Glutamatergic supramammillary nucleus neurons respond to threatening stressors and promote active coping
- Department of Anesthesiology, Washington University in St. Louis, United States
- Department of Medicine, Northwestern University Feinberg School of Medicine, United States
- Department of Neuroscience, Washington University in St. Louis, United States
- Department of Psychiatry, Washington University in St. Louis, United States
- Medical College of Wisconsin, United States
- Department of Obstetrics and Gynecology, Washington University in St. Louis, United States
- Center for Neurobiology of Addiction, Pain, and Emotion University of Washington, United States
- Department of Anesthesiology and Pain Medicine University of Washington, United States
- Department of Pharmacology University of Washington, United States
- Department of Bioengineering University of Washington, United States
Figures
Figure 1 with 3 supplements
SuMVGLUT2+::POA arborize widely in the brain.
(A) Schematic of injections of Retro-AAV-DIO-eYFP into the POA and AAV-fDIO-Cre into the SuM of VGLUT2-Flp mice to label only VGLUT + SuM neurons that project to the POA. (B) Projections of VGLUT2 + …
Figure 1—figure supplement 1
SuMVGLUT2+ neurons project to the preoptic area of the hypothalamus and are not VGAT+.
(A) Schematic of injections into VGLUT2-Cre mice of Retro-AAV-DIO-tdTomato into the POA and AAV-DIO-ChR2eYFP into the SuM. (B and C) Projections of VGLUT2 +SuM neurons are evident in the POA with …
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Figure 1—figure supplement 1—source data 1
Quantification of labeling of Cre +and Cre- cells in SuM following injections in POA in VGLUT2-Cre and VGAT-Cre mice.
- https://cdn.elifesciences.org/articles/90972/elife-90972-fig1-figsupp1-data1-v1.xlsx
Figure 1—figure supplement 2
Retrograde verification of projection targets of SuMVGLUT2+::POA.
(A, D, G, and J) Schematic showing the injection sites of Retro-AAV’s in VGLUT2-Cre mice. (B–C) In VGLUT2-Cre mice, injection of Retro-AAV-DIO-eYFP unilaterally in the (B) accumbens nucleus resulted …
Figure 1—figure supplement 3
Combinatorial viral and genetic approach is effective with minimal background.
(A) Schematic of injection of Retro-AAV-Cre into the POA and AAV-Nuc-flox(mCherry)-eGFP into the SuM of WT mice. (B) In SuM, eGFP (green) Cre + and mCherry (red) Cre- labeled neurons are seen based …
Figure 2
SuMVGLUT2+::POA neurons project to a subset of brain regions compared to all SuMVGLUT2+ neurons.
(A) Schematic of injections in VGLUT2-Cre mice of AAV-DIO-ChR2eYFP into SuM. (B) eYFP (green) labeled neurons present in SuM and (C) processes were observed in the POA. (D) Processes from SuM VGLUT2 …
Figure 3 with 2 supplements
Acute stressors recruit SuMVGLUT2+::POA neurons.
(A) Schematic of VGLUT2-Flp mice injected with AAV-DIO-GCaMP7s in SuM and Retro-AAV-fDIO-Cre in the POA with a (B) fiber placed over SuM for fiber photometry recordings from SuMVGLUT2+::POA neurons. …
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Figure 3—source data 1
Photometry data for Figure 3F and G.
- https://cdn.elifesciences.org/articles/90972/elife-90972-fig3-data1-v1.xlsx
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Figure 3—source data 2
Photometry data for Figure 3H and I.
- https://cdn.elifesciences.org/articles/90972/elife-90972-fig3-data2-v1.xlsx
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Figure 3—source data 3
Photometry data for Figure 3J and K.
- https://cdn.elifesciences.org/articles/90972/elife-90972-fig3-data3-v1.xlsx
Figure 3—figure supplement 1
SuMVGLUT2+::POA neurons show Fos induction after FST, and dunk test evokes active coping.
Sections of brains from mice (A) left in the home cage or (B) subjected to 15 min of forced swimming 90 min prior to sacrifice were probed with anti-Fos antibodies (cyan) showed (scale bars 500 μm) …
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Figure 3—figure supplement 1—source data 1
cFos quantification and Dunk FST data.
