Single-cell in vivo imaging of adult neural stem cells in the zebrafish telencephalon
Nature Protocols. 2016 August. 11(8):1360-70. DOI: 10.1038/nprot.2016.077. Epub 2016 Jun 30.
|Authors/Editors:||Barbosa J S, Di Giaimo R, Götz M, Ninkovic J|
Adult neural stem cells (aNSCNSCNSCs) in zebrafish produce mature neurons throughout their entire life span in both the intact and regenerating brain. An understanding of the behavior of aNSCNSCNSCs in their intact niche and during regeneration in vivo should facilitate the identification of the molecular mechanisms controlling regeneration-specific cellular events. A greater understanding of the process in regeneration-competent species may enable regeneration to be achieved in regeneration-incompetent species, including humans. Here we describe a protocol for labeling and repetitive imaging of aNSCNSCNSCs in vivo. We label single aNSCNSCNSCs, allowing nonambiguous re-identification of single cells in repetitive imaging sessions using electroporation of a red-reporter plasmid in Tg(gfap:GFP)mi2001 transgenic fish expressing GFP in aNSCNSCNSCs. We image using two-photon microscopy through the thinned skull of anesthetized and immobilized fish. Our protocol allows imaging every 2 d for a period of up to 1 month. This methodology allowed the visualization of aNSCNSCNSC behavior in vivo in their natural niche, in contrast to previously available technologies, which rely on the imaging of either dissociated cells or tissue slices. We used this protocol to follow the mode of aNSCNSCNSC division, fate changes and cell death in both the intact and injured zebrafish telencephalon. This experimental setup can be widely used, with minimal prior experience, to assess key factors for processes that modulate aNSCNSCNSC behavior. A typical experiment with data analysis takes up to 1.5 months.