Chronic Unpredictable Stress Protocol

Mice were randomly subdivided into 8 treatment groups including untreated control (Control), BDPP-only (BDPP), probiotic or synbiotic and each treatment group was treated with one condition, either stressed or non-stressed. During the stress protocol, mice were submitted to 28 days of random mild stressors (CUS), allowed to rest for 28 days (CUS+Rest) followed by re-stimulation to a subthreshold mild unpredictable stress (US) for 7 days (CUS+US) to model recurrent stress. The CUS+US timepoint was controlled to a 7 day US without prior stress exposure as demonstrated in previous studied ( Figure 1A ) (15). For each treatment, condition (stressed or non-stressed) and timepoint, the group size included n=16 animals, combined over three independent experiments where tissues were allocated towards the biochemical and imaging studies equally. This group size was determined based on power analyses from previous data conducted by us (28) and others (15). During the stress periods, animals in all study groups, including the mice for the immunophenotyping, were subjected twice daily to random mild stressors ( Table S2 ). Stressors included 45° cage tilt for 12 h, wet bedding for 10-12 h, no bedding for 10-12 h, food and/or water deprivation for 12 h, 4°C cold exposure for 1 h, cold water swim for 5 min, cage shaking for 20 min, reversed light schedule, restraint stress for 1 h, predator scent exposure for 8 h, or crowding with 12 animals/cage for 1 h. Consistent with our ongoing and published (28) experiments, no significant changes between groups were observed for weight, water or food consumption throughout the testing period. Behavioral assessment on all animals (n =16 per group) was conducted following each timepoint. From the 16 animals per group, 6 mice were perfused and used for immunofluorescent analysis, 6 mice were used for tissue RNA, protein (brain and peripheral tissues), blood and feces collection and the remaining 4 for other purposes not relevant to this manuscript. Animals were sacrificed immediately following behavior, blood was collected by cardiac puncture into heparinized tubes while tissues were frozen on dry ice and stored at -80°C until analysis.

A Synbiotic Attenuates Chronic-Stress Induced Psychological Deficits. (A) Chronic and recurrent stress are modelled using the chronic unpredictable stress (CUS) protocol. Following 2 weeks of rest and 2 weeks of pretreatment with the BDPP, probiotics or synbiotics, mice are exposed to 28 days of random mild unpredictable stressors (CUS) following by 28 days of rest (CUS+Rest) and a re-stimulation of 7 days of unpredictable stress (US) modelling recurrent stress (CUS+US). The final CUS+US timepoint can be compared to the 7-day subthreshold US. Each group contained n = 16 animals. Behavior phenotypes were assessed using the (B) forced swim test for depressive-like behavior and (C) open field test for anxiety-like behavior where for each group there are n = 16 mice +/- SEM and significance is determined with a two-way ANOVA and Tukey’s post-hoc analysis. DC, dendritic cell; ILC, innate lymphoid cell; BC, B cell; Treg, regulatory T cell; Th17, T helper 17 cell; TNFβ, tumor necrosis factor β; TLR4, toll-like receptor 4; NFκB, nuclear factor kappa light chain enhancer of B cells; Casp1, caspase1; IL-1β, interleukin 1 beta; CCL2(MCP1), monocyte chemoattractant protein 1; CCL5(RANTES), C-C motif 5; ICAM, intercellular adhesion molecule; VCAM, vascular cell adhesion molecule. In all cases, *p < 0.05, **p < 0.01, ***p < 0.001.