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Fig. 4 Effects of H89 on L/M function and brain O-GlcNAcylation in zebrafish. H89 (100 ng/g) was intracerebroventricularly injected into adult zebrafish daily for three consecutive days. A: fear-context memory test graphs depict the crossing time of each group in the learning and memory task (n = 10 fish/group). For statistical analysis, a two-way ANOVA test followed by FDR correction was used (**P < 0.01, ****P < 0.0001). The experiments were independently replicated three times. B: representative IF staining images (×40) and quantification graphs of PKAcα and p-CREB in the Dl region of the zebrafish brain 6 h after fear conditioning. PKAcα (n = 4 fish/group) and p-CREB (n = 3 fish/group) were stained in green, and nuclei were counterstained with DAPI in blue (**P < 0.01, ***P < 0.001). Enlarged images are presented in white boxes. Scale bar = 20 μm. C: representative confocal images (×40) and quantification graphs of anti-O-GlcNAc, OGT, and OGA. O-GlcNAc (n = 3 fish/group), OGT (n = 4 fish/group), and OGA (n = 4 fish/group) were stained in green, and nuclei were stained with DAPI (blue) (**P < 0.01, ***P < 0.001). Enlarged images are presented in white boxes. Scale bar = 20 μm. The Kruskal–Wallis test with FDR correction was used to assess the statistical significance of the confocal quantification data. Con, control; DI, dorsal telencephalic area; FDR, false discovery rate; IF, immunofluorescence; L/M, learning and memory; OGA, O-GlcNAcase; OGT, O-GlcNAc transferase; p-CREB, Phosphorylated-cAMP response element-binding protein.