Green Fluorescent Protein Labeling of Dopaminergic Neurons in Zebrafish for the Study of Parkinson’s Diseases

Abstract

The loss of dopaminergic neurons in the substantia nigra is the pathological hallmark of Parkinson's disease (PD) associated with the characteristic motor deficits. Despite all the recent progress in the identification of genes and pathways associated with PD, we still lack a complete understanding of the molecular mechanisms underpinning the disease. Our goals were to develop a new transgenic zebra fish line where tyrosine hydroxylase (TH)-positive neurons were labeled with green fluorescent protein (EGFP) under the control of the zebra fish tyrosine hydroxylase (th1) promoter and to demonstrate the usefulness of the model for the study of the mechanisms underlying cell death in a toxin induced model of PD.A 2.5 kb gene fragment of the th1 promoter was isolated and cloned into a miniTol2vector followed by EGFP and microinjected to originate the Tg (-2.5th: EGFP) zebra fish line. Tg (-2.5th: EGFP) were visualized by fluorescence microscopy for EGFP and TH expression during development. The levels of EGFP mRNA were detected by RT-qPCR and protein expression was examined by western blot analysis. The model of neurotoxicity was generated by treating transgenic zebra fish with 6-hydroxydopamine (6-OHDA), followed by the analysis of cell death markers and behavior.EGFP expression was detected mainly in the posterior tuberculum of the ventral diencephalon (vDC). In the vDC, TH-labeled neurons of groups 1-were correctly labeled with EGFP. Further, we demonstrated that these neurons in larvae and adults are sensitive to 6-OHDA treatment, resulting in cell loss and behavioral deficits. In conclusion, this transgenic line constitutes a powerful tool to visualize and study dopaminergic neurons throughout the zebra fish life-cycle and enables high-throughput screening of molecules of interest for PD.