- https://cdn.elifesciences.org/articles/90972/elife-90972-fig3-figsupp1-data1-v1.xlsx
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Figure 3—figure supplement 1—source data 2
This file contains information for cell counts broken out by animal by condition and the underling quantification by animal by trial of time spent mobile in the dunk test.
- https://cdn.elifesciences.org/articles/90972/elife-90972-fig3-figsupp1-data2-v1.xlsx
Figure 3—figure supplement 2
SuMVGLUT2+::POA neurons are not recruited during increased locomotor activity in the absence of acute stressor.
(A) Heat maps showing velocity for 12 episodes of rapid locomotion from a representative animal during free movement in open field with mean ± 95% CI for the trials. (B) For that same animal, heat …
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Figure 3—figure supplement 2—source data 1
Photometry data and velocity values.
- https://cdn.elifesciences.org/articles/90972/elife-90972-fig3-figsupp2-data1-v1.xlsx
Figure 4
Photostimulation of SuMVGLUT2+::POA neurons evokes active coping behaviors.
(A) Illustration of injections and fiber implant in VGLUT2 +Cre mice and schematic of photostimulation paradigm of 5 min pre, 5 min stimulation, and 5 min post. Behaviors were evaluated during each …
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Figure 4—source data 1
Behaviors induced by photostimulation of SuMVGLUT2+::POA neurons.
- https://cdn.elifesciences.org/articles/90972/elife-90972-fig4-data1-v1.xlsx
Figure 5 with 1 supplement
Activation of SuMVGLUT2+::POA neurons drives aversion but does not promote anxiety-like behavior.
(A) Schematic of injection of Retro-AAV-ChR2eYFP into the POA and placement of a midline optic fiber over SuM. (B) Representative heat maps for Cre +and Cre- mice at baseline (no stim) and 10 Hz …
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Figure 5—source data 1
Photostimulation of SuMVGLUT2+::POA neurons drive aversion.
- https://cdn.elifesciences.org/articles/90972/elife-90972-fig5-data1-v1.xlsx
Figure 5—figure supplement 1
GABAergic neurons in SuM do not drive place preference.
(A) Schematic of viral injection into VGAT-Cre mice in SuM. (B) VGAT +neurons labeled and outline of implanted fiber. (C) Real-time arena showing the pair of photostimulation on one side of the …
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Figure 5—figure supplement 1—source data 1
Effects of photostimulation of GABAergic neurons in SuM.
- https://cdn.elifesciences.org/articles/90972/elife-90972-fig5-figsupp1-data1-v1.xlsx
Figure 6 with 1 supplement
SuMVGLUT2+::POA neurons can drive instrumental action-outcome operant behavior.
(A) Schematic of injection and implant in VGLUT2 +Cre mice and paradigm of testing in 10 min trials with 4 days before a progressive ratio (PR) trial on day 5. (B) Illustration of the testing …
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Figure 6—source data 1
Photostimulation of SuMVGLUT2+::POA neurons can drive instrumental action-outcome operant behavior.
- https://cdn.elifesciences.org/articles/90972/elife-90972-fig6-data1-v1.xlsx
Figure 6—figure supplement 1
SuMVGLUT2+::POA stimulation drives active port activation.
(A) Cre +mice activated the active port significantly more (**p=0.001) compared to the inactive port. (B) In Cre- mice, no significant change in the number of port activations of both the active and …
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Figure 6—figure supplement 1—source data 1
Number of activate vs inactive port activations in Cre +and Cre- mice.
- https://cdn.elifesciences.org/articles/90972/elife-90972-fig6-figsupp1-data1-v1.xlsx
Figure 7
SuMVGLUT2+::POA neurons are recruited during active coping, and activation is sufficient drive a switch to active coping.
(A) Color-coded per video frame (dot color) behavior combined with normalized Ca2+-dependent and isosbestic fiber photometry signals. Scoring and analysis of climbing, swimming, hindpaw swimming, …
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Figure 7—source data 1
Photometry data for Figure A-E.
