Glutamatergic supramammillary nucleus neurons respond to threatening stressors and promote active coping
Figures
![](https://iiif.elifesciences.org/lax:90972%2Felife-90972-fig1-v1.tif/full/617,/0/default.jpg)
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 + SuM neurons labeled with eYFP seen in the POA. (C) Cell bodies labeled with eYFP in the SuM. (D–L) Arborizing processes from the SuMVGLUT2+::POA neurons are seen in multiple brain regions including the (D) AcbSh and AcbC; (E) lateral septum; (F) multiple hypothalamic areas; (G) lHb and PVT; (H) the PAG and the DRD; (I) VLPAG and LPB; (J) DRI and LDTg; (K) and DRI, and MVePC. (M) Light sheet microscopy image of a cleared mouse brain hemisphere, viewed from medial to lateral, showing eYFP labeled neurons in SuMVGLUT2+::POA with cell bodies in SuM and processes in areas corresponding to septum, hippocampus, Acb, and POA. (500 μm scale bars). Abbreviations- MPA- medial preotic, VLPO- ventral lateral preoptic, LPO- lateral preotic, HDB- nucleus of the horizontal limb of the diagonal band AcbSh- Accumbens shell, AcbC- Accumbens core, Shi- septohippocampal nucleus, LSI- lateral septal nucleus, intermediate part, LSV- lateral septal nucleus, ventral part, LV- lateral ventricle, MS- medial septal nucleus, lHb- lateral habenula, mHb- medial habenula, PVT- paraventricular thalamus, DM- dorsomedial hypothalamic nucleus, LH- lateral hypothalamic area, VMH- ventromedial hypothalamic nucleus, PAG- periaqueductal gray, DMPAG- dorsomedial periaqueductal gray, DLPAG- dorsolateral periaqueductal gray, LDTg- laterodorsal tegmental nucleus, LPAG- lateral periaqueductal gray, DRD- dorsal raphe nucleus, dorsal part, LPB- lateral parabrachial nucleus, MPB- medial parabrachial, scp- superior cerebellar peduncle, Bar- Barrington’s nucleus, DTg- dorsal tegmental nucleus, DRI- dorsal raphe, interfascicular part, LC- locus coeruleus, MVe- medial vestibular nucleus, 4V- fourth ventricle.
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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 dense labeling in LPO and the site of viral injection in SuM (500 µm scale bar). (D) The brain regions, POA and SuM, shown in (B, C, E and F). (E and F) Cell bodies of VGLUT2 +SuM neurons are evident in SuM with injection of Retro-AAVs in POA. (G) Schematic of injection in POA of fluorophore switching construct in Retro-AAAV in VGLUT2-Cre or VGAT-Cre mice. (H and I) Labeling of Cre- (red) and Cre + neurons in POA but only Cre + seen in SuM of VLGUT2-Cre mice showing all SuM::POA neurons are VGLUT2+. (J) Quantification of labeling of Cre + and Cre- cells in SuM following injections in POA in VGLUT2-Cre and VGAT-Cre mice. (K and L) Labeling of Cre- (red) and Cre + neurons in POA but only Cre- seen in SuM of VGAT-Cre mice. (100 µm scale bar).
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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
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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 in labeling of cell bodies in (C) SuM. (E–F) Similarly, injection of Retro-AVV-DIO-tdTomato into (E) septum resulted in labeling of cell bodies in (F) SuM. (H–I) injection of Retro-AAV-DIO-eYFP targeting the (H) PV and lHb resulted in cell bodies in (I) SuM being labeled with eYFP. (K–L) Retro-AVV-DIO-tdTomato injected into (K) PAG-labeled cells in (L) SuM. (M) The cleared brain hemisphere from a VGLUT2-Flp mouse injected in the POA with injected Retro-AAV-DIO-eYFP shown in Figure 2M displayed from a perspective of ventral to dorsal shows cells labeled in SuM and processes extending widely, including in the hippocampus and POA. (100 and 500 μm scale bars).
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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 on expression of Cre mediated by POA injected retrograde AAV constructs showing SuM::POA neurons are a subset of all cells in SuM. (C and D) In wildtype mice injected in POA with Retro-AAV-fDIO-Cre and in SuM with AAV-DIO-ChR2eYFP, expression of ChR2eYFP (green) is not seen. (E–H) In Ai14 reporter mice injected with AAV-fDIO-Cre into SuM, tdTomato expression is not observed unless Retro-AAV2-Flp is injected. (100 μm scale bars).
