutaneously) were administered to each rat in a counterbalanced order. Baseline ethanol intake, derived from the mean ethanol intakes of the three previous non-drug days, was approximately 8 g/kg/day. Results indicated that 0.05, 0.1, and 0.5 mg/kg doses of WAY reduced 24-hr ethanol drinking by 25-30% (p < 0.01) without affecting 24-hr water intake or body weight. In the second experiment, the effects of WAY (0.5 mg/kg), fluoxetine (1.0 mg/kg), or a combination of both were tested in another group of female P rats. WAY and fluoxetine, each alone, reduced ethanol drinking by around 20% and, when combined, decreased ethanol intake by 50%, whereas the body weight and the total fluid intake were not significantly affected. Taken together, these results indicate that both fluoxetine and WAY preferentially reduce ethanol drinking in the P line of rats and, when administered together, reduce ethanol intake in an additive manner. It is proposed that coadministration of these two compounds with distinct mechanisms of action may be a new strategy for reducing alcohol intake.
Asunto(s)
Consumo de Bebidas Alcohólicas/fisiopatología , Alcoholismo/fisiopatología , Fluoxetina/farmacología , Piperazinas/farmacología , Piridinas/farmacología , Receptores de Serotonina/efectos de los fármacos , Inhibidores Selectivos de la Recaptación de Serotonina/farmacología , Antagonistas de la Serotonina/farmacología , Animales , Relación Dosis-Respuesta a Droga , Sinergismo Farmacológico , Femenino , Ratas , Ratas Endogámicas , Receptores de Serotonina/fisiología , Receptores de Serotonina 5-HT1RESUMEN
Current knowledge of genes that regulate pattern formation and differentiation processes during mammalian embryonic development is limited. In an effort to isolate developmentally relevant genes, 20 novel, end-sequenced cDNAs selected from a Day 10.5 postcoitum mouse embryo library were genetically mapped in intersubspecific backcross mice. Eleven of 20 cDNA clones mapped to three mouse autosomes (Chr 5, 11, and 14), a result that was unlikely (P < 0.03) if the distribution of genes expressed in embryos is random within the mouse genome. Several clones were candidates for mouse developmental mutations by virtue of genetic colocalization and concordance of embryonic expression patterns with the distribution of defects in mutant mice: Estm11 was a candidate for the mouse mutation wabbler-lethal (wl), since Estm11 mapped in the vicinity of wl on mouse Chr 14 and was expressed in those regions of embryonic brain that exhibit axonal degeneration in wl. End-sequence analysis, genetic mapping, and in situ hybridization appeared to be an effective combination of methods for identification and characterization of genes with potential regulatory functions during mammalian embryogenesis.
Asunto(s)
Mapeo Cromosómico , ADN Complementario , Familia de Multigenes , Animales , Secuencia de Bases , Embrión de Mamíferos , Femenino , Expresión Génica , Masculino , Ratones , Ratones Endogámicos C57BL , Datos de Secuencia MolecularRESUMEN
Serotonin (5-HT) projections from the ascending raphe nuclei reach the dorsal hippocampus via the cingulum bundle (CB) and fimbria-fornix (FF). Microinjection of the serotonergic neurotoxin 5,7-dihydroxytryptamine (5,7-DHT) into the CB and FF produces a significant decrease in the density of 5-HT immunoreactive fibers in the hippocampus as early as 3 days postlesion (Zhou, F.C. and Azmitia, E.C. (1983) Brain Res. Bull., 373, 337-348). In the present study we used an anti-peptide antibody against the second extracellular loop of the 5-HT1A receptor and employed immunocytochemistry to examine changes in the expression and distribution of the 5-HT1A receptor in the hippocampus 14 days following administration of 5,7-DHT into the CB and FF. The density of 5-HT immunoreactive fibers was greatly reduced 14 days following the lesions. 5-HT1A immunoreactivity (IR) was localized to the proximal axon near the axon hillock of cells in the pyramidal cell layer of the cornu Ammonus and in the granule cell layer of the dentate gyrus. The intensity of 5-HT1A-IR was increased in the CA1 and dentate gyrus following 5,7-DHT lesions. Intensity in the CA3 also increased but not to a significant level. These findings demonstrate that 5-HT denervation in the hippocampus is followed by increased expression of the 5-HT1A receptor protein. These changes in receptor expression 14 days postlesion may represent adaptive changes by postsynaptic cells following reduced 5-HT innervation and may be the molecular basis for 5-HT1A receptor supersensitivity.