* * * * * * * * * * * * * * * Figure S1 A. B. C. D. E. F. G. H. I. J. Pre-limbic Cortex (PrL) Primary Motor Cortex (MCX) Central Amydgala (CeA) Hippocampus CA3 Region (CA3) Control Stress B. C. 200 Control Stress 160 120 * ΔFosB+ cells (#) * Prelimbic Cortex 80 * 40 ΔFosB PrL CeA CA3 MCX D. E. 20 Control Stress * 15 Prelimbic Cortex Iba-1 Area (%) 10 * * 5 Iba-1 PrL CeA CA3 MCX F. G. 20 Control Stress 15 * * Prelimbic Cortex VCAM-1 Area (%) 10 * 5 VCAM/CD45 PrL CeA CA3 MCX H. I. 20 Control Stress 15 * Prelimbic Cortex ICAM-1 Area (%) 10 * * 5 ICAM/CD45 PrL CeA CA3 MCX J. K. L. CD45+ Cells (#) 4 8 12 16 * Con Stress 4 8 * Con Stress 8 * VCAM-1 Associated CD45+ Cells (#) ICAM-1 Associated CD45+ Cells (#) 4 Con Stress

Figure S1. Social stress caused the co-occurrence of neuronal activation, microglial restructuring, and neurovascular adhesion of monocytes within threat appraisal brain regions. Male C57BL/6 mice were subjected to 6 cycles of repeated social defeat (Stress) or left undisturbed as controls. Mice were perfused and the brain was PFA fixed 14 h after the last cycle of stress. Neuronal activation (ΔFosB), microglial activation (Iba-1), vascular endothelial activation (VCAM-1 & ICAM-1), and the presence of monocytes (CD45+) in the vasculature (ICAM-1/CD45 or VCAM-1/CD45) were assessed. A) Illustration of threat appraisal centers used in the study. Representative images within the prelimbic cortex of B) ΔFosB, D) Iba-1, F) VCAM-1/CD45, and H) ICAM-1/CD45 labeling. Insets show enlarged images of positive labeling. C) Number of FosB+ cells (Stress x Region interaction; F(3,29)=4.97, p=0.008), E) Iba-1 proportional area (Stress x Region interaction; F(3,31)=20.98, p<0.0001), G) VCAM-1 (Stress x Region interaction; F(3,34)=6.22, p=0.0022), and I) ICAM-1 (Stress x Region interaction; F(3,32)=5.39, p=0.0049) proportional area within threat appraisal centers after social stress. J) The number of CD45+ cells (F(1,10)=9.42, p=0.0134), the number of CD45+ cells associated with K) VCAM-1 (F(1,9)=25.20, p=0.001) and L) ICAM-1 positive blood vessels (F(1,9)=7.47, p=0.0257). Bars represent the mean ± SEM. Means with asterisk (*) are significantly different from the corresponding control mice (p<0.05), according to F-protected post hoc analysis.

* * * * * * * * * * * * * * * * * Figure S2 A. B. C. D. 14 12 8 4 E. 280 Control Stress 240 * 200 160 ΔFosB+ cells (#) 120 * # 80 # 40 Veh CZP Mino Veh CZP Mino Veh CZP Mino B. CeA CA3 Motor 10 Control Stress 8 # * 6 Iba-1 Area (%) * 4 2 Veh CZP Mino Veh CZP Mino Veh CZP Mino CeA CA3 Motor C. D. 10 Control Stress 4 8 12 14 Control Stress 8 * * 6 * * VCAM-1 Area (%) * VCAM-1 Area (%) * 4 * 2 Veh CZP Mino Veh CZP Mino Veh CZP Mino Veh CZP Mino CeA CA3 Motor PrL E. 20 Control Stress 15 * * * ICAM-1 Area (%) 10 * * 5 * Veh CZP Mino Veh CZP Mino Veh CZP Mino CeA CA3 Motor

