Primary Neuron Culture for Nerve Growth and Axon Guidance Studies in Zebrafish (Danio rerio)

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.

Flexible and Organic Neural Interfaces: A Review

“…This would permit to check the presence of eventual inflammations in vivo [87,88], or to simply follow growth and differentiation of neurons by means of confocal microscopy in vitro [89,90], as well using illumination to perform cell stimulation (both in vitro and in vivo) [91].…”

A Novel Long Non-coding RNA, durga Modulates Dendrite Density and Expression of kalirin in Zebrafish

“…Zebrafish is emerged as a convenient model to explore axonal growth (Chen et al, 2013), signaling by neurotropic factors during development and in human diseases. Thus, we studied the kalrna locus in detail on chromosome 9 of the zebrafish genome (Zv10).…”

Isolation and Culture of Adult Zebrafish Brain-derived Neurospheres

“…The zebrafish adult brain displays multiple neurogenic niches harboring neural stem/progenitor cells making zebrafish a powerful model organism for understanding the stem cell activity in the brain as well as the molecular programs required for central nervous system regeneration. Over the past 17 years, several research groups developed methodologies for isolating and culturing zebrafish neural cells 8,9 . These studies were aimed at producing embryonic neuronal and glial cells in vitro, but not at maintaining NSCs and investigating their properties.…”