Isolation and culture of primary embryonic zebrafish neural tissue

• Published in: Journal of neuroscience methods Vol. 328; p. 108419
• Main Authors: Patel, Bhavika B., Clark, Kendra L., Kozik, Emily M., Dash, Linkan, Kuhlman, Julie A., Sakaguchi, Donald S.
• Format: Journal Article
• Language: English
• Published: Netherlands Elsevier B.V 01.12.2019
• Subjects: Cell migration; Primary neuron culture; Time-lapse microscopy; Zebrafish; Zebrafish; Time-lapse microscopy; Primary neuron culture; Cell migration; cell migration; zebrafish; time-lapse microscopy
• ISSN: 0165-0270; 1872-678X
• DOI: 10.1016/j.jneumeth.2019.108419

•An efficient method for isolating embryonic zebrafish neurons from neural tissue.•Primary cells can survive 9 days in vitro, expressing markers for neurons and glia.•85% of dissociated cells expressed serotonin, indicating neuronal subtypes present.•Cell migration was studied using time-lapse microscopy of glial cells and neurons.•The primary culture can be utilized to study cell-cell interactions. Primary cell culture is a valuable tool to utilize in parallel with in vivo studies in order to maximize our understanding of the mechanisms surrounding neurogenesis and central nervous system (CNS) regeneration and plasticity. The zebrafish is an important model for biomedical research and primary neural cells are readily obtainable from their embryonic stages viatissue dissociation. Further, transgenic reporter lines with cell type-specific expression allows for observation of distinct cell populations within the dissociated tissue. Here, we define an efficient method for ex vivo quantification and characterization of neuronal and glial tissue dissociated from embryonic zebrafish. Zebrafish brain dissociated cells have been documented to survive in culture for at least 9 days in vitro (div). Anti-HuC/D and anti-Acetylated Tubulin antibodies were used to identify neurons in culture; at 3 div approximately 48% of cells were HuC/D positive and 85% expressed serotonin, suggesting our protocol can efficiently isolate neurons from whole embryonic zebrafish brains. Live time-lapse imaging was also carried out to analyze cell migration in vitro. Primary cultures of zebrafish neural cells typically have low rates of survivability in vitro. We have developed a culture system that has long term cell viability, enabling direct analysis of cell-cell and cell-extracellular matrix interactions. These results demonstrate a practical method for isolating, dissociating and culturing of embryonic zebrafish neural tissue. This approach could further be utilized to better understand zebrafish regeneration in vitro.