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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 +neurons were also present in mHb, lHb, and PVT. (E) In hippocampus, processes were observed in DG and CA2. (F) Schematic of injections in VGLUT2-Flp mice of Retro-AAV-fDIO-Cre and AAV-DIO-ChR2eYFP into SuM. (G–H) Cells in SuM and processes in POA were labeled by ChR2eYFP. (I) Labeled processes in lHb were evident but none seen in mHb. (J) In hippocampus, labeled processes were present in CA2 but not observed in DG. (500 μm scale bars) (K) Schematic summary showing the projection to DG and mHb present in the total SuM VGLUT2 +population (outlined arrows) but absent in the SuMVGLUT2+::POA population (filled arrows).
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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. (C) While mice were connected for fiber photometry, they were subjected to ten 30 s trials of forced swimming, (D) to a 2 s foot shock following a 30 s tone for five trials, or (E) to ambush by a mock predator via remote-controlled spider. (F) Heat map and mean ± 95% CI Z-score for recordings obtained from a single animal during the repeated forced swim showing increase Ca2+ signal during the swim session. (G) The mean ± 95% CI Z-score of 10 trials for all animals (n=8) in the dunk assay. (H) Heat map and mean ± 95% CI Z-score for recordings from a single animal during the five shock trials showing increase Ca2+ signal. (I) Mean ± 95% CI Z-score for recordings of five trials for all animals (n=6) in the foot shock assay. (J) Heat map, mean ± 95% CI Z-score (blue), mean ± 95% CI velocity (gold), for recordings obtained from animals (n=9) during the ambush showing a significant increase (***99.9% CI) in Ca2+ signal as the animals flee from the remote-controlled spider with the mean ± 95% confidence interval Z- score of 1 trial (ambush) for all 9 animals. (K) Heat map, mean (±95% CI) Z-score (blue), mean (±95% CI) velocity (gold), for the same mice but ambush in the predator assay. Time frame gated for spontaneous locomotion. Ca2+ signal does not increase significantly during spontaneous locomotion. Mean peak velocity was not significantly different. For Ca2+ signal differences: *=95% CI, **=99% CI, ***=99.9% CI, ns = not significant.
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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
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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) (C) a significant (p=0.029) increase in the mean ± SEM number of anti-Fos labeled neurons in brains from mice subjected to force swim (n=4) compared to control mice (n=4). (D) In VLGUT2-Cre mice, Retro-AAV-DIO-mCherry was injected in the POA of mice (n=5) subjected to 15 min forced swim. (E) Region of SuM shown with higher magnification in (F) where SuMVGLUT2+::POA neurons labeled by mCherry show overlap with cells (arrows) labeled by anti-Fos. (Scale bar 100 μm) (G) A significant increase (p=0.016) in the number of VGLUT2 + cells labeled with mCherry and anti-Fos is seen in mice subjected to forced swim. (H) Mice subjected to ten 30 s trials of ‘dunk’ forced swimming. (I) Quantitation of average time mobile for every 30-s trial during ‘dunk’ forced swim.
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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
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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 maps of Ca2+-dependent signal were analyzed during average peak velocity, showing no change in activity during peak velocity. Representative heat map for a single animal with the mean ± 95% confidence interval of the 12 trials for same animal. (C) Mean ± 95% confidence interval for Z-score of 12 trials for all 13 animals and Boxplot for average Z-score. (D) Cross-correlation analysis shows no correlation between Ca2+ signal from SuMVGLUT2+::POA neurons and spontaneous velocity increases.
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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
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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 epoch. (B) For Cre+ (n=16) and Cre- (n=16) mice, the behavior in 10 s bins was scored based on the predominant behavior displayed during each 10 s period for the 15-min trial into grooming, stationary, walking, chewing, rearing, rapid locomotion, digging (moving bedding toward the tail), treading (move beading forward), and jumping. The graphic shows a by-animal scoring of the 15-min trial color-coded for each of the behavior categories. (C) During the stimulation period, Cre+ (n=15) mice show significantly (*p=0.012) greater jumps than Cre- (n=16) mice, and jumping behaviors were not significantly different during pre and post stim periods. (D) Behavior was also scored for time spent moving beading (digging or treading). Cre +mice showed significantly (**p=0.004) greater time engaging in digging/treading behaviors during the stimulation period, and significantly (*p=0.014) less time during the post-stimulation period compared to Cre- mice. (E) Behavior was also scored for time spent engaging in grooming behaviors, and Cre +mice spent significantly (*p=0.016) less time grooming during the stimulation period and significantly (**p=0.005) more time grooming during the post stimulation period compared to Cre- mice. All data plotted as mean ± SEM.
