Effects of CSC on absolute adrenal weight and plasma CORT and ACTH concentrations.

<p>Following decapitation on day 20 of CSC between 0800 and 1000 h under basal conditions the left and right adrenals of SHC (n = 36) and CSC (n = 42) mice were removed, pruned of fat, pooled, and weighed. Depicted is the absolute weight [mg] of the left and right adrenal (sum of both, A). Furthermore, plasma CORT [ng/ml] (SHC: n = 47; CSC: n = 53; B) and ACTH [pg/ml] (SHC and CSC: n = 5; C) concentrations were determined in trunk blood following decapitation. Grey bars represent SHC, black bars CSC mice. Data represent the mean + SEM. * represent <i>P</i> < 0.05, *** represent <i>P</i> < 0.001 vs. respective SHC mice.</p

Effects of CSC on hippocampal GR and MR mRNA and cytoplasmic protein expression as well as on nuclear GR protein expression under basal conditions and in response to EPF exposure.

<p>Following decapitation on day 20 hippocampi of both SHC and CSC mice were removed. Afterwards, either RNA of SHC (n = 13) and CSC (n = 19) mice was extracted and reversely transcribed into cDNA for quantification of GR and MR mRNA expression [arbitrary units] via qPCR using TaqMan technology normalized to the mRNA expression of the housekeeping gene GAPDH (A/D), or protein was extracted from the hippocampi of SHC (GR: n = 8; MR: n = 6) and CSC (GR: n = 6; MR: n = 6) mice for determination of cytoplasmic GR (B/C) and MR (E/F) protein expression [grey density] normalized to the loading control ß-Tubulin and for determination of basal nuclear GR (G/H) protein expression (SHC: n = 6, CSC: n = 5) normalized to the loading control TATA binding protein (TBP). Another set of SHC and CSC mice was exposed to an elevated platform (EPF) for 5 min on day 20 of CSC. 10 min after termination of EPF exposure, SHC (n = 6) and CSC (n = 7) mice were decapitated, hippocampi were removed and protein was extracted for determination of nuclear GR protein expression [grey density] normalized to the loading control TBP (G/H). Grey bars represent SHC, black bars CSC mice. Data represent the mean + SEM. * represent <i>P</i> < 0.05 vs. respective SHC mice; ^# represent <i>P</i> < 0.05 vs. respective basal. Representative images of bands detected for cytoplasmic GR (~ 86 kDa; C) or MR (~ 107 kDa; F) and respective loading control ß-Tubulin (~ 50 kDa; C/F) are shown for SHC and CSC mice. Representative images of bands detected for nuclear GR (~ 86 kDa; H) and the loading control TBP (~ 36 kDa; B) are shown for SHC and CSC mice under basal and EPF conditions.</p

Effects of CSC on FKBP51 protein expression in the hippocampus.

<p>Following decapitation on day 20 hippocampi of both SHC (n = 8) and CSC (n = 6) mice were removed and protein was extracted for determination of FKBP51 protein expression [grey density] normalized to the loading control ß-Tubulin (A/B). Grey bars represent SHC, black bars CSC mice. Data represent the mean + SEM. Representative images of bands detected for FKBP51 (~ 51 kDa; B) and the loading control ß-Tubulin (~ 50 kDa; B) are shown for SHC and CSC mice.</p

Effects of CSC on the basal morning and forced swim (FS)-induced plasma ACTH concentrations in trunk blood.

<p>Following decapitation on day 20 of CSC between 0800 and 1000[pg/ml] concentrations were determined in trunk blood of SHC (n = 42) and CSC (n = 61) mice (A). Another set of SHC (n = 7) and CSC (n = 6) mice was exposed to 6 min forced swim (FS) on day 20 of CSC and decapitated 10 min following FS exposure whereby trunk blood was collected for determination of plasma ACTH [pg/ml] concentrations (B). Grey bars represent SHC, black bars CSC mice. Data represent the mean + SEM. * represent <i>P</i><0.05, ** represent <i>P</i><0.01 <i>vs.</i> respective SHC mice.</p

Effects of CSC on the plasma ACTH concentrations in trunk blood following DST.

<p>On day 20 of CSC, between 0700 and 0800(30 µg/kg, ip). Four hours later all animals were exposed to forced swim (FS) for 6 min. 10 min after termination of the acute stressor exposure all mice were decapitated and trunk blood was collected for determination of plasma ACTH [pg/ml] (SHC: n = 11; CSC: n = 12). Grey bars represent SHC, black bars CSC mice. Data represent the mean + SEM. ** represent <i>P</i><0.01 <i>vs.</i> respective SHC mice; # represent <i>P</i><0.05, ### represent <i>P</i><0.001 <i>vs.</i> respective vehicle group.</p

Effects of CSC on pituitary weight, pituitary cell number and the number of ACTH positive cells.

<p>Following decapitation on day 20 of CSC the pituitaries of SHC (n = 42) and CSC (n = 61) mice were removed and absolute pituitary weight [mg] was determined (A). The pituitary cells of one set of SHC (n = 6) and CSC (n = 14) mice were isolated and subsequently quantified (per ml) by means of a Cell Viability Analyzer (B). Pituitaries from another set of SHC (n = 6) and CSC (n = 6) mice were cut on a cryostat and subsequently one series of cry-sections was stained with an antibody against ACTH. Per animal, three to six different sections were analyzed and averaged for the percentage of ACTH positive anterior pituitary tissue in relation to the total anterior pituitary tissue (C). Moreover, the number of ACTH positive pituitary cells [n] was counted in the same three to six digitized images and averaged per animal to provide individual means (D). Grey bars represent SHC, black bars CSC mice. Data represent the mean + SEM. * represent <i>P</i><0.05, ** represent <i>P</i><0.01 <i>vs.</i> respective SHC mice. (E) Representative images of cryo-sections stained with antibody against ACTH showing anterior and intermediate pituitary.</p