Figure 4

Activation of reporter gene expression after doxycycline withdrawal. Double transgenic CaMKIIα-tTA/EGFP-Ptetbi-Luc rats (4.5 × 66.1) were treated with Dox during development until E18, P0 and P10, respectively (10 μg/mL). After 60 days of Dox withdrawal (±Dox), reporter gene expression was analyzed by luciferase measurements of brain regions (A) and EGFP IHC (B-J). (A) A highly significant difference in luciferase activity was found between the different groups (olfactory bulb: F (4,22) = 143, P < 0.001; cortex: F (4,22) = 79.4, P < 0.001; hippocampus: F (4,22) = 125, P < 0.001; cerebellum: F (4,22) = 31.5, P < 0.001). In forebrain regions (olfactory bulb, cortex, hippocampus), strong luciferase expression was induced by Dox withdrawal at E18, P0 and P10 (±Dox) compared with Dox-treated animals (+Dox). However, in rats which had received Dox until birth (P0) or until P10, luciferase expression did not reach the level of untreated animals (-Dox). This suggests that prolonged Dox treatment during development, in particular after birth, impairs activation of Ptet-controlled gene expression after Dox withdrawal. Data are presented as mean + standard error of the mean. Logarithmic data transformation was performed prior to statistical analysis. Characters (*, +, #) represent P-values obtained by one-way analysis of variance followed by Bonferroni post hoc analysis. * represents significance levels versus +Dox (***P ≤ 0.001); ⁺ versus P10 (⁺P ≤ 0.05; ⁺⁺⁺P ≤ 0.001) and ^# versus P0 (^#P ≤ 0.05; ^##P ≤ 0.01; ^###P ≤ 0.001). Light units are normalized to the protein content of the lysates. (B-J) IHC confirms and extends the findings of the luciferase assays. Again, Dox-treated rats which had received Dox only until E18 (B-D) showed a strong forebrain-specific reporter protein expression pattern which was comparable to untreated rats (-Dox, Figure 3A-C) whereas the level of expression in all forebrain regions was significantly reduced when Dox was given until P0 (E-G) or P10 (H-J). Dox: doxycycline hydrochloride; E: embryonic day; IHC: immunohistochemistry; P: postnatal day.