S-Sulfocysteine Induces Seizure-Like Behaviors in Zebrafish.

Sulfite is a neurotoxin, which is detoxified by the molybdenum cofactor (Moco)-dependent enzyme sulfite oxidase (SOX). In humans, SOX deficiency causes the formation of the glutamate analog S-Sulfocysteine (SSC) resulting in a constant overstimulation of ionotropic glutamatergic receptors. Overstimulation leads to seizures, severe brain damage, and early childhood death. SOX deficiency may be caused either by a mutated sox gene or by mutations in one of the genes of the multi-step Moco biosynthesis pathway. While patients affected in the first step of Moco biosynthesis can be treated by a substitution therapy, no therapy is available for patients affected either in the second or third step of Moco biosynthesis or with isolated SOX deficiency. In the present study, we used a combination of behavior analysis and vital dye staining to show that SSC induces increased swimming, seizure-like movements, and increased cell death in the central nervous system of zebrafish larvae. Seizure-like movements were fully revertible upon removal of SSC or could be alleviated by a glutamatergic receptor antagonist. We conclude that in zebrafish SSC can chemically induce phenotypic characteristics comparable to the disease condition of human patients lacking SOX activity.

Culture and Transfection of Zebrafish Primary Cells.

Zebrafish embryos are transparent and develop rapidly outside the mother, thus allowing for excellent in vivo imaging of dynamic biological processes in an intact and developing vertebrate. However, the detailed imaging of the morphologies of distinct cell types and subcellular structures is limited in whole mounts. Therefore, we established an efficient and easy-to-use protocol to culture live primary cells from zebrafish embryos and adult tissue. In brief, 2 dpf zebrafish embryos are dechorionated, deyolked, sterilized, and dissociated to single cells with collagenase. After a filtration step, primary cells are plated onto glass bottom dishes and cultivated for several days. Fresh cultures, as much as long term differenciated ones, can be used for high resolution confocal imaging studies. The culture contains different cell types, with striated myocytes and neurons being prominent on poly-L-lysine coating. To specifically label subcellular structures by fluorescent marker proteins, we also established an electroporation protocol which allows the transfection of plasmid DNA into different cell types, including neurons. Thus, in the presence of operator defined stimuli, complex cell behavior, and intracellular dynamics of primary zebrafish cells can be assessed with high spatial and temporal resolution. In addition, by using adult zebrafish brain, we demonstrate that the described dissociation technique, as well as the basic culturing conditions, also work for adult zebrafish tissue.

Embryonic zebrafish primary cell culture for transfection and live cellular and subcellular imaging.

Although having great potential for live cell imaging to address numerous cell biological questions with high spatial and temporal resolution, primary cell cultures of zebrafish embryos are not widely used. We present an easy-to-use protocol for preparing primary cell cultures of 2 dpf zebrafish embryos allowing for live cell imaging of fully differentiated cells such as neurons and myocytes. We demonstrate that different cell types can be identified by morphology and expression of transgenic cell type-specific fluorescent reporters and that fluorescent cells can be sorted by flow cytometry to prepare an enriched culture. To facilitate subcellular imaging in live primary cells, we successfully tested a selection of fluorescent vital dyes. Most importantly, we demonstrate that zebrafish primary cells can be transfected efficiently with expression constructs allowing for visualizing subcellular structures with fluorescent marker proteins for time lapse imaging. We propose zebrafish primary cell culture as a versatile tool to address cell biological questions in combination with a powerful in vivo model.