Ontogenesis of visual processing circuits in the zebrafish: analysis of specific neuronal subpopulations in development

• Autor(a) principal: Martins, João Júlio
• Data de Publicação: 2018
• Tipo de documento: Dissertação
• Idioma: eng
• Título da fonte: Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos)
• Texto Completo: http://hdl.handle.net/10362/82974
• topic: Zebrafish; olig2; chrna4; pcp4a pitx2c; chat; Confocal; Domínio/Área Científica::Engenharia e Tecnologia::Outras Engenharias e Tecnologias

The Zebrafish (Danio rerio) larva is an ideal model for identification of neuronal circuits such as those associated with visually guided behaviors like the optomotor and the optokinetic responses. The use of larvae expressing the calcium level indicator GCaMP in particular neuronal subpopulations allows monitoring of activity of these neurons and facilitates their anatomical and functional characterization. Our goal, in this context, is to characterize some of these subpopulations taking advantage of the expression of GFP using the Gal4-UAS system during the first 6 days of development. To examine and characterize the GFP expression in zebrafish larvae, we have applied an immunofluorescence protocol and recorded anatomical stacks using confocal imaging. This information gathered from several equivalent individuals and registered to a reference brain may then be used to establish anatomical atlases at these early stages of development that will complement those already developed for the 6 dpf larvae. Characterization of the transgenic lines at the early stages will help us to locate the functional clusters identified at 6 dpf with more anatomical accuracy and precision. We will present our work focusing on zebrafish lines, generated using a BAC transgenic approach, that expresses GFF or GFP from an insertion next to the olig2, chrna4 and pcp4a promoter regions. Two of these lines (olig2: GFP and chrna4: GFF) are shown to represent a similar pattern of expression has the original gene, whilst the pcp4a: GFF line does not, although its characterization is still of use to complement functional maps of neuron activity. In addition, we are using time lapse imaging with Lightsheet microscopy to follow the dynamics of neuronal differentiation and extension of projections during the 20-48 hours post fertilization period on an already characterized line(Chat:GFF).