Development of Optogenetic Tools for Manipulating Neuronal Activity and Behaviour in Zebrafish

• Autor(a) principal: Esteves, Rita Maria Santos
• Data de Publicação: 2016
• Tipo de documento: Dissertação
• Idioma: eng
• Título da fonte: Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos)
• Texto Completo: http://hdl.handle.net/10362/21501
• topic: Optogenetics; GECI; Zebrafish; Neurobiology; Neural circuits; Domínio/Área Científica::Engenharia e Tecnologia::Outras Engenharias e Tecnologias

Integration of sensory input and computation of behavioural output is a dynamic process involving diverse populations of cells often distributed throughout the brain. To study this topic, monitoring neuronal activity from a large population of cells and manipulating targeted neuronal activity in a behaving animal is crucial. This is possible in zebrafish, due to its small and transparent larval brain and its genetic malleability, by making use of optogenetic tools that allow reversible light-dependent activation and inhibition of neuronal activity, and genetically encoded calcium indicators (GECI) that enable non-invasive activity recording. State-of-the-art optogenetic tools with faster kinetics and higher sensitivity facilitate reliable manipulation of activity with high temporal precision during behaviour. Such tools have been developed to be compatible with better calcium indicators to successfully manipulate and optically record neuronal activity simultaneously. In this project, the latest developed optogenetic tools - activators ChrimsonR, C1V1(t/t) and Chronos, inhibitor Jaws, red calcium sensor jRCaMP1b and nuclear markers H2B-RFP and H2B-mCherry - were optimized to be expressed in zebrafish. Behavioural assays to characterize the activating and inhibitory optogenetic tools ChrimsonR and Jaws were established. An escape response of short latency could reliably be evoked in transgenic animals with stable expression of ChrimsonR in trigeminal neurons. Combination of this fast, sensitive and red-shifted tool with GCaMP calcium imaging opens the possibility to simultaneously manipulate and record activity with high spatial and temporal precision from a large population of neurons to study their dynamic interactions during behaviour.