- https://cdn.elifesciences.org/articles/90972/elife-90972-fig7-data1-v1.xlsx
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Figure 7—source data 2
Data for photostimulation of SuM VGLUT2 and VGAT neurons during FST.
- https://cdn.elifesciences.org/articles/90972/elife-90972-fig7-data2-v1.xlsx
Figure 8
Consummatory behavior suppresses SuMVGLUT2+::POA neuron activity, and SuMVGLUT2+::POA neuron blocks food consumption.
(A) Schematic of injections in VGLUT2-Flp mice with AAV-DIO-GCaMP7s in SuM and Retro-AAV-fDIO-Cre in the POA and a fiber placed over SuM. (B) Mice were given ad lib food access or food deprived for …
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Figure 8—source data 1
Food interaction and food eaten data for Figure 8C and D.
- https://cdn.elifesciences.org/articles/90972/elife-90972-fig8-data1-v1.xlsx
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Figure 8—source data 2
Photometry data for Figure 8E and F.
- https://cdn.elifesciences.org/articles/90972/elife-90972-fig8-data2-v1.xlsx
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Figure 8—source data 3
Food interaction and food eaten data for Figure 8H and I.
- https://cdn.elifesciences.org/articles/90972/elife-90972-fig8-data3-v1.xlsx
Figure 9
Graphical summary.
The presented studies show SuMVGLUT2+::POA neurons and a subset of neurons in SuM with broad arborizations to multiple brain regions involved in responding to threat and are recruited by multiple …
Author response image 1
Videos
Video 1
Predator ambush.
Example of a mouse fleeing after being ambushed by a remote-controlled mechanical spider hidden in an enclosure.
Video 2
Example of photostimulation of SuMVGLUT2+::POA neurons evoking active coping behaviors.
Mice show an increase in active coping behaviors (e.g. jumping, digging) during photostimulation of SuMVGLUT2+::POA neurons.
Video 3
Example of photostimulation of SuMVGLUT2+::POA neurons driving aversion.
Mice avoid the photostimulation side with higher frequencies showing greater aversion.
Video 4
Annotated video showing color-coded behaviors with concurrent calcium activity recording during forced swim test.
Climbing is shown in red, floating in blue, hindpaw swimming in green, and swimming in yellow.
Video 5
Photostimulation of SuMVGLUT2+::POA neurons during forced swim test elicits active coping behaviors.
Tables
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Strain, strain background (Slc17a6tm2(cre)Lowl/J, C57BL/6;FVB;129S6) | Slc17a6tm2(cre)Lowl/J (VGLUT2-Cre) | The Jackson Laboratory | RRID:IMSR_JAX:016963 | Males and females |
| Strain, strain background (B6;129S-Slc17a6tm1.1(flpo)Hze/J, C57BL/6 J) | B6;129S-Slc17a6tm1.1(flpo)Hze/J (VGLUT2-Flp) | The Jackson Laboratory | RRID: IMSR_JAX: 030212 | Males and females |
| Strain, strain background (B6.Cg-Gt(ROSA)26Sortm14(CAG-tdTomato)Hze/J, C57BL/6 J) | B6.Cg-Gt(ROSA)26Sortm14(CAG-tdTomato)Hze/J (Ai14) | The Jackson Laboratory | RRID: IMSR_JAX: 007914 | Males and females |
| Strain, strain background (B6J.129S6(FVB)-Slc32a1tm2(cre)Lowl/MwarJ, C57BL/6;FVB;129S6) | B6J.129S6(FVB)-Slc32a1tm2(cre)Lowl/MwarJ (VGAT-Cre) | The Jackson Laboratory | RRID: IMSR_JAX: 028862 | Males and females |
| Strain, strain background (C57BL/6 J, C57BL/6 J) | C57BL/6 J | The Jackson Laboratory | RRID: IMSR_JAX: 000664 | Males and females |
