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

Abstract

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 24 hours 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.

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

“…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].…”

Analysis of Embryonic and Larval Zebrafish Skeletal Myofibers from Dissociated Preparations

“…In mice this has allowed for investigations and analyses that are impractical or impossible in whole animals, including subcellular protein localization studies 30 , live cell imaging 31 , and electrophysiologic measurements 32 . In the zebrafish, similar dissociation techniques have been previously developed to specifically identify and examine motorneurons 33 as well as Rohon-Beard sensory neurons 34 , and these techniques have enabled detailed analysis of these neuron subtypes.…”

Zebrafish disease model of human RNASET2 deficient cystic leukoencephalopathy displays abnormalities in early microglia

“…To detect apoptosis or a different mode of cell death in WT and mutant larvae we used annexinV (aV)/propidium iodide (PI) cell staining following a modified protocol that involves dissociation of the larvae and quantification by flow cytometry (Sakowski et al, 2012). Notably, this approach allows precise comparisons of staining intensities between WT and mutants with aV (apoptosis) or aV and PI (necrosis) revealing the proportions of necrotic and apoptotic cells within the total population.…”