The pineal C-type property based on PP1 is transmitted to tegmentum neurons. (A–E) C-type neuron-visualized images in different genotypes: PP1+/+ (A), PP1+/− (B), and PP1−/− (C), eye-enucleated fish (D), and pineal-ablated fish (E) in whole-brain imaging. (Scale bar: 100 μm.) (F) Quantification of the C-type area in the tegmentum (Dunnett’s multiple comparison test). (Gi–Gvi) Six separate optical brain sections showing that C-type pineal ganglion cells (Gii) and C-type tegmentum neurons (Giii) are present in different planes and that C-type cells were not found in other planes (Gi and Giv–vi). (H and I) Comparison of calcium change profiles in C-type pineal ganglion cells (H) and tegmentum neurons (I) before (intact, black and blue) and after (enucleated, gray and light blue) surgical enucleation (n = 5) in whole-brain imaging. The open arrowhead in (I) shows the decrease in calcium levels upon the onset of two-photon excitation laser in the tegmentum neurons. (J, K, and L) Comparison of C-type-visualized images (J and K) and calcium change profiles (L) before and after surgical enucleation (n = 9) in tegmentum imaging. Data are normalized to the average of frames 26-50 (25 frames before the initial UV exposure in H, I, and L). (Scale bar: 100 μm.) Experiments were performed using 5 to 6 dpf zebrafish larvae.
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