Pineal ganglion cells showing chromatic properties originating from PP1 cells. (A) Calcium change profile in a single pineal ganglion cell exhibiting a pattern similar to that of PP1 cells in Tg(elavl3:GCaMP6s) larvae. (B) Image analyses showing the calcium level decrease by UV (image ΔF/F405) and the calcium level increase by visible light (image ΔF/F588) across different genotypes: PP1+/+ (Bi–Biii), PP1+/− (Biv–Bvi), and PP1−/− (Bvii–Bix). T-averaged images represent the average of all frames. Yellow arrowheads represent the pineal ganglion cell showing the calcium change profile in (A). White arrowheads indicate cells showing a decrease by UV and an increase by visible light. Cyan arrowheads indicate cells showing only a UV-induced decrease. Red arrowheads indicate cells showing a small visible light-induced calcium increase. The dotted landmarks indicate the ROIs used for the pixel-based analyses in (C–F). (C and D) Comparison of pixel-based histograms for ΔF/F405 and ΔF/F588 across different genotypes, PP1+/+ [n = 5, blue (C) and red (D) circles], PP1+/− [n = 6, light blue (C) and brown (D) squares], and PP1−/− (n = 5, black triangles). The vertical axis indicates the percentage of pixels within the pineal ROI shown in (B) (e.g., Bi, Biv, and Bvii) that fall into each ΔF/F range. (E and F) Quantification of pixel values in images ΔF/F405 and ΔF/F588 (Dunnett’s multiple comparison test). (G, H, I, and J) Visualization of chromatic-type (C-type) ganglion cells (G), followed by a binary image (H) used for quantitative analyses of the C-type area (I, Dunnett’s multiple comparison test) and the ROI for obtaining the C-type calcium change profile (J). (Scale bar: 25 μm.) Experiments were performed using 5 to 6 dpf zebrafish larvae.
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