Figure S2. Effect of clonazepam and minocycline on neuronal, microglial, and endothelial activation in stress-responsive regions. Male C57BL/6 mice received either minocycline in their drinking water, clonazepam injections i.p., or vehicle treatment daily two days before and during social defeat (Stress). Mice were perfused and the brain was PFA fixed 14 h after the last cycle of stress. Neuronal activation (ΔFosB), microglial activation (Iba-1) and vascular endothelial activation (VCAM-1 & ICAM-1) were assessed. A) Quantification of ΔFosB+ cells in the CeA (Stress x Drug interaction, F(2,34)=4.46, p=0.0202), CA3 (Stress x Drug interaction, F(2,36)=7.94, p=0.0016) and Motor Cortex is shown. B) Iba-1 proportional area in the CeA, CA3 (Stress x Drug interaction, F(2,31)=13.37, p<.0001) and Motor Cortex is shown. C) VCAM-1 proportional area in the CeA (Stress x Drug interaction, F(2,31)=17.52, p<.0001), CA3 (Stress x Drug interaction, F(2,29)=9.08, p=0.0011) Motor Cortex, and D) PrL (Stress x Intervention interaction, F(2,31)=8.39, p=0.002) are shown. E) ICAM-1 proportional area in the CeA (Stress x Drug interaction, F(2,33)=6.90, p=0.0035), CA3 (Stress x Drug interaction, F(2,31)=4.01, p=0.0298) and Motor cortex (Stress x Drug interaction, F(2,32)=6.74, p=0.0041). Bars represent the mean ± SEM. Means with asterisk (*) are significantly different from the corresponding control mice (p<0.05), and means with (#) tended to be different from control mice (p<0.1), according to F-protected post hoc analysis.

Figure S3 Vehicle Minocycline 100 200 300 Con Stress 400 * A. B. Time in Corner Zone (s) Social Interaction Time in Interaction Zone (s) 100 200 300 Vehicle PLX Con Stress C. D. * # Time in Interaction Zone (s) Social Interaction Time in Corner Zone (s) Figure S3. Inhibiting microglial activation or depleting microglia during stress does not prevent social avoidance behavior. Male C57BL/6 mice received minocycline or vehicle in their drinking water two days before and during social defeat (Stress). Mice were tested for social avoidance 14 h after the last cycle of stress. Time spent in the A) interaction zone (main effect of Stress; F(1,29)=163.62, p<.0001) and B) corner zone (main effect of Stress; F(1,29)=186.38, p<.0001). In a separate experiment, male C57BL/6 mice were provided with a diet of PLX5622 (1200 ppm chow) or vehicle chow for 14 days and then exposed to repeated social defeat (Stress) or left undisturbed as controls. Mice were tested for social avoidance 14 h after the last cycle of stress. Time spent in the C) interaction zone (main effect of Stress; F(1,12)=8.31, p=0.0181) and D) corner zone (main effect of Stress; F(1,12)=20.51, p=0.0014). Bars represent the mean ± SEM. Means with asterisk (*) are significantly different from the corresponding control mice (p<0.05), and means with (#) tended to be different from control mice (p<0.1), according to F-protected post hoc analysis.

* * * * * * * * * * * * * * Figure S4 A. ΔFosB+ cells (#) B. 100 Control Stress 80 * * 60 ΔFosB+ cells (#) 40 20 Veh PLX Veh PLX Veh PLX CeA CA3 Motor B. 15 Control Stress * 10 * Iba-1 Area (%) 5 Veh PLX Veh PLX Veh PLX CeA CA3 Motor C. D. 20 Control Stress 20 Control Stress 15 * 15 * VCAM-1 Area (%) VCAM-1 Area (%) 10 * * 10 * * 5 5 Veh PLX Veh PLX Veh PLX Veh PLX CeA CA3 Motor PrL E. 30 Control Stress 25 * * 20 * ICAM-1 Area (%) 15 * 10 5 Veh PLX Veh PLX Veh PLX CeA CA3 Motor

