SuMVGLUT2+::POA arborize widely in the brain.

(A) Schematic of injections of Retro-AAV-DIO-eYFP in to 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 neurons 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

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) Process from SuM VGLUT2+ neurons were also present in MHb, LHb and PVT. (E) In hippocampus process 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 process 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).

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 ced over SuM for fiber photometry recordings from SuMVGLUT2+::POA neurons. (C) While mice were connected for fiber photometry they were (C) subjected to ten 30-second trials of forced swimming, (D) to a 2-second foot shock following a 30 second tone for five trials, or (E) to mock predator ambushed by remote-controlled spider. (F) Heat map and mean ± 95% CI Z-score for recordings obtained from 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 an ± 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 an increase in Ca2+ signal as the animals flee from the remote-controlled spider with the mean ±95% confidence interval Z-score of 1 trial bush) 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 during spontaneous locomotion. Mean peak velocity was not significantly different.

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 mins post. Behaviors was evaluated during each epoch. (B) For Cre+ (n=16) and Cre- (n=16) mice the behavior in 10 seconds bins was scored based on the predominate behavior displayed during each 10 period for the 15-minute trial into grooming, stationary, walking, chewing, rearing, rapid locomotion, digging (moving bedding towards the tail), treading (move beading forward), and jumping. The graphic show a by animal scoring of the 15-minute trial color coded for each of the behavior categories. (C) During the stimulation period Cre+ (n=15) mice show significantly (p=0.0012) greater jumps than Cre- (n=16) mice and jumping behaviors was 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=0.002) 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<0.001) less time grooming during the stimulation period and significantly (p<0.001) more during the post stimulation period compared to Cre- mice. All data plotted as mean ±SEM

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 10Hz photostimulation in two-sided arena with photostimulation associated with one side. (C) Quantification of time spent of 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-minute bins during 10 Hz stimulation trials was significantly (p<0.0001) lower in Cre+ mice. (F) (Inset) Diagram of light/dark arena with photo stimulation 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 10Hz photostimulation in open field test. (H) In 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 10Hz 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.

SuMVGLUT2+::POA neurons drive can drive instrumental actionoutcome operant behavior

(A) Schematic of injection and implant in VGLUT2+ Cre mice and paradigm of testing in 10-minute 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 trigger the house light and paused stimulation for 10 seconds. 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 Cremice (n=11) mice on all 4 days of testing. (D) Cre+ mice activated the port significantly (****p<0.0001, ***p<0.001) than the Cre- mice on all 4 trials. Cre+ activated the active port significantly (p<0.001) on during the fourth trial than the first. (E) The time to first activation of the active port was not significantly (p=0.32) different during the first trial but Cre+ mice became significantly (**p<0.01, ***p<0.001) on subsequent trials. (F) On the fifth day after four 10-minute trials mice were tested for 30 minutes using a progressive ratio. Cre+ performed significantly (p<0.0001) more active port activations that Cre- mice and activated the inactive port significantly more (p<0.005) times. Cre+ mice also triggered significantly (p<0.001) more pauses in photostimulation that Cre- mice. (G) Individual data for a representative cohort of Cre+ mice showing cumulative active port activations by Cre+ mice (n=7) as function of time during the progressive ratio test are shown illustrated ongoing engagement of the active port throughout the 30-minute trial. (H) The cumulative pauses in photostimulation (rewards) earned as a function of time during the progressive ratio trial are show for individual Cre+ mice. Mice earned between 7 and 11 pause reward during the trial. All data plotted as means ±SEM.

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 was completed using deep learning based classification and quantification (LabGym) of recorded behavior. Inset shows injection and implants and behavioral assay. Box highlights the time frame shown in B. (B) Heat maps for recordings obtained 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. (C) Representative heat map, averaged behavior, and mean ±SEM Z-score of Ca2+ dependent signal for 20-second 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-second window around onset of hindpaw swim from for events from nine mice. Engaging in hindpaw swim correlates with brief increase in Ca2+ signal. (E) Representative heat map and mean ±SEM Z-score of Ca2+ dependent signal for 20-second 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 of forced swim test following 15-minute swim on first day. (H) The average time spent immobile during the pre-stimulation period was not significantly (p=0.48) 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 (*=0.016) lower in Cre+ mice compared to Cre-. (I) In VGAT-Cre+ mice expressing ChR2 (n=10), the average time spent immobile during the pre-stimulation period was not significantly (p=0.08) different compered to eYFP controls. Photostimulation did not significantly increase time immobile (p=0.64). However, there was a significant (*** p=0.0004) decrease in the time spent immobile compared in the post-stimulation period.

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 hours prior to testing. After being placed in arena and allowed to habituate mice were given a 20-minute trial divided into 5 mins baseline and 15 mins 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 compered to a sharp decrease in Ca2+ activity after the introduction of the food pellet following addition in (F) food deprivation. (G) Mice were fasted for 24 hours prior to testing and the schematize 20 min paradigm was used. Following food deprivation animals were given access a chow pellet. The trial was divided in to a 5-minute pre- and post-stimulation periods with 10-minute 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=0.37) different. During the stimulation period and the post stimulation period the average time interacting with the food was significantly (p=0.0002, p=0.0001) 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+ mice ate significantly (p=0.001) less food during the trial than Cre- mice. (**p=0.001, ***p<0.001, ****p<0.0001)

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 and evident in the POA with dense labeling in LPO and the site of viral injection in SuM (500um 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. (100um scale bar)

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 show in Figure 2M displayed from a perspective of ventral to dorsal showing cells labeled in SuM and processes extending widely including in the hippocampus and POA. (100 and 500 μm scale bars)

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 positive and mCherry (red) Cre negative 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)

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 minutes of forced swimming 90 min prior to sacrifice were probed with anti-Fos antibodies (cyan) showed (scale bars 500μm) (C) a significant (p= 0.029) increase in them mean ±SEM number of anti-Fos labeled neurons in brains from mice subjected to force swim (n=5) 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 minutes 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 100μm) (G) A significant increase (p= 0.016) in the number of VGLUT2+ cells labeled with mCherry and anti-Fos in mice subjected to forced swim. (H) Mice subjected to 10 30-second trials of “dunk” forced swimming. (I) Quantitation of average time mobile for every 30-second trial during “dunk” forced swim.

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 map of Ca2+ dependent signal was 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 spontaneous velocity increases.

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 10Hz did not drive a significant place preference. (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 or perimeter 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.