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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
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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 photostimulation in two-sided arena with photostimulation associated with one side. (C) Quantification of time spent on stimulation side for Cre+ (n=19) and control Cre- (n=21) mice showing frequency-dependent increase in avoidance of the stimulation side of the area (***p<0.001, ****p<0.0001). (D) Mean ± SEM entrances per min (in 1 min bins) to the during 10 Hz stimulation trials were not significantly (p=0.87) different between Cre+ (n=14) and Cre- (n=13) mice. (E) Mean time spent on the stimulation side for Cre- and Cre +in 1 min bins during 10 Hz stimulation trials was significantly (p<0.0001) lower in Cre +mice. (F) (Inset) Diagram of light/dark arena with photostimulation provided on the dark side of the arena and quantification of time spent on dark (stimulation) side, demonstrating that Cre- (n=12) animals show a baseline preference for the dark side of arena that is overcome by photostimulation with Cre+ (n=13) mice spending significantly (**p=0.002, ***p<0.001, ****p<0.0001) less time on the stimulation side. (G) Representative heat map of Cre- and Cre +during 10 Hz photostimulation in open field test. (H) In the open field test, time spent in center and perimeter were not significantly different (p=0.19) between Cre+ (n=18) and Cre- (n=17) during 10 Hz photostimulation. (I) Distance traveled during open field testing was significantly (p<0.001) increased in Cre +compared to Cre- mice. (J) Schematic of real time light/dark choice testing with stimulation provided at 10 Hz throughout the arena. (K) Both Cre+ (n=8) and Cre- (n=9) mice showed a preference for the dark portion of the arena but were not significantly (p=0.9) different. (L) The total distance traveled by Cre +mice were significantly (p=0.003) greater than Cre- mice. All data plotted as mean ± SEM.
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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
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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 arena. (D) Photostimulation at 10 Hz did not drive a significant place preference (p=0.67). (eYFP (control) n=26 and ChR2=26). All data plotted as mean ± SEM. (E) Representative path traces of VGAT-Cre mice with ChR2 or eYFP (control) in open field testing. (F) No significant differences in center (p=0.254) or perimeter (p=0.287) time when comparing the ChR2eYFP expressing to controls VGAT-Cre mice. (G) Chr2eYFP expressing mice traveled slightly but significantly (p=0.034) less distance compered to control. All data plotted as mean ± SEM.
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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
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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 paradigm and set up. 10 Hz photostimulation was applied during the trials. Activation of the active port triggered the house light and paused stimulation for 10 s. Also shown is the progressive ratio used with number of required port activations per reward on vertical axis and reward number on the horizontal. (C) Cre +mice (n=11) activated the active port triggering significantly (****p<0.0001) more pauses in stimulation than Cre- mice (n=11) mice on all 4 days of testing. (D) Cre +mice activated the port significantly (****p<0.0001, ***p<0.001) more than the Cre- mice on all four trials. (E) The time to first activation of the active port was significantly (*p=0.046) different during the first trial, and Cre +mice activated the active port significantly (**p<0.01, **p<0.01, **p=0.034) faster on subsequent trials. (F) On the fifth day after four 10-min trials, mice were tested for 30 min using a progressive ratio. Cre +performed significantly (****p<0.001) more active port activations than Cre- mice and activated the inactive port significantly more (**p=0.001) times. Cre +mice also triggered significantly (****p<0.001) more pauses in photostimulation than Cre- mice. (G) Individual data for a representative (n=7) cohort of Cre +mice showing cumulative active port activations as a function of time during the progressive ratio test show on-going engagement of the active port throughout the 30 min trial. (H) The cumulative pauses in photostimulation (rewards) earned as a function of time during the progressive ratio trial are shown for individual Cre +mice. Mice earned between 7 and 11 pause rewards during the trial. All data plotted as mean ± SEM.
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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
![](https://iiif.elifesciences.org/lax:90972%2Felife-90972-fig6-figsupp1-v1.tif/full/617,/0/default.jpg)
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 inactive ports. All data plotted as mean ± SEM.
