A stress-activated neuronal ensemble in the supramammillary nucleus produces anxiety-like behavior in male mice
- Key Laboratory of Modern Teaching Technology, Ministry of Education, Shaanxi Normal University, China
- Department of Histology and Embryology, School of Basic Medical Science, Xi'an Medical University, China
- Laboratory Animal Center, Shaanxi Normal University, China
- School of Psychology, Shaanxi Normal University, China
- Faculty of Education, Shaanxi Normal University, China
Figures
Figure 1 with 1 supplement
Stress activates the supramammillary nucleus (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 regular-spiking neurons (RNs) at baseline and after CSDS exposure. n=16‒23 per group; two-way ANOVA followed by Sidak’s post hoc test. The bars in C and F indicate the Min to Max of all data points, and the ‘+’ indicates mean value of all data points. ‘**’, p<0.01; ‘***’, p<0.001. CSDS: chronic social stress; FS: foot shock.
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Figure 1—source data 1
The number of c-Fos+ cells in panel C and normalized firing rate in panel F, and corresponding statistical results.
- https://cdn.elifesciences.org/articles/108593/elife-108593-fig1-data1-v1.xlsx
Figure 1—figure supplement 1
The effect of CSDS on the local field potential (LFP) in the supramammillary nucleus (SuM).
(A) Representative images of the location of electrodes. (B) Statistical comparison of the firing rate of fast-spiking neurons (FNs) between baseline and after CSDS. n=4–12 neurons from 2 to 4 mice per group, two-way ANOVA, Sidak’s post hoc test. (C–D) Power spectrum of LFPs. Data in B is presented as mean ± SEM. ‘ns’, p>0.05. CSDS: chronic social stress.
Figure 2 with 1 supplement
Activation of the supramammillary nucleus (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 elevated zero maze (EZM). n=8 per group; unpaired t test. (H) Statistical analysis of sucrose pellets consumed. n=8–10 per group; Mann-Whitney test. The bars in D–H indicate the Min to Max of all data points, and the ‘+’ indicates mean value of all data points. ‘ns’, p>0.05; ‘**’, p<0.01.
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Figure 2—source data 1
The number of c-Fos+ cells in panel D, and behavioral test data in panel E-H, and corresponding statistical results.
- https://cdn.elifesciences.org/articles/108593/elife-108593-fig2-data1-v1.xlsx
Figure 2—figure supplement 1
The chemogenetic manipulation of supramammillary nucleus (SuM) and stress-activated neurons (SANs) has effects on the performance of mice in the open field (OF) and the elevated zero maze (EZM).
(A) Representative images of virus expression. (B) Statistical comparison of the total distance that wild-type (WT) mice traveled in OF. (C) Statistical comparison of the total distance that WT mice traveled in EZM. (D) Statistical comparison of the total distance that TRAP2 mice traveled in OF. (E) Statistical comparison of the total distance that TRAP2 mice traveled in EZM. Data in B–I are presented as mean ± SEM. ‘ns’, p>0.05, ‘**’, p<0.01, ‘***’, p<0.001.
Figure 3
Stress-activated neurons (SANs) in the supramammillary nucleus (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’s 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 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.
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Figure 3—source data 1
The number of TRAPed cells in panel C and panel F, c-Fos+ cells in panel G and the reactivation ratio in panel H, and corresponding statistical results.
- https://cdn.elifesciences.org/articles/108593/elife-108593-fig3-data1-v1.xlsx
Figure 4
Selective chemogenetic activation of stress-activated neurons (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 elevated zero maze (EZM). n=10‒11 per group; two-way ANOVA followed by Sidak’s 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 clozapine N-oxide (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 supramammillary nucleus (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 open field (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 D and F are presented as the means ± SEMs. The bars in B and I–L indicate the Min to Max of all data points, and the ‘+’ indicates mean value of all data points. ‘*’, p<0.05; ‘**’, p<0.01; ‘***’, p<0.001.
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Figure 4—source data 1
The behavioral test data in panel B and I-L, corticosterone level in panel D, co-stain ratio in panel F and corresponding statistical results.
- https://cdn.elifesciences.org/articles/108593/elife-108593-fig4-data1-v1.xlsx
Figure 5 with 3 supplements
Ventral subiculum (vSub)-supramammillary nucleus (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 optically induced postsynaptic currents (oPSCs) in the SuM. (E) Representative traces of oPSCs. (F) Workflow of Ca2+ imaging. (G) Schematic of Ca2+ imaging of dorsal subiculum (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 ∆F/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 ∆F/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. The bars in L–M and Q–R indicate the Min to Max of all data points, and the ‘+’ indicates mean value of all data points. ‘ns’, p>0.05; ‘*’, p<0.05.
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Figure 5—source data 1
The Ca2+ peak of ΔF/F in panel L and Q, the area under curve of the Ca2+ trace in panel M and R, and corresponding statistical results.
- https://cdn.elifesciences.org/articles/108593/elife-108593-fig5-data1-v1.xlsx
Figure 5—figure supplement 1
Specific retrograde neuronal tracing of the upstream of the stress-activated neuron (SAN) in the supramammillary nucleus (SuM).
