title: Isolation and Culture of Adult Zebrafish Brain-derived Neurospheres.
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.
authors.affiliation: Yale Cardiovascular Research Center, Internal Medicine, Yale University; Department of Medicine, University of California, San Diego.
authors.forename: Miguel A
authors.initials: MA
authors.lastname: Lopez-Ramirez
authors.affiliation: APHP Groupe Hospitalier Pitié-Salpètrière, Université Pierre and Marie Curie.
authors.forename: Charles-Félix
authors.initials: CF
authors.lastname: Calvo
authors.affiliation: Yale Cardiovascular Research Center, Internal Medicine, Yale University.
authors.forename: Emma
authors.initials: E
authors.lastname: Ristori
authors.affiliation: Yale Cardiovascular Research Center, Internal Medicine, Yale University; APHP Groupe Hospitalier Pitié-Salpètrière, Université Pierre and Marie Curie.
authors.forename: Jean-Léon
authors.initials: JL
authors.lastname: Thomas
authors.affiliation: Yale Cardiovascular Research Center, Internal Medicine, Yale University; stefania.nicoli@yale.edu.
authors.forename: Stefania
authors.initials: S
authors.lastname: Nicoli
mesh_terms: D053687:Adult Stem Cells / Q000166:cytology*; D000818:Animals; D001921:Brain / Q000166:cytology* / Q000502:physiology; D018929:Cell Culture Techniques / Q000379:methods; D002454:Cell Differentiation / Q000502:physiology; D049109:Cell Proliferation / Q000502:physiology; D002531:Cerebellum / Q000166:cytology; D039902:Multipotent Stem Cells / Q000166:cytology*; D058953:Neural Stem Cells / Q000166:cytology*; D055495:Neurogenesis / Q000502:physiology; D012038:Regeneration / Q000502:physiology; D013687:Telencephalon / Q000166:cytology; D015027:Zebrafish