Pituitaries of basal and stressed Crhr1 wild type and knockout mice

Pituitaries of basal and stressed Crhr1-wildtype (wt) and -knockout (ko) mice (Timpl et al., 1998) As the Crh-system and the HPA-axis are known to be crucially invovled in the onset, development and maintainance of psychiatric disorders like anxiety and depression and regulate the behavioural and endocrine stress responses the further analysis of Crhr1-dependent signaling cascades is essential to understand the molecular mechanisms behind these psychiatric diseases. In this project, new candidate genes involved in Crhr1-dependent signaling cascades were dissected using metyrapone treated stressed Crhr1-wt and -ko (Timpl et al., 1998) mice using 30 min restraint stress. Changes in mRNA expression of pituitaries of basal and stressed Crhr1-wt and -ko mice were analysed 1 and 3h, respectively, after stress begin on the Sentrix Mouse-6 Expression BeadChip. To activate the CRHR1-dependent signaling in the pituitary of CRHR1-wt and -ko mice animals were stressed using 30 min restraint stress in 50 ml Falcon tubes. 12 h before stress mice were injected intraperitonally (i.p.) with 150 mg/kg bodyweight (b.w.) metyrapone. Metyrapone is an inhibitor of the steroid beta-hydroxylase, an enzyme catalyzing the biosynthesis of corticosterone. By this way, the negative feedback of corticosterone on the HPA-axis was blocked. A second metyrapone injection (100 mg/kg b.w.) was applied directly before stress. 1 and 3h, respectively, after stress, mice were anesthetized, decapitated and the pituitaries prepared and flash-frozen. mRNA was isolated according to the TRIzol protocol (Invitrogen). Pooled amplified RNA samples were then hybridised on Illumina mouse BeadChips (N=2 per group) and detected in the Illumina BeadArray Reader (Illumina, Inc., San Diego, CA). The experiment was performed in technical duplicates.