(A) Workflow of rabies virus (RV)-based retrograde neuronal tracing. (B) Representative image of virus expression in SuM. (C) Representative images of traced upstreaming brain area of SuM.
Figure 5—figure supplement 2
Non-virus and virus-based retrograde neuronal tracing of the upstream of the supramammillary nucleus (SuM).
(A) Representative images of retrograde neuronal tracing using CTB-647, injection site, dorsal subiculum (dSub), and ventral subiculum (vSub). (B) Representative images of retrograde neuronal tracing using AAV2/Retro, injection site, dSub, and vSub.
Figure 5—figure supplement 3
Calcium fiber photometry during elevated plus maze (EPM) test.
(A) Heatmap of the Ca2+ fluorescence intensity during the transition from the open arms to the closed arms. (B) Representative Ca2+ activity during the transition from the open to the closed arms. (C) Average ∆F/F of Ca2+ recorded in the dorsal subiculum (dSub) and ventral subiculum (vSub). (D) Statistical analysis of the peak Ca2+ activity. n=5 per group; unpaired t test.
Figure 6
Selective inhibition of ventral subiculum (vSub)-supramammillary nucleus (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=6–10 per group; two-way ANOVA followed by Sidak’s post hoc test. (F) Statistical analysis of the distance that the mice traveled in the elevated zero maze (EZM). n=6–10 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 bars in E–G indicate the Min to Max of all data points, and the ‘+’ indicates mean value of all data points. ‘ns’, p>0.05; ‘*’, p<0.05; ‘**’, p<0.01; ‘***’, p<0.001. CSDS: chronic social stress.
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Figure 6—source data 1
The body weight in panel C, and behavioral test data in panel E-G, and corresponding statistical results.
- https://cdn.elifesciences.org/articles/108593/elife-108593-fig6-data1-v1.xlsx
Tables
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Strain, strain background (Mus musculus) | C57BL/6J | Key Laboratory of Modern Teaching Technology, Ministry of Education | RRID:IMSR_JAX:000664 | |
| Strain, strain background (Mus musculus) | Fos2A-iCreERT2 | JAX | 030323, RRID:IMSR_JAX:030323 | |
| Strain, strain background (Mus musculus) | Rosa26-CAG-LSL-tdTomato (Ai14) | Shanghai Model Organisms Center | NM-KI-225042 | |
| Strain, strain background (Mus musculus) | CD-1 | Charles River | #201 | |
| Recombinant DNA reagent | AAV2/9-hSyn-hM3Dq-EGFP | BrainVTA | PT-0891 | |
| Recombinant DNA reagent | AAV2/9-hSyn-EGFP | BrainVTA | PT-1990 | |
| Recombinant DNA reagent | AAV2/9-hSyn-DIO-hM3Dq -EGFP | BrainVTA | PT-0891 | |
| Recombinant DNA reagent | AAV2/9-hSyn-DIO-EGFP | BrainVTA | PT-1103 | |
| Recombinant DNA reagent | AAV2/Retro-hSyn-Cre | Taitool | S0278 | |
| Recombinant DNA reagent | AAV2/9-hSyn-DIO-hM4Di-mCherry | Taitool | S0193 | |
| Recombinant DNA reagent | AAV2/9-hSyn-DIO-mCherry | Braincase | BC-0025 | |
| Recombinant DNA reagent | AAV2/9-hSyn-DIO-GCaMP7b | BrainVTA | PT-2892 | |
| Recombinant DNA reagent | AAV2/9-hSyn-ChR2-mCherry | BrainVTA | PT-0150 | |
| Recombinant DNA reagent | AAV2/Retro-hSyn-EGFP | Taitool | S0237 | |
| Recombinant DNA reagent | AAV2/9-Ef1α-DIO-RVG | BrainVTA | PT-0061 | |
| Recombinant DNA reagent | AAV2/9-Ef1α-DIO-mCherry-F2A-TVA | BrainVTA | PT-0207 | |
| Recombinant DNA reagent | RV-ENVA-ΔG-EGFP | BrainVTA | R01001 | |
| Antibody | Anti-c-Fos (Rabbit Monoclonal) | Cell Signaling Technology | Cat#2250, RRID:AB_2247211 | 1:500 |
| Antibody | Donkey anti-rabbit conjugated to AF647 | Jackson ImmunoResearch | Cat#706-605-148, RRID:AB_2340476 | 1:500 |
| Commercial assay or kit | RNAscope | ACDbio | Cat#323100 | |
| Chemical compound, drug | 4-Hydroxytamoxifen | Sigma-Aldrich | Cat#H6278 | |
| Chemical compound, drug | 4-Hydroxytamoxifen | Bidepharm | Cat# BD00958757 | |
| Chemical compound, drug | Clozapine N-oxide | Cayman | Cat#25780 | |
| Software, algorithm | SpikeInterface | SpikeInterface | RRID:SCR_021150 | |
| Other | Antifade reagent | Invitrogen | P36981 |
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A stress-activated neuronal ensemble in the supramammillary nucleus produces anxiety-like behavior in male mice
eLife 14:RP108593.
https://doi.org/10.7554/eLife.108593.3
