RESUMO
Neural plasticity following brain injury illustrates the potential for regeneration in the central nervous system. Lesioning of the perforant path, which innervates the outer two-thirds of the molecular layer of the dentate gyrus, was one of the first models to demonstrate structural plasticity of mature granule cells (Parnavelas et al., 1974; Caceres and Steward, 1983; Diekmann et al., 1996). The dentate gyrus also harbors a continuously proliferating population of neuronal precursors that can integrate into functional circuits and show enhanced short-term plasticity (Schmidt-Hieber et al., 2004; Abrous et al., 2005). To examine the response of adult-generated granule cells to unilateral complete transection of the perforant path in vivo, we tracked these cells using transgenic POMC-EGFP mice or by retroviral expression of GFP. Lesioning triggered a marked proliferation of newborn neurons. Subsequently, the dendrites of newborn neurons showed reduced complexity within the denervated zone, but dendritic spines still formed in the absence of glutamatergic nerve terminals. Electron micrographs confirmed the lack of intact presynaptic terminals apposing spines on mature cells and on newborn neurons. Newborn neurons, but not mature granule cells, had a higher density of dendritic spines in the inner molecular layer postlesion accompanied by an increase in miniature EPSC amplitudes and rise times. Our results indicate that injury causes an increase in newborn neurons and lamina-specific synaptic reorganization indicative of enhanced plasticity. The presence of de novo dendritic spines in the denervated zone suggests that the postlesion environment provides the necessary signals for spine formation.
Assuntos
Lesões Encefálicas/patologia , Lesões Encefálicas/fisiopatologia , Proliferação de Células , Giro Denteado/citologia , Neurônios/fisiologia , Animais , Animais Recém-Nascidos , Lesões Encefálicas/prevenção & controle , Bromodesoxiuridina/metabolismo , Movimento Celular/genética , Espinhas Dendríticas/metabolismo , Espinhas Dendríticas/ultraestrutura , Maleato de Dizocilpina/administração & dosagem , Potenciais Evocados/efeitos dos fármacos , Antagonistas de Aminoácidos Excitatórios/administração & dosagem , Potenciais Pós-Sinápticos Excitadores/efeitos dos fármacos , Vetores Genéticos/fisiologia , Proteínas de Fluorescência Verde/genética , Técnicas In Vitro , Modelos Lineares , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Microscopia Eletrônica de Transmissão , Neurônios/ultraestrutura , Técnicas de Patch-Clamp/métodos , Via Perfurante/lesões , Pró-Opiomelanocortina/genética , Proteínas Proto-Oncogênicas c-fos/metabolismo , Coloração pela Prata , Estatísticas não Paramétricas , Sinapses/metabolismo , Sinapses/ultraestrutura , Fatores de Tempo , Proteína Vesicular 2 de Transporte de Glutamato/metabolismoRESUMO
Adult-born dentate granule cells integrate into the hippocampal network, extend neurites and form synapses in otherwise mature tissue. Excitatory and inhibitory inputs innervate these new granule cells in a stereotyped, temporally segregated manner, which presents a unique opportunity to study synapse development in the adult brain. To examine the role of neuroligins as synapse-inducing molecules in vivo, we infected dividing neural precursors in adult mice with a retroviral construct that increased neuroligin-1 levels during granule cell differentiation. By 21 days post-mitosis, exogenous neuroligin-1 was expressed at the tips of dendritic spines and increased the number of dendritic spines. Neuroligin-1-overexpressing cells showed a selective increase in functional excitatory synapses and connection multiplicity by single afferent fibers, as well as an increase in the synaptic AMPA/NMDA receptor ratio. In contrast to its synapse-inducing ability in vitro, neuroligin-1 overexpression did not induce precocious synapse formation in adult-born neurons. However, the dendrites of neuroligin-1-overexpressing cells did have more thin protrusions during an early period of dendritic outgrowth, suggesting enhanced filopodium formation or stabilization. Our results indicate that neuroligin-1 expression selectively increases the degree, but not the onset, of excitatory synapse formation in adult-born neurons.
