Stress activates the SuM

(A) Workflow of the c-Fos staining. (B) Representative images of c-Fos staining (DAPI: blue; c-Fos: white; scale bar: 100 µm). (C) Statistical analysis of the number of c-Fos-positive cells displayed in panel B. n = 10–13 per group; unpaired t test. (D) Workflow of CSDS exposure and in vivo recording. (E) Representative spikes acquired by multichannel recording. (F) Statistical analysis of the firing rates of RNs at baseline and after CSDS exposure. n = 16‒23 per group; two-way ANOVA followed by Sidak’s post hoc test. The data in C and F are presented as the means ± SEMs. “**”, p <0.01; “***”, p < 0.001. CSDS: chronic social stress; FS: foot shock. Also see Supplemental Figure 1.

Activation of SuM produces anxiety-like behavior

(A-B) Virus injection information (A) and workflow for chemogenetic manipulation (B). (C) Representative c-Fos images. (D) Statistical analysis of the number of c-Fos-positive cells displayed in panel C. n = 4 per group; unpaired t test. (E-F) Statistical analysis of the distance traveled in the central area (E) and the time that the mice spent in the central area (F) in the OF test. n = 10 per group; unpaired t test for the data in (E) and the Mann‒Whitney test for the data in (F). (G) Statistical analysis of the time that the mice spent in the open arms of the EZM. n = 8 per group; unpaired t test. (H) Statistical analysis of sucrose pellets consumed. n = 8–10 per group; Mann‒Whitney test. The data in D-H are presented as the means ± SEMs. “ns”, p >0.05; “**”, p <0.01. Also see Supplemental Figure 2.

SANs in the SuM selectively respond to social stress but not reward

(A) Workflow of neuronal tagging. (B) Representative image of SANs in the SuM (DAPI: blue, tagged cells: red). (C) Quantitative statistics of stress-tagged cells in several brain areas. n = 3 per area; one-way ANOVA followed by Tukey’4s post hoc test. (D) Workflow of neuronal tagging and c-Fos staining. (E) Representative images of stress-tagged cells and c-Fos expression induced by sucrose and social stress (DAPI: blue, tdTomato: red, c-Fos: green). (F) Statistical analysis of the number of stress-tagged cells in the SuM. n = 3 per group; one-way ANOVA followed by Tukey’s post hoc test. (G) Statistical analysis of the number of c-Fos+ cells after sucrose or social stress exposure. n = 3 per group; one-way ANOVA followed by Tukey’s post hoc test. (H) Statistical analysis of the likelihood of reactivation of stress-tagged cells. n = 3 per group; one-way ANOVA followed by Tukey’s post hoc test. The data in C and F-H are presented as the means ± SEMs. “ns”, p >0.05; “*”, p <0.05; “**”, p <0.01; “***”, p < 0.001.

Selective chemogenetic activation of SANs elevates corticosterone level and produces anxiety-like behavior

(A) Workflow of the acute stress and anxiety tests. (B) Statistical analysis of the time that the mice spent in the open arms of the EZM. n = 10‒11 per group; two-way ANOVA followed by Sidak post hoc test. (C) Workflow of the CORT assay and c-Fos staining. (D) Statistical analysis of the serum concentration of corticosterone after the application of CNO. n = 4-5 per group; unpaired t test. (E) Representative images of stress-tagged cells and c-Fos expression induced by chemogenetic manipulation (DAPI: blue, EGFP: green, c-Fos: violet). (F) Statistical analysis of the percentage of costained cells relative to EGPF+ cells in the SuM. n = 4-5 per group; unpaired t test. (G-H) Virus injection information and workflow of chemogenetic manipulation. (I-J) Statistical analysis of the distance traveled in the central area (I) and the time that the mice spent in the central area (J) in the OF test. n = 14–15 per group; unpaired t test. (K) Statistical analysis of the time that the mice spent in the open arms of the EZM. n = 14–15 per group; Mann‒Whitney test. (L) Statistical analysis of sucrose pellets consumed. n = 13–15 per group; unpaired t test. The data in B, D, E and I-L are presented as the means ± SEMs. “*”, p <0.05; “**”, p <0.01; “***”, p < 0.001. Also see Supplemental Figure 2.

vSub-SuM projections encoding anxiety-like behavior

(A) Workflow of virus-based retrograde neuronal tracing. (B) Representative images of in situ RNA staining (DAPI: blue, Slc32a1: green, Slc17a7: red, EGFP: white). (C) Workflow of ex vivo electrophysiological recording. (D) Schematic of oPSCs in the SuM. (E) Representative traces of oPSCs. (F) Workflow of Ca2+ imaging. (G) Schematic of Ca2+ imaging of dSub and vSub projection neurons. (H) Representative images of GCaMP7b expression in the dSub, vSub and SuM (DAPI: blue, GCaMP7b: green). (I) Heatmap of the Ca2+ fluorescence intensity during the transition from the closed to the open arms. (J) Representative Ca2+ activity during the transition from the closed arms to the open arms. (K) Average ΔF/F of Ca2+ recorded in the dSub and vSub. (L) Statistical analysis of the peak Ca2+ activity. n = 5 per group; unpaired t test. (M) Statistical analysis of the area under the curve of Ca2+ activity. n = 5 per group; unpaired t test. (N) Heatmap of the Ca2+ fluorescence intensity during exposure to foot shocks. (O) Representative Ca2+ activity during exposure to foot shocks. (P) Average ΔF/F of Ca2+ recorded in the dSub and vSub. (Q) Statistical analysis of the peak Ca2+ activity. n = 5‒6 per group; unpaired t test. (R) Statistical analysis of the area under the curve of Ca2+ activity. n = 5‒6 per group; unpaired t test. Data in L-M and Q-R are presented as the mean ± SEM. “ns”, p >0.05; “*”, p <0.05. Also see Supplemental Figure 4 & Supplemental Figure 5.

Selective inhibition of vSub-SuM projections alleviated CSDS-induced anxiety

(A) Workflow of CSDS and chemogenetic manipulation. (B) Schematic of chemogenetic manipulation of specific projections. (C) Body weight during CSDS exposure. (D) Representative images of virus expression. (E) Statistical analysis of the social interaction ratio after CSDS exposure. n = 4‒5 per group; two-way ANOVA followed by Sidak’s post hoc test. (F) Statistical analysis of the distance that the mice traveled in the EZM. n = 4‒5 per group; two-way ANOVA followed by Sidak’s post hoc test. (G) Statistical analysis of the time that the mice spent in the open arms of the EZM; two-way ANOVA followed by Sidak post hoc test. The data in C and E-Gare presented as the means ± SEMs. “ns”, p >0.05; “*”, p <0.05; “**”, p <0.01; “***”, p < 0.001.