Fig. 1.

Description of the model. a Schematic diagram showing the most relevant elements in the plasmid used for retroviral production. The rtTA-IRES-EGFP was purchased from AddGene (reference number PMA2640). This plasmid permits the selective expression of the advanced rtTA element and EGFP as a reporter due to the presence of a rtTA-IRES-EGFP element. b The general basis of the experiments is shown. In tTO-GSK-3β mice, the expression of GSK-3β takes place after the binding of the active rtTA element to the tetO bidirectional promoter. The rtTA element will undergo a conformational change and thus become activated by the presence of doxycycline. Upon activation, the rtTA element will allow the expression of both GSK-3β and β-galactosidase (β-Gal) in targeted cells. c, d Representative images and their corresponding high-power magnification and orthogonal views showing the staining against β-Gal 48 h after retrovirus injection. As shown in c, in tet-OFF mice (no doxycycline treatment), no β-Gal expression was detected in EGFP⁺ cells. In contrast, in tet-ON mice treated during 48 h after retrovirus injection with doxycycline, a strong and selective induction of the reporter protein was observed (d). e Quantification of the percentage of EGFP⁺ cells that were also β-Gal⁺ (time course). The percentage of EGFP⁺ cells that were β-Gal⁺ cells rapidly increased as the post-injection interval increased until reaching a plateau at 48 h post-injection. f–h To obtain a physiological indicator of GSK-3β activity in infected newborn neurons, phosphorylated (PHF-1 epitopes) Tau (one of the main substrates of GSK-3β activity) was measured in individual EGFP⁺ cells. f–g Representative images of EGFP and PHF-1 immunohistochemistry in tet-OFF (f) and tet-ON (g) mice. As shown, the fluorescence intensity of PHF-1 phospho-Tau was significantly increased in the tet-ON as compared to tet-OFF mice (h). ML molecular layer, GL granule layer, H hilus. Yellow scale bar 20 µm, purple scale bar 10 µm, purple triangle EGFP⁺ β-galactosidase⁻ cell, yellow triangle EGFP⁺ β-galactosidase⁺ cell, white arrows EGFP⁺ cell. Asterisks indicate significant differences compared to tet-OFF mice (**0.001 < p < 0.01) (***p < 0.001)