RESUMO
Synapses established during central nervous system development can be modified through synapse elimination and formation. These processes are, in part, activity dependent and require regulated trafficking of post-synaptic components. Here, we investigate the activity-driven remodeling of cultured rat hippocampal neurons at 14 days in vitro, focusing on the post-synaptic proteins PSD-95, Shank, neuroligin (NL)1 and actin. Using live imaging and photoconductive stimulation, we found that high-frequency activity altered the trajectory, but not velocity, of PSD-95-GFP and Shank-YFP clusters, whereas it reduced the speed and increased the number of NL1 clusters. Actin-CFP reorganized into puncta following activity and approximately 50% of new puncta colocalized with NL1 clusters. Actin reorganization was enhanced by the overexpression of NL1 and decreased by the expression of an NL1 mutant, NL1-R473C. These results demonstrate activity-dependent changes that may result in the formation of new post-synaptic sites and suggest that NL1 modulates actin reorganization. The results also suggest that a common mechanism underlies both the developmental and activity-dependent remodeling of excitatory synapses.
Assuntos
Actinas/metabolismo , Molécula de Adesão de Leucócito Ativado/metabolismo , Regulação da Expressão Gênica/fisiologia , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas de Membrana/metabolismo , Neurônios/metabolismo , Actinas/genética , Molécula de Adesão de Leucócito Ativado/genética , Análise de Variância , Animais , Animais Recém-Nascidos , Contagem de Células/métodos , Proteína 4 Homóloga a Disks-Large , Relação Dose-Resposta à Radiação , Regulação da Expressão Gênica/efeitos da radiação , Hipocampo/citologia , Peptídeos e Proteínas de Sinalização Intracelular/genética , Proteínas Luminescentes/genética , Proteínas de Membrana/genética , Mutação/genética , Proteínas do Tecido Nervoso , Estimulação Luminosa/métodos , Transporte Proteico/fisiologia , Transporte Proteico/efeitos da radiação , Proteínas R-SNARE/genética , Proteínas R-SNARE/metabolismo , Ratos , Ratos Sprague-Dawley , Transfecção/métodosRESUMO
Loss of the ability to regulate calcium is a central event leading to neuronal cell death during a wide range of pathological conditions including stroke and seizure. Here we present a new dissociated hippocampal cell culture model of acute electrical activity which incorporates the photoconductive stimulation of neuronal networks grown on silicon wafers. This technology allows precise modeling of user defined neuronal activity patterns, and the study of their effect on neuronal physiology. Here, seizure-like conditions were created by continuous stimulation, causing hundreds of neurons to fire synchronously at 50Hz for 4min. This stimulation protocol induced cell death as monitored by propidium iodide staining. The number of dead cells per stimulation region increased from 3.6+/-2.1 preceding stimulation to 81+/-21 30min following stimulation. Excitotoxicity primarily affected excitatory rather than inhibitory neurons, and was preceded by an increase in intracellular calcium as well as changes in the mitochondrial membrane potential, as measured by a tetramethylrhodamine methyl ester (TMRM) assay. Cyclosporin A (CsA), a mitochondrial permeability transition pore (PTP) blocker, was effective in preventing cell death. We propose that photoconductive stimulation is a useful tool for investigating the pathogenesis of excitotoxicity in vitro.