Fig. 3

Effects of rolipram on L/M deficits and PKA/CREB signaling, neuroinflammation, and O-GlcNAc cycling induced by sleep deprivation (SD) in zebrafish. Zebrafish were subjected to SD for 3 days, with or without daily intraperitoneal injection of rolipram (20 ng/g) over the same 3-day period. A: fear-conditioned (FC) memory test graphs depict the crossing time of each group during the L/M task (n = 6–8 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 vs. Con; ##P < 0.01, ###P < 0.001 vs. SD) were used for statistical analysis. The experiments were independently replicated three times. B: representative IF staining images (×40) and quantification graphs of p-CREB and c-Fos in the Dl region of zebrafish brains 6 h after FC. p-CREB (n = 4 fish/group) and c-Fos (n = 3 fish/group) were stained in green, and nuclei were counterstained with DAPI in blue (*P < 0.05 vs. Con; #P < 0.05 vs. SD). Enlarged images are presented in white boxes. Scale bar = 20 μm. C: representative IF images (×40) of DAPI (blue), GFAP (green), and merged staining of the Dl (scale bar = 20 μm) and Vd (scale bar = 50 μm) regions of zebrafish brains. Enlarged images are shown in the white box. Graphs show densitometric quantification (n = 3 fish/group, *P < 0.05 vs. Con; #P < 0.05 vs. SD). D: representative confocal images and quantification graphs of O-GlcNAc, OGT, and OGA. O-GlcNAc (n = 4 fish/group) and OGT (n = 3 fish/group) were stained in green, OGA (n = 3 fish/group) was stained in red, and nuclei were stained with DAPI (blue) (**P < 0.01 vs. Con; #P < 0.05 vs. SD). Enlarged images are presented in white boxes. Scale bar = 20 μm. The Kruskal–Wallis test was used to determine the statistical significance of the immunofluorescence staining quantification data. Con, control; CREB, cAMP response element-binding protein; DI, dorsal telencephalic area; GFAP, glial fibrillary acidic protein; IF, immunofluorescence; L/M, learning and memory; OGA, O-GlcNAcase; OGT, O-GlcNAc transferase; p-CREB, Phosphorylated-cAMP response element-binding protein; PKA, protein kinase A; Rol, Rolipram.