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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
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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, and immobility were completed using deep learning-based classification and quantification (LabGym) of recorded behavior. Inset shows injection, implants, and behavioral assay. Box highlights the time frame shown in B. (B) Heat maps for recordings obtained from mice (n=9) during forced swim, averaged color-coded behaviors, and mean ± SEM Z-score of Ca2+-dependent signal for 2-min period around behavioral shift from climbing and swimming to hindpaw swimming and immobility, showing significant (*95% CI) decline in the Ca2+-dependent signal. (C) Representative heat map, averaged behavior, and mean ± SEM Z-score of Ca2+-dependent signal for 20 s window around onset of hindpaw swim from a representative animal. (D) Representative heat map and mean ± SEM Z-score of Ca2+-dependent signal for 20 s window around onset of hindpaw swim from for events from nine mice. Engaging in hindpaw swim correlates with a brief, significant (***99.9% CI) increase in Ca2+ signal. (E) Representative heat map and mean ± SEM Z-score of Ca2+-dependent signal for 20 s window around random time points. (F) Schematic of injection and implant in VGLUT2-Cre or VGAT-Cre mice. (G) Illustration of testing paradigm on second forced swim test following 15 min swim on first day. (H) The average time spent immobile during the pre-stimulation period was not significantly (p>0.999) different. During 10 Hz photostimulation VGLUT2-Cre+mice engaged in vigorous swimming and the time spent immobile was significantly (****=p < 0.001) less than Cre- mice. In the post-stimulation period, the time spent immobile remained significantly (*=0.047) lower in Cre +mice compared to Cre-. (I) In VGAT-Cre + mice expressing ChR2 (n=11), the average time spent immobile during the pre-stimulation period was not significantly (p=0.218) different compared to eYFP controls. Photostimulation did not significantly increase time immobile (p>0.999). However, there was a significant (**p=0.001) decrease in the time spent immobile compared to the post-stimulation period. For Ca2+ signal differences: *=95% CI, **=99% CI, ***=99.9% CI, ns = not significant. All data plotted as mean ± SEM unless otherwise noted.
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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
![](https://iiif.elifesciences.org/lax:90972%2Felife-90972-fig8-v1.tif/full/617,/0/default.jpg)
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 24 hr prior to testing. After being placed in the arena and allowed to habituate, mice were given a 20 min trial divided into 5 min of baseline and 15 min after a chow pellet was added to the arena. (C) Food deprived mice (n=9) spent significantly (p<0.001) more time interacting with the food pellet and (D) ate significantly (p<0.001) more than in the fed state. (E) Heat map and mean ± SEM Z-scores for Ca2+-dependent and isosbestic signals for recordings obtained from 9 animals show a small drop in (E) fed mice state (*95% CI) compared to a sharp decrease in Ca2+ activity after the introduction of the food pellet following addition in (F) food deprivation (***99.9% CI). (G) Mice were fasted for 24 hr prior to testing, and the schematized 20 min paradigm was used. Following food deprivation, animals were given access to a chow pellet. The trial was divided into 5 min pre- and post-stimulation periods, with a 10 min period of stimulation. (H) The time spent interacting with the food was quantified for each period. Cre+ (n=16) and Cre- (n=17) mice both rapidly engaged with the food pellet, and the average time spent interacting was not significantly (p=0.47) different. During the stimulation period and the post-stimulation period, the average time interacting with the food was significantly (p=0.0006, p=0.0002) lower in Cre +mice compared to Cre- mice. (I) The food eaten during the total trial was calculated based on pellet weights, and, on average, Cre+ (n=16) mice ate significantly (p=0.001) less food during the trial than Cre- mice (n=17). (**p=0.001, ***p<0.001, ****p<0.0001). For Ca2+ signal differences: *=95% CI, **=99% CI, ***=99.9% CI, ns = not significant. All data plotted as mean ± SEM unless otherwise noted.
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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
![](https://iiif.elifesciences.org/lax:90972%2Felife-90972-fig9-v1.tif/full/617,/0/default.jpg)
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 threating stressors including foot shock. Activation of SuMVGLUT2+::POA neurons is sufficient to evoke active coping-like behaviors in the absence of threating stressors, convert passive coping strategies to active coping strategies, drive aversion, and evoke performance of an operant task under a negative reinforcement paradigm.
Videos
Predator ambush.
Example of a mouse fleeing after being ambushed by a remote-controlled mechanical spider hidden in an enclosure.
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.
Example of photostimulation of SuMVGLUT2+::POA neurons driving aversion.
Mice avoid the photostimulation side with higher frequencies showing greater aversion.
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.
Photostimulation of SuMVGLUT2+::POA neurons during forced swim test elicits active coping behaviors.
Tables
Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
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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