Figure 7.

Social isolation stress induces ATF-7 phosphorylation and release of ATF-7 from the 5-HT receptor 5B (Htr5b) promoter. (A) Brain sections containing dorsal raphe nuclei of group- or isolation-reared wild-type (WT) mice were stained with antibodies which recognize P-ATF-7 and P-ATF-2 (green), or NeuN (red), a neuronal specific nuclear protein. DNA was stained with TOTO-3 (blue). The merged images are shown in the right panels. The average number of neurons expressing P-ATF-7 or P-ATF-2 in three independent experiments is indicated by the bar graph±s.d. (B) Nuclear extracts were prepared from the brainstem of group- or isolation-reared WT mice, which were perfused with PFA, and analyzed by SDS–PAGE after decrosslinking, followed by western blotting with anti-P-ATF-2/7, anti-ATF-7, and anti-ATF-2. The ratio of the density of each band in isolation-reared mice to that in group-reared mice is indicated in the bar graph. (C) Release of ATF-7 from the Htr5b promoter by isolation stress. Chromatin immunoprecipitation (ChIP) assays were carried out using chromatin from the brainstem of group- or isolation-reared WT mice, and anti-ATF-7. Extracted DNA was amplified by real-time PCR using primers that cover the ATF-7-binding sites of Htr5b (n=3). (D) Brain sections containing dorsal raphe nuclei of group- or isolation-reared WT mice were stained with antibodies that recognize P-p38, as described above. The number of neurons expressing P-p38 is quantified at the right (n=3). ^*P<0.05, ^**P<0.01.