Figure 3

Figure 3. Direct injection of Pgk1 into the brain chamber prevents MPTP-induced dopaminergic cell death in the ventral diencephalon of zebrafish embryo at 6 dpf. (A) Schematic diagram depicts the distribution of dopaminergic neuronal cells in the brain of zebrafish embryo. The vDC region is marked with a square. (B) Schedule of MPTP treatment for embryos from 2 to 6 dpf. Negative control: embryos were injected with DsRed protein at 2 dpf without MPTP treatment. Mock control: embryos were injected with DsRed protein at 2 dpf and treated with 45 µM MPTP from 2 through 6 dpf. Experimental groups: Pgk1 was individually injected at 2, 4, and 2 plus 4 (2 + 4) dpf and then treated with MPTP from 2 to 6 dpf. Immunostaining assay was performed at 6 dpf. (C–G) Immunostaining of zebrafish embryos at 6 dpf. Using confocal microscopy, Tyrosine hydroxylase (TH)-specific antibody, labeled with a green fluorescence signal, could be observed in the vDC region of embryos with different treatments, as indicated. (H) Statistical analysis of the average number of the green fluorescent signal-labeled TH cells located in vDC. Number of examined embryos was indicated at the lower left corner of each panel. Projections of Z-stack images were generated with 2 µm. (I) Western blot analysis of the TH and GFP proteins expressed in the head of 10 larvae from transgenic line Tg(dat:EGFP) treated as indicated. The α-tubulin served as an internal control. The relative expression values of (J) TH and (K) GFP quantified from different groups after normalization of the expression level of α-tubulin. The level of each examined protein in the negative group set was expressed as 1. All data were averaged from three independent experiments and represented as mean ± S.D. Student’s t-test was used to determine significant differences between each group (**, p < 0.01; *, p < 0.05). OB: olfactory bulb; Tel: telencephalon; vDC: ventral diencephalon; LC: locus coeruleus. Scale bar, 50 μm.