Introduction

Threat-response neural circuits are conserved across species and have roles in normal behaviors and psychiatric diseases[1–4]. Identifying and responding to threatening stressors is critical for survival, but maladaptive changes in underlying neural circuits can contribute to stress, mood, and anxiety disorders[5, 6]. Active coping in response to stressors is a psychosocial factor associated with resilience against stress induced mood and anxiety disorders [7]. Available evidence indicates that active (e.g., escape, fighting) and passive (e.g., freezing, immobility) coping responses to stressors are governed by separable neural circuits[1, 8, 9]. The functioning of circuits underlying active coping could be key for overcoming anxiety and related disorders[10, 11].The neural circuits, cells, and mechanisms underlying active coping strategies remain unclear[12, 13].

The supramammillary nucleus (SuM) has been suggested to be engaged by threatening stressors and has efferent connections to stress-sensitive brain regions so may be an important regulator of responses to stressors[14–16]. Research on SuM has focused on connections to the hippocampus and septum while SuM projections to other brain areas remain less understood[15, 17, 18]. SuM contains distinct populations with functionally diverse roles including regulation of hippocampal activity during REM, spatial memory, arousal, and environmental interactions[19–23]. In addition to functional diversity, anatomical and molecular-cellular diversity is present in SuM[24–26]. Divergent populations in SuM have been defined by differential to the dentate gyrus and CA2 regions of the hippocampus, and neurochemically based on neurotransmitter expression[27, 28]. Major projections from the SuM, which have yet to be examined, include the preoptic hypothalamus area (POA). We examined if SuM neurons projected to the POA and if this projection could be used to aid in separation of populations in SuM.

We used retrograde adeno associated viral (AAV) and combinatorial genetic tools to identify and characterize a population of glutamatergic SuM neurons with projections to the POA (SuM^VGLUT2+::POA). We found that SuM^VGLUT2+::POA neurons represented an anatomical subset of SuM neurons with extensive arborizations to brain regions including those that mediate stress and threat responses including the paraventricular nucleus (PVT), periaqueductal gray (PAG), and the habenula (Hb). Thus, SuM^VGLUT2+::POA neurons are positioned as hubs with spokes to many areas regulating responses to threatening stressors. We hypothesized this population could respond to and regulate responses to stressors. We found SuM^VGLUT2+::POA neurons are recruited by multiple types of acute threatening stressors, encode a negative valance, do not promote anxiety-like behaviors, and evoke active coping behaviors. Further, activation of these SuM^VGLUT2+::POA neurons was sufficient to convert passive coping strategies to active behaviors. These findings indicate SuM^VGLUT2+::POA neurons are a central hub linked to multiple stress and threat responsive areas and can drive state transitions between passive and active responses to stress.

Results

VGLUT2+ SuM neurons projecting to the POA (SuM^VGLUT2+::POA) arborize to multiple stress-engaged brain regions

The SuM contains functionally diverse and anatomically distinct populations with efferent projections to many brain regions[16, 24, 29, 30]. The diversity includes glutamatergic, GABAergic, and co-expressing GABAergic/Glutamatergic populations[21, 31]. Using retrograde adeno associated virus (Retro-AAV) (Retro-AAV2-DIO-tdTomato) and anterograde (AAV5-DIO-ChR2eYFP) tracing we identified a population of VGLUT2+ expressing neurons in SuM that project to the POA (SuM^VGLUT2+::POA) with dense projections in the lateral preoptic area (LPO) within the POA (Supplemental Figure 1A-F) in VGLUT2-Cre mice (n=4). Using a viral construct encoding for a nuclear restricted fluorophore that switches from mCherry (red) to eGFP (green) in a Cre- dependent manner (Retro-AAV2-Nuc-flox(mCherry)-eGFP), we found SuM^VGLUT2+::POA neurons were positive for VGLUT2+ in VGLUT2-Cre mice (n=4) and negative for VGAT expression in VGAT-Cre mice (n=3) (Supplemental Figure 1G-L) indicating that SuM^VGLUT2::POA neurons do not belong to a GABAergic/Glutamatergic populations.

