Reduction in tyrosine hydroxylase immunoreactivity upon Mn exposure. (A–D) Confocal images of catecholaminergic neurons comprising of dopaminergic and noradrenergic neurons in 5-dpf larvae (dorsal view) stained with an antibody against tyrosine hydroxylase showing a reduction in staining upon 48 hours of exposure to Mn. Maximum intensity projection (z-axis) images of tyrosine hydroxylase immunoreactivity in (A) telencephalon, (B) pretectum, (C) ventral diencephalon and (D) area postrema. (E) A single optical slice showing tyrosine hydroxylase immunoreactivity in the telencephalon of an Mn-exposed larva before (top) and after (bottom) image intensity multiplication. Scale bars: 30 µm. (F) The number of tyrosine-hydroxylase-immunoreactive cell bodies in different regions of 5-dpf larvae exposed to the indicated concentrations of Mn showed no significant differences in cell counts (n=4; telencephalon P=0.50; pretectum P=0.23; ventral diencephalon P=0.057 for control and those treated with 0.8 mM Mn, and P=0.23 for control and those treated with 1 mM Mn; and area postrema P=0.31). Error bars represent s.e.m. Statistical tests were performed using ANOVA and Wilcoxon rank sum test with Bonferroni correction for multiple comparisons. (G) Bar plots indicating the average intensities of tyrosine hydroxylase immunostaining in control larvae and Mn-treated larvae from identical ROIs drawn in the regions indicated on top of each plot. Error bars represent s.e.m. RMANOVA and Freidman tests were performed to calculate statistical significance. *Values that are significantly different from control at an α level of 0.05.
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