RESUMO
mRNA localization and regulated translation provide a means of spatially restricting gene expression within neurons during axon guidance and long-term synaptic plasticity. Here we show that synapse formation specifically alters the localization of the mRNA encoding sensorin, a peptide neurotransmitter with neurotrophin-like properties. In isolated Aplysia sensory neurons, which do not form chemical synapses, sensorin mRNA is diffusely distributed throughout distal neurites. Upon contact with a target motor neuron, sensorin mRNA rapidly concentrates at synapses. This redistribution only occurs in the presence of a target motor neuron and parallels the distribution of sensorin protein. Reduction of sensorin mRNA, but not protein, with dsRNA inhibits synapse formation. Our results indicate that synapse formation can alter mRNA localization within individual neurons. They further suggest that translation of a specific localized mRNA, encoding the neuropeptide sensorin, is required for synapse formation between sensory and motor neurons.
Assuntos
Aplysia/citologia , Neurônios/fisiologia , RNA Mensageiro/metabolismo , Sinapses/fisiologia , Análise de Variância , Animais , Western Blotting/métodos , Células Cultivadas , Técnicas de Cocultura/métodos , Dactinomicina/farmacologia , Diagnóstico por Imagem/métodos , Estimulação Elétrica/métodos , Técnicas Eletrofisiológicas Cardíacas/métodos , Potenciais Pós-Sinápticos Excitadores/fisiologia , Regulação da Expressão Gênica/fisiologia , Proteínas de Fluorescência Verde/metabolismo , Imuno-Histoquímica/métodos , Hibridização In Situ/métodos , Microinjeções/métodos , Neurônios Motores/metabolismo , Neurônios Motores/fisiologia , Neuritos/efeitos dos fármacos , Neuritos/metabolismo , Neurônios/citologia , Neurônios/efeitos dos fármacos , Neurônios/efeitos da radiação , Neurônios Aferentes/classificação , Neurônios Aferentes/citologia , Neurônios Aferentes/efeitos dos fármacos , Neurônios Aferentes/fisiologia , Neurônios Aferentes/efeitos da radiação , Neuropeptídeos/genética , Neuropeptídeos/metabolismo , Cloreto de Potássio/farmacologia , Inibidores da Síntese de Proteínas/farmacologia , RNA de Cadeia Dupla/farmacologiaRESUMO
Local protein synthesis is required for long-lasting synapse-specific plasticity in cultured Aplysia sensorimotor synapses. To identify synaptically localized mRNAs, we prepared a cDNA library from isolated sensory neurites. By sequence analysis, we estimate that the library contains 263 distinct mRNAs, with 98 of these mRNAs constituting 70% of all clones. The localized transcripts are enriched for mRNAs encoding cytoskeletal elements and components of the translational machinery. In situ hybridization confirms that the mRNAs for at least eight of these transcripts are present in distal neurites. Immunocytochemistry reveals that serotonin regulates the translation of one of the localized mRNAs, that encoding alpha1-tubulin. Our identification of mRNAs encoding cytoskeletal elements suggests that local protein synthesis is required for the growth of new synaptic connections associated with persistent synaptic strengthening. Our finding of mRNAs encoding components of the translational machinery suggests that local protein synthesis serves to increase the translational capacity of synapses.