Behavioural consequences of two chronic psychosocial stress paradigms: Anxiety without depression  David A. Slattery, Nicole Uschold, Mauro Magoni, Julia Bär, Maurizio Popoli, Inga D. Neumann, Stefan O. Reber  Psychoneuroendocrinology  Volume 37, Issue 5, Pages 702-714 (May 2012) DOI: 10.1016/j.psyneuen.2011.09.002 Copyright © 2011 Elsevier Ltd Terms and Conditions

Figure 1 Experimental design. One week after arrival, experimental mice were chronically stressed by 19 d exposure to either the social defeat/overcrowding (SD/OC) paradigm or the chronic subordinate colony housing (CSC) paradigm or were kept as non-stressed single housed controls (SHC). SHC, SDOC, and CSC mice were exposed to either the saccharin preference test (SPT), the elevated plus-maze (EPM), or the social preference/avoidance test (SPAT) on day 20, to the tail suspension test (TST) or the EPM on day 24, or the forced swim test (FST), EPM, or SPAT on day 28. Importantly, every behavioural test was performed in a separate set of animals to avoid possible interactions between the different tests. In another set of animals 24h homecage locomotion was tracked (using NOLDUS EthoVision system) on days −1/0, days 20/21, and days 26/27 before mice were killed on day 28 to reveal changes in adrenal and pituitary weights. SD/OC and CSC mice that were tested/killed on day 28 were single housed after termination of respective chronic psychosocial paradigm (day 20 to day 28). Psychoneuroendocrinology 2012 37, 702-714DOI: (10.1016/j.psyneuen.2011.09.002) Copyright © 2011 Elsevier Ltd Terms and Conditions

Figure 2 Effects of chronic psychosocial stress on body weight development. To assess chronic psychosocial stress effects on body weight development, delta body weight gain was calculated for day 1 to day 20 (SHC: n=94; SD/OC: n=93; CSC: n=94; A), for day 20 to day 28 (SHC: n=45; SD/OC: n=45; CSC: n=47; B), and for day 1 to day 28 (SHC: n=45; SD/OC: n=45; CSC: n=47; C), during/following chronic psychosocial stressor exposure. Changes in absolute body weight from day 1 to day 20 (SHC: n=94, white squares; SD/OC: n=93, grey circles; CSC: n=94, black triangles) are shown in D. Compared with SHC mice SD/OC mice showed a reduced body weight on day 10, 15, 17, and 19, whereas CSC mice only on day 3 and day 10. Data represent mean+SEM; *P≤0.05, **P≤0.01, ***P≤0.001 vs. SHC mice; ##P≤ 0.01, ###P≤ 0.001 vs. SD/OC mice. Psychoneuroendocrinology 2012 37, 702-714DOI: (10.1016/j.psyneuen.2011.09.002) Copyright © 2011 Elsevier Ltd Terms and Conditions

Figure 3 Effects of chronic psychosocial stress on adrenal and pituitary weight. To assess chronic psychosocial stress effects on absolute (A and B) and relative (C and D) adrenal (A and C) and pituitary (B and D) weight, SHC (n=10, 9), SD/OC (n=8, 9), and CSC (n=12, 10) mice were killed one week following termination of chronic psychosocial stressor exposure on day 28. Afterwards the pituitary and both adrenal glands (pooled) were taken out, pruned from fat tissue, and weighed. Data represent mean+SEM; **P≤0.01 vs. SHC mice; ##P≤0.01 vs. SD/OC mice. Psychoneuroendocrinology 2012 37, 702-714DOI: (10.1016/j.psyneuen.2011.09.002) Copyright © 2011 Elsevier Ltd Terms and Conditions

Figure 4 Effects of chronic psychosocial stress on 24h home cage locomotion. To assess chronic psychosocial stress effects on locomotion, distance travelled [cm] in the home cage over 24h was recorded on days −1/0 (A and B), 20/21 (C and D), and 26/27 (E and F) in SHC (white bars and squares), SD/OC (grey bars and circles), and CSC (black bars and triangles) mice using EthoVision XT. Data are presented as mean+SEM of absolute distance travelled per 1h-intervals (A, C, and E; SHC: n=7, 8, 9; SD/OC: n=10, 9, 8; CSC: n=9, 8, 12) and during last 6h of light and first 6h of dark phase (B, D, and F; SHC: n=6, 8, 8; SD/OC: n=8, 8, 8; CSC: n=8, 9, 8). *P≤0.05; **P≤0.01 vs. respective SHC mice; #P≤0.05; ##P≤0.01 vs. respective SD/OC mice; $$P≤0.01; $$$p≤0.001 vs. respective bright phase. Psychoneuroendocrinology 2012 37, 702-714DOI: (10.1016/j.psyneuen.2011.09.002) Copyright © 2011 Elsevier Ltd Terms and Conditions

Figure 5 Effects of chronic psychosocial stress on depression-like behaviour. To assess chronic psychosocial stress effects on depression-like behaviour a different set of SHC (n=19, 18, 18), SD/OC (n=12, 11, 18), and CSC (n=7, 7, 18) mice was exposed to the saccharin preference test (SPT) on day 20/21 (A), to the tail suspension test (TST) on day 24 (B), and to the forced swim test (FST) on day 28 (C). Data represent mean+SEM. Psychoneuroendocrinology 2012 37, 702-714DOI: (10.1016/j.psyneuen.2011.09.002) Copyright © 2011 Elsevier Ltd Terms and Conditions

Figure 6 Effects of chronic psychosocial stress on anxiety-related behaviour. To assess chronic psychosocial stress effects on anxiety-related behaviour a different set of SHC (n=56, 16, 13), SD/OC (n=56, 8, 8), and CSC (n=24, 8, 11) mice was exposed to the elevated plus-maze (EPM) on day 20 (A), day 24 (B), and day 28 (C). Data represent mean+SEM. *P≤0.05; ***P≤0.001 vs. respective SHC mice. Psychoneuroendocrinology 2012 37, 702-714DOI: (10.1016/j.psyneuen.2011.09.002) Copyright © 2011 Elsevier Ltd Terms and Conditions

Figure 7 Effects of chronic psychosocial stress on social anxiety. To assess chronic psychosocial stress effects on social anxiety a different set of SHC (n=9, 12), SD/OC (n=10, 6), and CSC (n=8, 14) mice was exposed to the social preference/avoidance test (SPAT) on day 20 and day 28. Figure A (day 20) and C (day 28) show the absolute time [s] spent in the 8cm broad contact zone (CZ) around the wire mesh cage with (left panel) and without (right panel) a con-specific mouse present, both trials lasting 150s each. Figure B (day 20) and D (day 28) show the calculated social preference (time in CZ with mouse [s]/time in CZ without mouse [s])×100%. Data represent mean+SEM. *P≤0.05, **P≤0.01, ***P≤0.001 vs. respective SHC mice. #P≤0.001 vs. respective “no mouse” trial; +p=0.047 vs. respective SHC mice obtained employing Mann–Whitney U comparison. Psychoneuroendocrinology 2012 37, 702-714DOI: (10.1016/j.psyneuen.2011.09.002) Copyright © 2011 Elsevier Ltd Terms and Conditions