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Fig. 5 Modulation of H89-induced L/M deficits, PKA/CREB signaling, and hypo-O-GlcNAcylation in the zebrafish brain by GlcN. Zebrafish were intracerebroventricularly injected with H89 (100 ng/g) for three consecutive days, with or without daily intracerebroventricular injection of GlcN (200 µg/g). A: Fear-context memory test graphs illustrate the crossing time of each group in the learning and memory tasks (n = 5 or 6 fish/group). The Kruskal–Wallis test (aP < 0.05, aaP < 0.01 vs. the 1st FC of Con) and two-way ANOVA followed by FDR test (**P < 0.01, ***P < 0.001, ****P < 0.0001 vs. Con; #P < 0.05, ###P < 0.001 vs. H89) were used for statistical analysis. The experiments were independently replicated three times. B: representative confocal images and quantification graphs of anti-O-GlcNAc, OGT, and OGA. O-GlcNAc (n = 4 fish/group), OGT (n = 4 fish/group), and OGA (n = 4 or 5 fish/group) were stained in green, and nuclei were stained with DAPI (blue) (**P < 0.01 vs. Control; #P < 0.05 vs. H89). Enlarged images are presented in white boxes. Scale bar = 20 μm. C: representative immunofluorescence staining images (×40) and quantification graphs of PKAcα and p-CREB at the Dl region of zebrafish brains 6 h after fear conditioning. PKAcα (n = 4 fish/group) and p-CREB (n = 4 or 5 fish/group) were stained in green, and nuclei were counterstained with DAPI in blue (***P < 0.001, ****P < 0.0001 vs. Control; #P < 0.05 vs. H89). Enlarged images are presented in white boxes. Scale bar = 20 μm. D: representative Western blot images and quantification graphs of Phospho-PKA substrate (n = 5 replicates/group), O-GlcNAc (n = 5 replicates/group), and β-actin of Con, H89, and HG (H89+GlcN) from protein lysate extracted from zebrafish brains (*P < 0.05, **P < 0.01 vs. Con; #P < 0.05 vs. H89). The Kruskal–Wallis test was used to determine the statistical significance of immunofluorescence staining and Western blot quantification. DI, dorsal telencephalic area; FDR, false discovery rate; L/M, learning and memory; OGA, O-GlcNAcase; OGT, O-GlcNAc transferase; p-CREB, Phosphorylated-cAMP response element-binding protein.