Single-cell in vivo imaging of adult neural stem cells in the zebrafish telencephalon

Developers

Joana S. Barbosa, Rossella Di Giaimo, Magdalena Götz and Jovica Ninkovic

Description of the technology

Adult neural stem cells (aNSCs) in zebrafish produce mature neurons throughout their entire life span in both the intact and regenerating brain. An understanding of the behavior of aNSCs 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.

In the frameworks of this technology was developed a protocol for labeling and repetitive imaging of aNSCs in vivo. Single aNSCs were labeled, 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 green fluorescent protein (GFP) in aNSCs. The image was prodiced 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 aNSC 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. This protocol was used to follow the mode of aNSC 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 aNSC behavior. A typical experiment with data analysis takes up to 1.5 months

Practical application

The technology can be further applied to study molecules and pathways that maintain the behavior of adult neural stem cells in the intact or the damaged brain in vivo, and to test compounds that can potentially modulate this behavior in vivo. This technology is highly promising for a use in experimental investigations in the fields of neuroscience, regenerative medicine, study of ageing processes, etc. The principles, supporting this technology, can be translated to animal models in other species.

Laboratories

• Institute of Stem Cell Research, Helmholtz Center Munich, Neuherberg/Munich (Germany)
• Center for Neuroscience and Cell Biology, University of Coimbra, Coimbra (Portugal)
• Department of Biology, University of Naples Federico II, Naples (Italy)
• Biomedical Center, University of Munich, Munich (Germany)
• Excellence Cluster of Systems Neurology SYNERGY, Ludwig Maximilian University of Munich, Munich (Germany)

Links

Publications

• Barbosa, J.S. et al. «Single-cell in vivo imaging of adult neural stem cells in the zebrafish telencephalon." 11 Nature Protocols (2016): 1360–1370.
• Barbosa, J.S. et al. «Live imaging of adult neural stem cell behavior in the intact and injured zebrafish brain." 348 Science (2015): 789–793.
• Baumgart, E.v. et al. «Stab wound injury of the zebrafish telencephalon: a model for comparative analysis of reactive gliosis." 60 Glia (2012): 343–357.