Article Figures & Data
Figures
- Figure 1.
BALB/c mice exhibit altered behavioral phenotypes immediately and two weeks after repeated SDS. A, Experimental timeline of repeated SDS. B, Body weight change on day 8 (left) and daily body weight change during the 7 d of SDS (right). C, Schematic of the SIT arena. D, Comparison of time spent in the avoidance and interaction zones during the SIT_0W between control and defeated mice (top), or among the three groups, stratifying defeated mice into nonavoidant and avoidant mice (bottom). E, Comparison of time spent in the open arms and the total distance during EPM_0W between control and defeated mice (top), or among the three groups (bottom). F, Comparison of time spent in the avoidance and interaction zones during the SIT_2W between control and defeated mice (top), or among the three groups (bottom). G, Comparison of time spent in the open arms and the total distance traveled during EPM_2W between control and defeated mice (top), or compared among the three groups (bottom). Control mice: n = 10, defeated mice: n = 15, data are presented as means ± SEM. B, ***/###p < 0.001, ****/####p < 0.0001, Unpaired t test for comparison between two groups and two-way repeated measures ANOVA followed by multiple comparison tests with Bonferroni correction for daily body weight change. D–G, ns, not significant, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, Unpaired t test with Welch’s correction for comparison between two groups and Welch ANOVA test followed by Games–Howell’s multiple comparisons test for comparison among the three groups. Orange triangles and blue squares indicate nonavoidant mice and avoidant mice in the SIT_0W, respectively. Detailed results of the statistical tests are described in Extended Data Figure 1-1, 2-1, 3-1, 4-1, 6-1.
- Figure 2.
Establishment and evaluation of the semi-automated c-Fos extraction and quantitation method. A, Example of a coronal section that includes the PAG. The pink shadow (left) shows the region outlined as PAG with the semi-automated method (PAG_auto), and the white shadow (middle) shows the PAG region that was manually outlined (PAG_manual). Scale bar: 1 mm. Red dots (right) indicate locations that were detected as c-Fos signals by the semi-automated method. B, The sensitivity and precision for PAG outlining using the semi-automated method. C, Comparison of c-Fos signal densities within PAG quantified either manually or by the semi-automated method, and the scatter plot showing correlations in c-Fos signal densities quantified by the manual and semi-automated methods. D, The sensitivity and precision for c-Fos signal detection within the PAG using the semi-automated method. Control: n = 10, defeat: n = 10 (10 defeated mice were randomly selected from 15 mice). Data are presented as means ± SEM. C, Paired t test and Pearson correlation coefficients. ns, not significant. Detailed results of the statistical tests are described in Extended Data Figure 2-1.
- Figure 3.
Identification of brain regions that exhibit differential activity between the control and defeated mice following social interaction. A–S, Comparison of c-Fos signal densities between control and defeated mice in various brain regions. PAG, periaqueductal gray; NAc, nucleus accumbens; CeA, central amygdala; mPFC, medial prefrontal cortex; VTA, ventral tegmental area; CoA, cortical amygdala; LSv, lateral septal nucleus ventral part; BNST, bed nucleus of the stria terminalis; BLA, basolateral amygdala; DG, dentate gyrus; LHb, lateral habenula; MHb, medial habenula; PBN, parabrachial nucleus; LC, locus coeruleus. Control: n = 10, defeat: n = 15, data are presented as means ± SEM, *p < 0.05, **p < 0.01, unpaired t test. Detailed results of the statistical tests are described in Extended Data Figure 3-1. T–W, Representative images of c-Fos signals in the PAG (T), CeA (U), NAc (V), and mPFC (W) of control and defeated mice. Scale bar: 0.05 mm.
- Figure 4.
Repeated SDS leads to the emergence of an activity correlation between various brain regions. A–ab, Scatter plots showing correlations in c-Fos signal densities among pairs of brain regions that showed altered activity between control and defeated mice in Figure 3. ac, ad, Heatmaps showing the overview of the r and p values of Pearson correlation coefficients. Control: n = 10, defeat: n = 15, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, Pearson correlation coefficients. Detailed results of the statistical tests are described in Extended Data Figure 4-1.
- Figure 5.
Hierarchical cluster analysis of subpopulations of defeated mice based on c-Fos expression following social interaction 18 d after the repeated SDS. Results of two-way hierarchical clustering analysis are shown together with heatmap visualization of normalized c-Fos signal densities. Av_both indicates mice that were avoidant mice in both SIT_0W and SIT_2W. Av_0W and Av_2W indicate mice that were avoidant in either SIT_0W or SIT_2W, respectively. Non-Av indicates mice that were nonavoidant in both SIT_0W and SIT_2W; * shows the brain regions that showed altered activity between control and defeated mice in Figure 3.
- Figure 6.
Identification of brain regions that showed altered c-Fos signal densities among subpopulations of defeated mice following social interaction 18 d after the repeated SDS. A–S, Comparison of c-Fos signal densities among the subpopulations of defeated mice defined in Figure 5. Av_both: n = 4, Av_0W: n = 4, Av_2W: n = 3, Non-Av: n = 4, data are presented as means ± SEM, */#p < 0.05, two-way ANOVA followed by multiple comparison tests with Bonferroni correction. Detailed results of the statistical tests are described in Extended Data Figure 6-1. T, Representative images of c-Fos signals in the LHb of Av_both and Av_2w mice. Scale bar: 0.05 mm.
Extended Data
Extended Data 1
A code for Semi-automated c-Fos extraction and quantitation method. Download Extended Data 1, ZIP file.
Extended Data Figure 1-1
Detailed results of the statistical analyses in Figure 1. Download Figure 1-1, XLS file.
Extended Data Figure 2-1
Detailed results of the statistical analyses in Figure 2. Download Figure 2-1, XLS file.
Extended Data Figure 3-1
Detailed results of the statistical analyses in Figure 3. Download Figure 3-1, XLS file.
Extended Data Figure 4-1
Detailed results of the statistical analyses in Figure 4. Download Figure 4-1, XLS file.
Extended Data Figure 6-1
Detailed results of the statistical analyses in Figure 6. Download Figure 6-1, XLS file.
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