To examine arborization of SuM^VGLUT2+::POA neurons we utilized a combinatorial genetic approach. Specifically, we used mice (VGLUT2-Flp) that express Flp recombinase in VGLUT2 expressing cells in a combination with Flp dependent expression (fDIO) of Cre and Cre (DIO) dependent fluorophore expression. We injected Retro-AAV2-DIO-eYFP into POA and AAV-fDIO-Cre in to SuM (Figure 1A). We thus expressed Cre in SuM^VGLUT2+ neurons and, of those, only neurons projecting to the POA (SuM^VGLUT2::POA neurons) were labeled with eYFP. We found SuM^VGLUT2::POA neurons arborize widely, projecting to multiple brain regions including: the nucleus accumbens (Acb), Septum, lateral hypothalamus (LH), ventral medial hypothalamus (VMH), paraventricular nucleus (PVT), lateral habenula (lHb), the ventral lateral periaqueductal gray (VLPAG), dorsal raphe (DRD), lateral parabrachial nucleus (LPBN), laterodorsal tegmental nucleus (LDTg), and medial vestibular nucleus (MVe) (Figure 1 D-M). We visualized arborization of SuM^VGLUT2::POA neurons in cleared tissue by using optical light sheet imaging. For these experiments we injected VGLUT2-Flp mice (n=3) as in Figure 1A and brains were actively cleared using SHIELD for optical light sheet imaging[32]. Compiled three-dimensional images of a brain hemisphere, viewed from the medial to lateral perspective (Figure 1M) or viewed from the ventral to dorsal perspective (Supplemental Figure 2M), showed projections labeled by eYFP from SuM^VGLUT2+::POA to POA, areas of hippocampus, septum, Acb, and regions in the pons and midbrain. The results further established projections from SuM^VGLUT2+::POA neurons to multiple brain areas and illustrate the broad arborization.

Figure 1.

We used tracing with Retro-AAV’s to further corroborate our findings from anterograde tracing by injecting unilaterally into the Acb, septum, PVT, or PAG in VGLUT2-Cre mice. Cell bodies in SuM were labeled eYFP or tdTomato (Supplemental Figure 2A-L). Each area was injected in three or more mice, yielding similar results. We examined the anatomic distribution of SuM::POA neurons in SuM by injecting Retro-AAV2-Cre into the POA and AAV5-Nuc-flox(mCherry)-eGFP into the SuM (Supplemental Figure 3 A-B) of wildtype (WT) Cre- mice (n=3) In mice injected with this combination of viruses, we observed neurons labeled by mCherry (Cre negative) or eGFP (Cre expressing) interspersed in the SuM. We verified the combinatorial viral selectively labeled SuM^VGLUT2+::POA neurons with minimal background[33]. As positive controls, we injected Retro-AVV-Flpo into the POA and AAV-fDIO-Cre into the SuM to label the cells in SuM (Supplement Figure 3 G and H). As negative controls, we injected only AVV-fDIO-Cre into the SuM in Ai14 mice, which did not yield tdTomato expression (Supplemental Figure 3C-D). To confirm that the combination of Retro-AAV2-fDIO-Cres and AAV5-DIO-ChR2eYFP did not lead to labeling of cells with eYFP in the absence of Flp, we injected Retro-AAV2-fDIO-Cre into the POA and AAV-DIO-hChR2eYFP into SuM of WT mice. In these mice we did not observe any expression of eYFP (Supplemental Figure 3E-F). The results demonstrate the specificity of our combinatorial viral strategy. All studies were replicated in a minimum of three mice.

SuM^VGLUT2+::POA neurons are an anatomical distinct subset of all SuM^VGLUT2+ neurons

