Abstract
Zebrafish (Danio rerio) is a widely used model organism in genetics and developmental biology research. Genetic screens have proven useful for studying embryonic development of the nervous system in vivo, but in vitro studies utilizing zebrafish have been limited. Here, we introduce a robust zebrafish primary neuron culture system for functional nerve growth and guidance assays. Distinct classes of central nervous system neurons from the spinal cord, hindbrain, forebrain, and retina from wild type zebrafish, and fluorescent motor neurons from transgenic reporter zebrafish lines, were dissociated and plated onto various biological and synthetic substrates to optimize conditions for axon outgrowth. Time-lapse microscopy revealed dynamically moving growth cones at the tips of extending axons. The mean rate of axon extension in vitro was 21.4±1.2 µm hr−1 s.e.m. for spinal cord neurons, which corresponds to the typical ∼0.5 mm day−1 growth rate of nerves in vivo. Fluorescence labeling and confocal microscopy demonstrated that bundled microtubules project along axons to the growth cone central domain, with filamentous actin enriched in the growth cone peripheral domain. Importantly, the growth cone surface membrane expresses receptors for chemotropic factors, as detected by immunofluorescence microscopy. Live-cell functional assays of axon extension and directional guidance demonstrated mammalian brain-derived neurotrophic factor (BDNF)-dependent stimulation of outgrowth and growth cone chemoattraction, whereas mammalian myelin-associated glycoprotein inhibited outgrowth. High-resolution live-cell Ca2+-imaging revealed local elevation of cytoplasmic Ca2+ concentration in the growth cone induced by BDNF application. Moreover, BDNF-induced axon outgrowth, but not basal outgrowth, was blocked by treatments to suppress cytoplasmic Ca2+ signals. Thus, this primary neuron culture model system may be useful for studies of neuronal development, chemotropic axon guidance, and mechanisms underlying inhibition of neural regeneration in vitro, and complement observations made in vivo.
Highlights
The translucent nature of embryonic and larval zebrafish has enabled neurobiological research to be pursued in vivo with this model organism [1,2,3,4,5,6,7]
Forward genetic screens utilizing zebrafish have significantly increased our understanding of embryonic development of the nervous system [8,9,10]
Axon guidance pathway homology To assess the zebrafish genome for complements of human axon guidance genes, we carried out homology analyses of several important chemotropic factors (Table 1)
Summary
The translucent nature of embryonic and larval zebrafish has enabled neurobiological research to be pursued in vivo with this model organism [1,2,3,4,5,6,7]. Forward genetic screens utilizing zebrafish have significantly increased our understanding of embryonic development of the nervous system [8,9,10]. Unlike other widely used model organisms, in vitro studies utilizing zebrafish have been limited [11,12], likely due to the suboptimal conditions and complexity of pioneering cell culture systems [13,14]. Strengths of the zebrafish model system Zebrafish possess genetic tractability and physical advantages usually only found in invertebrate systems, yet have extensive conserved synteny to the human genome. They share most human genes, and gene functions are well conserved [15]. The average lifespan of zebrafish is 2–3 years, which is longer or equivalent to that of mice [22], and generation time is about 3 months [21]
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
