RESUMO
Although fluorescence microscopy is ubiquitous in biomedical research, microscopy methods reporting is inconsistent and perhaps undervalued. We emphasize the importance of appropriate microscopy methods reporting and seek to educate researchers about how microscopy metadata impact data interpretation. We provide comprehensive guidelines and resources to enable accurate reporting for the most common fluorescence light microscopy modalities. We aim to improve microscopy reporting, thus improving the quality, rigor and reproducibility of image-based science.
Assuntos
Pesquisa Biomédica/métodos , Pesquisa Biomédica/normas , Processamento de Imagem Assistida por Computador/métodos , Microscopia de Fluorescência/métodos , Microscopia de Fluorescência/normas , Convallaria , Escherichia coli/metabolismo , Corantes Fluorescentes , Proteínas de Fluorescência Verde/metabolismo , Humanos , Imageamento Tridimensional , Microscopia Confocal/métodos , Reprodutibilidade dos Testes , Projetos de Pesquisa , Razão Sinal-Ruído , SoftwareRESUMO
UNLABELLED: Dendritic filopodia are actin-rich structures that are thought to contribute to early spine synapse formation; however, the actin regulatory proteins important for early synaptogenesis are poorly defined. Using organotypic hippocampal slice cultures and primary neuron hippocampal cultures from Arp2/3 conditional knock-out mice, we analyze the roles of the Arp2/3 complex, an actin regulator that creates branched actin networks, and demonstrate it is essential for distinct stages of both structural and functional maturation of excitatory spine synapses. Our data show that initially the Arp2/3 complex inhibits the formation of dendritic filopodia but that later during development, the Arp2/3 complex drives the morphological maturation from filopodia to typical spine morphology. Furthermore, we demonstrate that although the Arp2/3 complex is not required for key spine maturation steps, such as presynaptic contact and recruitment of MAGUK (membrane-associated guanylate kinase) scaffolding proteins or NMDA receptors, it is necessary for the recruitment of AMPA receptors. This latter process, also known as synapse unsilencing, is a final and essential step in the neurodevelopment of excitatory postsynaptic synaptogenesis, setting the stage for neuronal interconnectivity. These findings provide the first evidence that the Arp2/3 complex is directly involved in functional maturation of dendritic spines during the developmental period of spinogenesis. SIGNIFICANCE STATEMENT: Excitatory spine synapse formation (spinogenesis) is a poorly understood yet pivotal period of neurodevelopment that occurs within 2-3 weeks after birth. Neurodevelopmental disorders such as intellectual disability and autism are characterized by abnormal spine structure, which may arise from abnormal excitatory synaptogenesis. The initial stage of spinogenesis is thought to begin with the emergence of actin-rich dendritic filopodia that initiate contact with presynaptic axonal boutons. However, it remains enigmatic how actin cytoskeletal regulation directs dendritic filopodial emergence or their subsequent maturation into dendritic spines during development and on into adulthood. In this study, we provide the first evidence that the Arp2/3 complex, a key actin nucleator, is involved in distinct stages of spine formation and is required for synapse unsilencing.
Assuntos
Complexo 2-3 de Proteínas Relacionadas à Actina/metabolismo , Espinhas Dendríticas/fisiologia , Neurônios/citologia , Sinapses/fisiologia , Complexo 2-3 de Proteínas Relacionadas à Actina/genética , Fatores Etários , Animais , Animais Recém-Nascidos , Células Cultivadas , Fármacos Atuantes sobre Aminoácidos Excitatórios/farmacologia , Potenciais Pós-Sinápticos Excitadores/efeitos dos fármacos , Potenciais Pós-Sinápticos Excitadores/genética , Feminino , Hipocampo/citologia , Masculino , Camundongos , Camundongos Knockout , Neuropeptídeos/genética , Neuropeptídeos/metabolismo , Fotodegradação , Pseudópodes/fisiologia , Receptores de AMPA/genética , Receptores de AMPA/metabolismo , Sinapses/ultraestrutura , Fatores de Tempo , Proteínas rac1 de Ligação ao GTP/genética , Proteínas rac1 de Ligação ao GTP/metabolismoRESUMO
Hydrocephalus is the most common developmental disability and leading cause of brain surgery for children. Current treatments are limited to surgical intervention, as the factors that contribute to the initiation of hydrocephalus are poorly understood. Here, we describe the development of obstructive hydrocephalus in mice that are null for Wrp (Srgap3). Wrp is highly expressed in the ventricular stem cell niche, and it is a gene required for cytoskeletal organization and is associated with syndromic and psychiatric disorders in humans. During the postnatal period of progenitor cell expansion and ventricular wall remodeling, loss of Wrp results in the abnormal migration of lineage-tagged cells from the ventricular region into the corpus callosum. Within this region, mutant progenitors appear to give rise to abnormal astroglial cells and induce periventricular lesions and hemorrhage that leads to cerebral aqueductal occlusion. These results indicate that periventricular abnormalities arising from abnormal migration from the ventricular niche can be an initiating cause of noncommunicating hydrocephalus.
Assuntos
Ventrículos Cerebrais/citologia , Proteínas Ativadoras de GTPase/metabolismo , Hidrocefalia/metabolismo , Células-Tronco/citologia , Animais , Encéfalo/patologia , Movimento Celular , Corantes Fluorescentes/farmacologia , Deleção de Genes , Humanos , Imuno-Histoquímica/métodos , Imageamento por Ressonância Magnética/métodos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Neurônios/metabolismoRESUMO
The WAVE-associated Rac GAP, WRP, is thought to regulate key aspects of synapse development and function and may be linked to mental retardation in humans. WRP contains a newly described inverse F-BAR (IF-BAR) domain of unknown function. Our studies show that this domain senses/facilitates outward protrusions analogous to filopodia and that the molecular basis for this is likely explained by a convex lipid-binding surface on the WRP IF-BAR domain. In dendrites the IF-BAR domain of WRP forms a bud on the shaft from which precursors to spines emerge. Loss of WRP in vivo and in vitro results in reduced density of spines. In vivo this is primarily a loss of mushroom-shaped spines. Developmentally, WRP function is critical at the onset of spinogenesis, when dendritic filopodia are prevalent. Finally, because WRP is implicated in mental retardation, behaviors of WRP heterozygous and null mice have been evaluated. Results from these studies confirm that loss of WRP is linked to impaired learning and memory.