Assuntos
Moléculas de Adesão Celular Neuronais/fisiologia , Espinhas Dendríticas/fisiologia , Neurônios/fisiologia , Sinapses/fisiologia , Animais , Animais Recém-Nascidos , Moléculas de Adesão Celular Neuronais/genética , Moléculas de Adesão Celular Neuronais/metabolismo , Células Cultivadas , Espinhas Dendríticas/metabolismo , Giro Denteado/citologia , Giro Denteado/metabolismo , Potenciais Pós-Sinápticos Excitadores/fisiologia , Vetores Genéticos/genética , Hipocampo/citologia , Hipocampo/metabolismo , Hipocampo/fisiologia , Imuno-Histoquímica , Camundongos , Microscopia Confocal , Vírus da Leucemia Murina de Moloney/genética , Neurônios/citologia , Neurônios/metabolismo , Técnicas de Patch-Clamp , Receptores de AMPA/metabolismo , Receptores de AMPA/fisiologia , Receptores de N-Metil-D-Aspartato/metabolismo , Receptores de N-Metil-D-Aspartato/fisiologia , Sinapses/metabolismo , Fatores de Tempo , Transdução GenéticaRESUMO
Neuronal activity enhances the elaboration of newborn neurons as they integrate into the synaptic circuitry of the adult brain. The role microRNAs play in the transduction of neuronal activity into growth and synapse formation is largely unknown. MicroRNAs can influence the expression of hundreds of genes and thus could regulate gene assemblies during processes like activity-dependent integration. Here, we developed viral-based methods for the in vivo detection and manipulation of the activity-dependent microRNA, miR-132, in the mouse hippocampus. We find, using lentiviral and retroviral reporters of miR-132 activity, that miR-132 is expressed at the right place and right time to influence the integration of newborn neurons. Retroviral knockdown of miR-132 using a specific 'sponge' containing multiple target sequences impaired the integration of newborn neurons into the excitatory synaptic circuitry of the adult brain. To assess potential miR-132 targets, we used a whole-genome microarray in PC12 cells, which have been used as a model of neuronal differentiation. miR-132 knockdown in PC12 cells resulted in the increased expression of hundreds of genes. Functional grouping indicated that genes involved in inflammatory/immune signaling were the most enriched class of genes induced by miR-132 knockdown. The correlation of miR-132 knockdown to increased proinflammatory molecular expression may indicate a mechanistic link whereby miR-132 functions as an endogenous mediator of activity-dependent integration in vivo.
Assuntos
Envelhecimento/metabolismo , Giro Denteado/citologia , Giro Denteado/metabolismo , MicroRNAs/metabolismo , Neurônios/metabolismo , Animais , Animais Recém-Nascidos , Diferenciação Celular/genética , Espinhas Dendríticas/metabolismo , Potenciais Pós-Sinápticos Excitadores , Regulação da Expressão Gênica no Desenvolvimento , Técnicas de Silenciamento de Genes , Genes Reporter/genética , Células HEK293 , Humanos , Inflamação/genética , Camundongos , MicroRNAs/genética , Neurônios/citologia , Células PC12 , Ratos , Receptores de AMPA/metabolismo , Retroviridae , Transdução de Sinais/genéticaRESUMO
Some cases of autism spectrum disorder have mutations in the lipid phosphatase, phosphatase and tensin homolog on chromosome 10 (Pten). Tissue specific deletion of Pten in the hippocampus and cortex of mice causes anatomical and behavioral abnormalities similar to human autism. However, the impact of reductions in Pten on synaptic and circuit function remains unexplored. We used in vivo stereotaxic injections of lentivirus expressing a short hairpin RNA to knock down Pten in mouse neonatal and young adult dentate granule cells. We then assessed the morphology and synaptic physiology between 2 weeks and 4 months later. Confocal imaging of the hippocampus revealed a marked increase in granule cell size and an increase in dendritic spine density. The onset of morphological changes occurred earlier in neonatal mice than in young adults. We used whole-cell recordings from granule cells in acute slices to assess synaptic function after Pten knockdown. Consistent with the increase in dendritic spines, the frequency of excitatory miniature and spontaneous postsynaptic currents increased. However, there was little or no effect on IPSCs. Thus, Pten knockdown results in an imbalance between excitatory and inhibitory synaptic activity. Because reductions in Pten affected mature granule cells as well as developing granule cells, we suggest that the disruption of circuit function by Pten hypofunction may be ongoing well beyond early development.