Figure S4. Microglial depletion with CSF1R antagonist does not prevent threat appraisal or endothelial activation within stress-responsive brain regions. Male C57BL/6 mice were provided with a diet of PLX5622 (1200 ppm chow) or vehicle chow for 14 days and then exposed to repeated social defeat (Stress). Mice were perfused and the brain was PFA fixed 14 h after the last cycle of stress. Neuronal activation (ΔFosB), microglial activation (Iba-1) and vascular endothelial activation (VCAM-1 & ICAM-1) were assessed. A) Quantification of ΔFosB+ cells in the CeA (main effect of Stress, F(1,16)=21.23, p=0.0006), CA3, and Motor Cortex. B) Iba-1 proportional area in the CeA (Stress x Drug interaction; F(1,16)=25.75, p=0.0002 & main effect of Drug, F(1,16)=171.75, p<.0001), CA3 (Stress x Drug interaction; F(1,16)=9.44, p=0.0089 & main effect of Drug F(1,16)=93.44, p<.0001) and Motor Cortex (main effect of Drug F(1,15)=197.84, p<.0001). C) Quantification of VCAM-1 positive area in the CeA (main effect of Stress; F(1,16)=43.79, p<.0001), CA3 (main effect of Stress; F(1,16)=31.94, p<.0001), Motor cortex, and D) PrL (Stress x PLX interaction; F(1,14)=23.84, p=0.0005 & main effect of PLX F(1,14)=21.91, p=0.0007). E) Quantification of ICAM-1 positive area in the CeA (main effect of Stress; F(1,16)=38.98, p<.0001), CA3 (main effect of Stress; F(1,17)=46.90, p<.0001 & main effect of Drug; F(1,16)=107.33, p<.0001) and Motor Cortex. Bars represent the mean ± SEM. Means with asterisk (*) are significantly different from the corresponding control mice (p<0.05), according to F-protected post hoc analysis.

Time to Enter Center (s) Figure S5 A. B. 2 4 6 * Con Stress 8 Casp1WT Casp1KO % Ly6Chi Monocytes in circulation 20 40 60 80 100 Lymphocytes Granulocytes Erythrocytes Monocytes % Cells in BM CD11b C. D. CD45 1.49% 2.68% 2.80% Con-Casp1WT Stress-Casp1WT Stress-Casp1KO 1 3 % Brain Macrophages # E. Casp1WT Casp1KO Enriched CD11b+ Cells IL-1b mRNA (Fold D) 2 4 6 8 10 Con Stress 12 * G. 20 40 60 Duration in Center (s) Open Field F. 80 120 Time to Enter Center (s) #

Figure S5. Peripheral immune activation during stress is not modulated by Caspase-1. Male C57BL/6 WT and Caspase-1KO (Casp1-/-) mice were exposed to repeated social defeat (Stress) or left undisturbed as controls. Anxiety-like behavior was determined 14 h after the last cycle of stress and then samples (bone marrow, blood, and brain) were collected. A) Percentage of bone marrow monocytes (main effect of Stress; F(1,15)=91.02, p<0.0001), granulocytes (main effect of Stress; F(1,15)=67.73, p<0.0001), lymphocytes (main effect of Stress; F(1,15)=155.55, p<0.0001) and erythrocytes (main effect of Stress; p<0.0001). B) Percentage of circulating Ly6Chi monocytes in the blood (main effect of Stress; F(1,15)=42.75, p<.0001). C) Representative bivariate dot plots of CD11b and CD45 labeling on enriched brain macrophages and microglia. D) Percentage of CD45hi macrophages in the brain (main effect of Stress; F(1,14)=8.40, p=0.0159). E) IL-1β mRNA levels in enriched CD11b+ cells from the brain (main effect of Stress; F(1,21)=24.69, p<.0001). F) Latency to enter the center of the open field (main effect of Stress; F(1,41)=1.73, p=0.1974) and G) time spent in the center of the open field (main effect of Stress; F(1,40)=8.37, p=0.0066). Bars represent the mean ± SEM. Means with asterisk (*) are significantly different from the corresponding control mice (p<0.05), and means with (#) tended to be different from control mice (p<0.1), according to F-protected post hoc analysis.