Anxiety-like Behavior in Female Sprague Dawley Rats Associated with Cecal Clostridiales

Abstract

The relationship between the microbiota profile and exposure to stress is not well understood. Therefore, we used a rat model of unpredictable chronic mild stress (UCMS) to investigate this relationship. Depressive-like behaviors were measured in Female Sprague Dawley rats using the sucrose preference test and the Porsolt swim test. Anxiety-like behaviors were measured with the light-dark box test. Fecal corticosterone, cecal microbiota (composition and organic acids), plasma gut permeability (lipopolysaccharide-binding protein, LBP) and plasma inflammation (12 cytokines) markers were measured. Atypical behaviors were observed in female rats following UCMS, but no depressive-like behaviors were observed. Circulating concentrations of cytokines granulocyte-macrophage colony-stimulating factor and cytokine-induced neutrophil chemoattractant 1 were higher in UCMS-exposed female rats; plasma LBP and cecal organic acid levels remained unchanged. Our results reflect a resilient and adaptive phenotype for female SD rats. The relative abundance of taxa from the Clostridiales order and Desulfovibrionaceae family did, however, correlate both positively and negatively with anxiety-like behaviors and plasma cytokine concentrations, regardless of UCMS exposure, supporting the brain-to-gut influence of mild anxiety with a microbiota profile that may involve inflammatory pathways.

Keywords: Clostridiales; anxiety; cytokines; microbiome; microbiome–gut–brain axis; stress.

Figure 1

Timeline of the study.

Figure 2

Results of behavioral testing unpredictable chronic mild stress in the UCM group and the control group. (a) Sucrose preference, measured in weekly 24 h sucrose preference tests (SPT) over six weeks of UCMS was not altered by time or exposure to UCMS, but (b) the total sucrose solution intake during the same tests increased in the UCMS group over six weeks. (c,d) Following six weeks of studying UCMS rats and control rats, no significant differences in individual scores in the light–dark box test were found. (e) There was no difference in scores in the Porstolt swim test between groups following six weeks of UCMS. (f) There were also no differences between the two groups in the coat state test. Data are presented as mean + SEM; control group, n = 10; UCMS group, n = 20. * indicates p < 0.05 between UCMS group and control group. # indicates p < 0.05 between timepoint and baseline.

Figure 3

Plasma cytokine concentrations. In female Sprague Dawley rats, following six weeks of unpredictable chronic mild stress (UCMS), a pattern of increased pro- and anti-inflammatory markers was observed compared with those of the control group (CON). Graphs shows the measured cytokines which were (a) monocyte chemoattractant protein-1 (MCP-1), (b) cytokine-induced neutrophil chemoattractant type-1 (CINC-1), (c) granulocyte-macrophage colony-stimulating factor (GM-CSF), (d) interferon-gamma (IFN-γ), (e) interleukin (IL)-18, (f) IL-12p70, (g) IL-33, (h) IL-1α, (i) IL-6, (j) tumor necrosis factor-alpha (TNF-α), (k) IL-17A, (l) IL-10. Data are presented mean +/− SEM; control group, n = 10; UCMS group, n = 20. * indicates a significantly different value from the control group at p < 0.05.

Figure 4

The alpha diversities (measured via four indexes: observed, Chao1, Shannon, and Simpson) of the cecal microbiota of Sprague Dawley rats did not differ after six-week exposure to unpredictable chronic mild stress (UCMS) between the UCMS group or the control group.

Figure 5

Beta diversity PCA plots of the cecal microbiota of Sprague Dawley rats after six-week exposure to unpredictable chronic mild stress (shown in blue) and control rats (shown in red). PCA plots show (a) Bray–Curtis distances, (b) weighted unifrac distances, and unweighted unifrac distances (c).

Figure 6

Relative abundance of cecal microbiota taxa identified via 16S rRNA next generation sequencing. Female Sprague Dawley rats exposed to six weeks of unpredictable chronic mild stress (UCMS) showed differences between groups in several taxa (a–e) than the control group did. Box and whisker plots show relative abundance of the microbial taxa for each rat, with the minimum value, first quartile, median, third quartile, and maximum value of each group shown. p = phylum; f = family; sp = species. * indicates significant difference at p value < 0.05. Data are presented mean +/− SEM; UCMS group, n = 20; control group, n = 10.