Supplementary Figure 9: Supplementary behavioral data for Evans blue (EB) extravasation and Ccr2 ^RFP::Cx3cr1 ^GFP mice after CSDS

A cohort of mice was behaviorally characterized to assess BBB permeability to circulating EB. SS mice spent less time in the interaction zone (A) and more time in the corners (B) when the social target (AGG) was present when compared to CTRL and RES mice. SS mice spent more time in the interaction zone when the social target was absent (A) but no difference was observed for the corners (B). C) Stressed mice displayed less locomotion versus unstressed CTRL when the social target was either present or absent. However no significant difference was measured between SS and RES groups. D) EB level in the NAc was significantly correlated with social avoidance. E) EB could be detected in hippocampus blood vessels 10 min after the retro-orbital injection was performed (left). No EB extravasation was observed after 16-h circulation followed by 5-min perfusion in the hippocampus (right). F) Similarly, EB was detectable in PFC blood vessels 10-min after the injection but not after 16-h circulation and 5-min perfusion. No difference was measured for the hippocampus (G) or PFC (H) after EB extraction. I) C-C chemokine receptor 2 (ccr2) mRNA expression is specifically elevated in the NAc of SS mice after 10-day CSDS and correlated with social avoidance. J) Stressed ccr2 ^RFP:: cx3cr1 ^GFP mice spent less time in the interaction zone when the social target (AGG) was present. Lower overall locomotion was also observed in stressed ccr2 ^RFP:: cx3cr1 ^GFP mice when compared to unstressed controls when the AGG was absent. K) Flow cytometry gating strategy for ccr2 ^RFP monocytes and cx3cr1 ^GFP microglia. L) No difference was measured between groups for percentage (%) of ccr2^-/cx3cr1⁺ cells (microglia). M) Immunohistochemical analysis of ccr2^RFP+ monocytes shows that they accumulate within blood vessels of the NAc (left) and lateral ventricle (right), but not in the parenchyma. Scale bar at 100 µm (50 µm for the insets). Data represent mean ± SEM, number of animals (n) is indicated on graphs. Correlations were evaluated with Pearson’s correlation coefficient, unpaired t-test for ccr2 ^RFP :: cx3cr1 ^GFP mice and one-way ANOVA followed by Bonferroni’s multiple comparison test for other graphs, *p < 0.05; **p < 0.01, ***p < 0.001