A novel approach to study motor neurons from zebrafish embryos and larvae in culture

Department of Neurology, University of Michigan, Ann Arbor, MI, USA.
Journal of Neuroscience Methods (Impact Factor: 2.05). 01/2012; 205(2):277-82. DOI: 10.1016/j.jneumeth.2012.01.007
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Zebrafish are becoming increasingly popular models for examining the mechanisms of and treatments for neurological diseases. The available methods and technology to examine disease processes in vivo are increasing, however, detailed observations of subcellular structures and processes are complex in whole organisms. To address this need, we developed a primary motor neuron (MN) culture technique for utilization with zebrafish neurological disease models. Our protocol enables the culturing of cells from embryos older than 24h post-fertilization, at points after MN axonal development and outgrowth begins, which enables MN axons to develop in vivo in the context of the normal endogenous cues of the model organism, while also providing the accessibility of an in vitro system. When utilized with the increasing number of genetically modified or transgenic models of neurological diseases, this approach provides a novel tool for the examination of cellular and subcellular disease mechanisms, and offers a new platform for therapeutic discoveries in zebrafish.

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Available from: Stacey A Sakowski, Oct 03, 2015
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    • "However, mechanisms such as high reparative activity in the nervous tissues of fish, including both anatomical and functional regeneration remain poorly explored. Some in vitro model systems have demonstrated their potential for studies on CNS injury and repair [4]. But, so far a model system of adult salmon primary brain neurons has not been described. "
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    ABSTRACT: Analysis of proliferative activity and the ability to neuron differentiation was performed in cultured cells of the brain and spinal cord of juvenile masu salmon Oncorhynchus masou. Proliferating cell nuclear antigen (PCNA) was used as a proliferative marker, while the markers of neuronal differentiation—a neuron protein HuCD, and a neuron-specific transcriptional factor with two DNA-binding sites Pax6—detected neurons. The results showed that cell proliferation occurred mainly in the suspension cell fraction. In monolayer, a few cells were only found to express PCNA. The results of morphological and immunohistochemical analysis allow us to conclude that proliferative activity in primary cultures from the O. masou brain is mainly connected with the suspension fraction of small cells. In contrast, a positive correlation between the cells expressing cystathionine β-synthase (CBS), a marker of H2S synthesis, and the cells expressing PCNA in the monolayer, indicates the participation of H2S in proliferative activity of neurons in primary cultures. The data obtained suggest that the hydrogen sulphide is also involved in the process of differentiation.
    Advances in Bioscience and Biotechnology 09/2015; 6(08):539-545. DOI:10.4236/abb.2015.68057
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    • "The approach is amendable to mechanistic studies of axon and dendrite development – including neuronal polarization, neurite initiation, outgrowth, axon guidance, synaptogenesis, and neural circuit formation – yet is also suitable for the classroom. Our method may complement not only the classic neuron culture systems utilizing chick, frog, mouse, and rat models, but also a recent method developed independently for the study of zebrafish motor neurons post-axogenesis [34]. "
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    ABSTRACT: Zebrafish () 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 , but 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 was 21.4±1.2 µm hr s.e.m. for spinal cord neurons, which corresponds to the typical ∼0.5 mm day growth rate of nerves . 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 Ca-imaging revealed local elevation of cytoplasmic Ca 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 Ca 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 and complement observations made .
    PLoS ONE 03/2013; 8(3):e57539. DOI:10.1371/journal.pone.0057539 · 3.23 Impact Factor
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    ABSTRACT: Adult zebrafish (Danio rerio) have a remarkable ability to restore function after an injury to the brain or spinal cord. The molecular and cellular mechanisms underlying this phenomenon are not fully understood. To enable investigation of these mechanisms we have developed an in vitro model system from the adult zebrafish brainstem, which can be maintained under serum-containing and serum-free conditions. While cultures are predominantly neuronal, they also contain glia and stem progenitor cells. Various stages of cellular differentiation are observed among both neuronal and non-neuronal populations. Quantitative morphological results revealed typical cellular growth over a two-week period. We argue that our novel brainstem culture model offers a powerful tool for the studies of axonal growth, neurogenesis, and regeneration in the adult zebrafish central nervous system.
    Journal of Neuroscience Methods 12/2013; 223. DOI:10.1016/j.jneumeth.2013.11.022 · 2.05 Impact Factor
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