| Antibody | Alexa Fluor 488 Goat anti-Rabbit IgG (Goat Polyclonal) | Invitrogen | Cat# A11008 | IHC (1:2500) |
| Antibody | Phospho-c-Fos (Ser32) (D82C12) XP (Rabbit Monoclonal) | Cell Signaling Technology | Cat# 5348 | IHC (1:500) |
| Recombinant DNA reagent | AAV5-EF1a-DIO-hChR2(H134R)-EYFP (2.5×1013 vg/ml) | Washington University Hope Center Viral Vector Core | N/A | |
| Recombinant DNA reagent | AAV2-Retro-DIO-ChR2-eYFP (2.8×1012 vg/ml) | Washington University Hope Center Viral Vector Core | N/A | |
| Recombinant DNA reagent | AAV2-retro-FLEX-tdTomato (7×1012 vg/ml) | Addgene | RRID: Addgene_ 28306-AAVrg | |
| Recombinant DNA reagent | AAV5-DIO-ChR2-eYFP (1.4×1013 vg/ml) | Washington University Hope Center Viral Vector Core | N/A | |
| Recombinant DNA reagent | AAV2-Retro-EF1a-fDIO-cre (7×1012 vg/ml) | Addgene | RRID: Addgene_ 121675-AAVrg | |
| Recombinant DNA reagent | AAV5-EF1a-Nuc-flox(mCherry)-EGFP (5.7×1012 vg/ml) | Addgene | RRID: Addgene_112677-AAV5 | |
| Recombinant DNA reagent | Retro-AAV2-EF1a-Nuc-flox(mCherry)-EGFP (7×10¹² vg/mL) | Addgene | RRID: Addgene_ 112677-AAVrg | |
| Recombinant DNA reagent | AAV2-retro-EF1a-DIO-eYFP (3×1013 vg/ml) | Washington University Hope Center Viral Vector Core | N/A | |
| Recombinant DNA reagent | AAV-retro-EF1a-Flpo (1.02×1013 GC/mL or 7×1012 vg/ml) | Addgene | RRID: Addgene_ 55637-AAVrg | |
| Recombinant DNA reagent | AAV9-EF1a-fDIO-cre (2.5×1013 GC/mL or 1×1013 vg/ml) | Addgene | RRID: Addgene_121675-AAV9 | |
| Recombinant DNA reagent | Retro-AAV2-EF1a-Cre (2.1×10^13 GC/mL) | Addgene | RRID: Addgene_55636-AAVrg | |
| Recombinant DNA reagent | AAV9-syn-FLEX-GCaMP7s-WPRE (1.2×1013 GC/ml) | Addgene | RRID: Addgene_104487-AAV9 | |
| Recombinant DNA reagent | AAV5-EF1a-DIO-eYFP (1.8x1013 vg/ml) | Washington University Hope Center Viral Vector Core | N/A | |
| Recombinant DNA reagent | AAV2 retro-Ef1a-DIO-mCherry 4.0×1012 vg/ml | University of Carolina Vector Core | N/A | |
| Chemical compound, drug | 4% paraformaldehyde | J.T. Baker, Avantor | S898-09 | |
| Chemical compound, drug | Goat Serum | Sigma Aldrich | G9023 | |
| Software, algorithm | Bonsai | https://bonsai-rx.org/ | N/A | |
| Software, algorithm | Ethovision | Noldus | N/A | |
| Software, algorithm | GraphPad Prism | GraphPad Software | N/A | |
| Software, algorithm | Labgym | https://github.com/umyelab/LabGym; Hu et al., 2023; Satpathy et al., 2024 | N/A | |
| Software, algorithm | DeepLabCut | https://github.com/DeepLabCut/DeepLabCut; Mathis et al., 2018; Nath et al., 2019; DeepLabCut, 2024 | N/A | Version 2.2 |
| Other | Vectashield Hardset Antifade Mounting Medium with DAPI | Vector Laboratories | Cat# NC9029229 | Mounting medium with DAPI for fluorescence microscopy. |
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For further information regarding data, reagents and resources, contact Aaron Norris, norrisa@wustl.edu.
Additional files
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Supplementary file 1
Statistical analyses and values.
Statistical analyses conducted for all figures including the type of analysis(es), p values, statistic (i.e. F, t statistic), and/or multiple comparisons.
- https://cdn.elifesciences.org/articles/90972/elife-90972-supp2-v1.docx
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MDAR checklist
- https://cdn.elifesciences.org/articles/90972/elife-90972-mdarchecklist1-v1.docx
