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J Vis Exp. 2016 Feb 29;(108):53617. doi: 10.3791/53617.

Isolation and Culture of Adult Zebrafish Brain-derived Neurospheres.

Author information

1
Yale Cardiovascular Research Center, Internal Medicine, Yale University; Department of Medicine, University of California, San Diego.
2
APHP Groupe Hospitalier Pitié-Salpètrière, Université Pierre and Marie Curie.
3
Yale Cardiovascular Research Center, Internal Medicine, Yale University.
4
Yale Cardiovascular Research Center, Internal Medicine, Yale University; APHP Groupe Hospitalier Pitié-Salpètrière, Université Pierre and Marie Curie.
5
Yale Cardiovascular Research Center, Internal Medicine, Yale University; stefania.nicoli@yale.edu.

Abstract

The zebrafish is a highly relevant model organism for understanding the cellular and molecular mechanisms involved in neurogenesis and brain regeneration in vertebrates. However, an in-depth analysis of the molecular mechanisms underlying zebrafish adult neurogenesis has been limited due to the lack of a reliable protocol for isolating and culturing neural adult stem/progenitor cells. Here we provide a reproducible method to examine adult neurogenesis using a neurosphere assay derived from zebrafish whole brain or from the telencephalon, tectum and cerebellum regions of the adult zebrafish brain. The protocol involves, first the microdissection of zebrafish adult brain, then single cell dissociation and isolation of self-renewing multipotent neural stem/progenitor cells. The entire procedure takes eight days. Additionally, we describe how to manipulate gene expression in zebrafish neurospheres, which will be particularly useful to test the role of specific signaling pathways during adult neural stem/progenitor cell proliferation and differentiation in zebrafish.

PMID:
26967835
PMCID:
PMC4828210
DOI:
10.3791/53617
[Indexed for MEDLINE]
Free PMC Article
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