RESUMO
Genes of the Eya family and of the Six1/2 subfamily are expressed throughout development of vertebrate cranial placodes and are required for their differentiation into ganglia and sense organs. How they regulate placodal neurogenesis, however, remains unclear. Through loss of function studies in Xenopus we show that Eya1 and Six1 are required for neuronal differentiation in all neurogenic placodes. The effects of overexpression of Eya1 or Six1 are dose dependent. At higher levels, Eya1 and Six1 expand the expression of SoxB1 genes (Sox2, Sox3), maintain cells in a proliferative state and block expression of neuronal determination and differentiation genes. At lower levels, Eya1 and Six1 promote neuronal differentiation, acting downstream of and/or parallel to Ngnr1. Our findings suggest that Eya1 and Six1 are required for both the regulation of placodal neuronal progenitor proliferation, through their effects on SoxB1 expression, and subsequent neuronal differentiation.
Assuntos
Diferenciação Celular , Proteínas de Ligação a DNA/metabolismo , Proteínas de Grupo de Alta Mobilidade/metabolismo , Proteínas de Homeodomínio/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Neurônios/citologia , Proteínas Nucleares/metabolismo , Proteínas Tirosina Fosfatases/metabolismo , Crânio/embriologia , Fatores de Transcrição/metabolismo , Proteínas de Xenopus/metabolismo , Xenopus/embriologia , Animais , Biomarcadores/metabolismo , Diferenciação Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Ectoderma/citologia , Ectoderma/efeitos dos fármacos , Embrião não Mamífero/citologia , Embrião não Mamífero/efeitos dos fármacos , Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , Humanos , Injeções , Modelos Biológicos , Neurônios/efeitos dos fármacos , Oligonucleotídeos Antissenso/farmacologia , Receptores de Glucocorticoides/metabolismo , Proteínas Recombinantes de Fusão/metabolismo , Fatores de Transcrição SOXB1 , Crânio/efeitos dos fármacos , Células-Tronco/citologia , Células-Tronco/efeitos dos fármacosRESUMO
We investigated the effect of in vitro exposure to nicotinic acetylcholine receptors (nAChRs), agonists, antagonists, and protein kinase A (PKA) modulators on the activity of the serotonin transporter (SERT) in prefrontocortical (PFC) synaptosomes. The plasma membrane SERT is an active transport mechanism specific for serotonin. Receptors and second messengers capable of altering transporter activity would be expected to have profound effects on serotonergic neurotransmission and on functions involving serotonergic input, such as cognition, anxiety, and mood. Our data suggest that activation of nAChRs, quite likely via PKA, increase the activity of the SERT in the PFC and, thereby, can alter 5-HT levels in a region important in the behavioral effects of nicotine and 5-HT. Nicotine at 4 microM increased [(3)H]5-HT uptake by 75%. Because the nAChR antagonists mecamylamine and dihydro-beta-erythrodine (DHbetaE) both decreased [(3)H]5-HT uptake into synaptosomes, it appeared that the SERT might be tonically activated by acetylcholine present within our synaptosomal preparations. Blocking PKA significantly decreased [(3)H]5-HT, while stimulation of PKA activity significantly increased the uptake. A 66% decrease compared with control was produced by 100 microM Rp-cAMP, and a 41% increase in 5-HT uptake over control was observed with 30 microM Sp-cAMPs. Furthermore, the enhancement in uptake produced by 4 microM nicotine was inhibited in a time-dependent fashion by preincubation with 10 microM Rp-cAMP. A better understanding of the influence of the cholinergic system and the receptors involved in the trafficking of SERT would help clarify the important relationship between the cholinergic and serotonergic systems and the role these systems play in behavior.
Assuntos
Proteínas Quinases Dependentes de AMP Cíclico/fisiologia , Córtex Pré-Frontal/metabolismo , Receptores Nicotínicos/fisiologia , Proteínas da Membrana Plasmática de Transporte de Serotonina/metabolismo , Animais , Atropina/farmacologia , Carbacol/farmacologia , Agonistas Colinérgicos/farmacologia , AMP Cíclico/análogos & derivados , AMP Cíclico/farmacologia , Di-Hidro-beta-Eritroidina/farmacologia , Relação Dose-Resposta a Droga , Interações Medicamentosas , Técnicas In Vitro , Mecamilamina/farmacologia , Antagonistas Muscarínicos/farmacologia , Nicotina/farmacologia , Agonistas Nicotínicos/farmacologia , Antagonistas Nicotínicos/farmacologia , Córtex Pré-Frontal/citologia , Córtex Pré-Frontal/efeitos dos fármacos , Inibidores de Proteínas Quinases/farmacologia , Ratos , Serotonina/metabolismo , Serotonina/farmacocinética , Sinaptossomos/efeitos dos fármacos , Tionucleotídeos/farmacologia , Fatores de Tempo , Trítio/farmacocinéticaRESUMO
We sought to investigate the effect of nicotine exposure (chronic and acute) on serotonin transporter (SERT) activity in two regions of the brain important for behavioral effects of nicotine. We first looked at the effects of chronic nicotine exposure (0.7 mg/kg nicotine, twice a day for 10 days) on [(3)H]5-HT uptake in prefrontal cortex (PFC) and hippocampus of rats. A significant increase in [(3)H]5-HT uptake was observed in synaptosomes prepared from both regions. To rule out the possibility that the increases were due to the last injection given, in a separate set of experiments a single injection of nicotine was administered the evening before sacrifice. No change in uptake occurred in either region, suggesting that the increases in uptake caused by nicotine was an effect of chronic exposure and not to an acute treatment. SERT binding studies, using prefrontocortical or hippocampal membrane preparations, revealed that chronic nicotine exposure significantly increased B(max) which correlated to an increase in SERT density. Lastly, we looked at the short-term effect of nicotine on [(3)H]5-HT uptake. Rats received a single nicotine injection 15-75 min before sacrifice. PFC synaptosomes displayed a time-dependent increase in uptake, whereas hippocampal synaptosomes showed an increase at only one time point.