Recent studies have highlighted functionally divergent roles of SuM neurons and suggested differential projections, particularly to regions of hippocampus and PVT, may identify functionally distinct populations [19, 20]. To examine if SuM^VGLUT2+::POA neurons are a subset of SuM^VGLUT2+ neurons, we qualitatively compared the projections of total SuM^VGLUT2+ neurons to SuM^VGLUT2+::POA neurons. Similar to projections for SuM^VGLUT2+::POA neurons (Figure 1), we observed labeled projections from the total SuM^VGLUT2+ neurons in the POA, PVT, lHB, and the CA2 field of the hippocampus (Figure 2 A-E). Importantly, we found areas that received projections from the total SuM^VGLUT2+ and not from SuM^VGLUT2+::POA neurons. As schematized (Figure 2K), projections from the total SuM^VGLUT2+ but not SuM^VGLUT2+::POA populations were present in dentate gyrus (DG) and medial habenula (mHb) (Figure 1G and Figure 2 F-J). Connection of the SuM to the dentate gyrus is well described and seen here for the SuM^VGLUT2+ population[34–38]. The lack of projections from SuM^VGLUT2+::POA neurons to dentate gyrus, but not to other structures is a notable difference (Figure 2J). Thus, the SuM^VGLUT2+::POA population represents a subset of the total SuM^VGLUT2+ neuronal population with distinct projection targets.

Figure 2.

Threatening stressors but not spontaneous higher velocity movement recruits SuM^VGLUT2+::POA neurons

Neurons in SuM can be activated by acute stressors, and we sought to examine the recruitment of SuM^VGLUT2+::POA neurons by threatening stressors [14, 39, 40]. We found that forced swimming induced cFos expression in SuM. Labeling SuM^VGLUT2+::POA neurons using Retro-AAV-DIO-mCherry revealed an increase in the number of mCherry and cFos labeled cells following forced swim (Supplemental Figure 4A-G). To further examine recruitment of SuM^VGLUT2+::POA neurons by acute stressors, we tested a diverse set of acute threatening stressors using in vivo Ca²⁺ detection via fiber photometry. We expressed GCaMP7s in SuM^VGLUT2+::POA neurons (Figure 3A-B) using the combinatorial viral genetic approach detailed for anatomic studies. We developed a dunk assay that utilized a moveable platform allowing mice to be placed into and removed from the water while obtaining fiber photometry recordings. Mice were dunked by lowering the platform below the water level forcing mice to swim for a 30-second trial every 2 minutes for a total of 10 trials (n=8 mice). During the 30-second swim time, mice exhibited active swimming and climbing behaviors reflected in the quantification of mean time mobile approaching 100 percent during the swim period without evidence for a shift in the behavioral strategy (Supplemental Figure 4I). These data show repeated exposure to an acute stressor, dunk in water, evoked active coping behavior (time mobile). A 95%, 99%, and 99.9% confidence interval (CI) were calculated to analyze the difference in the Ca2+-dependent signal preceding and subsequently after the dunk. Analysis of Ca²⁺dependent (470 nm excitation) and isosbestic control (415 nm excitation) signals from fiber photometry recordings revealed a significant (99.9% CI) rapid rise of approximately 5 standard deviations in GCaMP Ca²⁺ dependent signal with start of the swim trial that was sustained through the 30-sec swim period. The Ca²⁺dependent signal returned to baseline when the platform was raised, removing the animal from the water (Figure 3F and G). Repeated exposure to an acute stressor evoking active coping behaviors robustly recruited SuM^VGLUT2+::POA neurons.

Figure 3.

To examine additional stressors, we tested a foot shock paradigm (Figure 3D) and a predator ambush assay (Figure 3E). In foot shock assays, VGLUT2-Flp mice (n=6), prepared as above (Figure 3A and B), were subjected to five pseudorandomly spaced trials with a 30 second tone preceding a two-second shock. We observed a significant rapid increase in the Ca²⁺ dependent GCaMP7s signal following the foot shock (99.9% CI; Figure 3H and I). For the predator ambush, we adapted a previously demonstrated paradigm using a mock mechanical spider attack [41]. In this assay a remote-controlled mechanical spider was hidden in a box with a swing door. The box was inside a larger walled arena. Mice were able to freely explore the arena, and at a moment when the mouse was in proximity to the box opening, the spider was moved out towards the mouse. The mice fled, often to a corner, stopped, and turned to face the spider (Supplemental video 1). In mice (n=9) subjected to the ambush paradigm, we observed a significant (99.9% CI) increase in Ca²⁺ dependent GCaMP signal at the time of the ambush followed by suppression below the initial baseline (Figure 3J). Populations of neurons in SuM have previously been found to correlate with future movement velocity [42], so we examined if recruitment of SuM^VGLUT2+::POA neurons was correlated to periods of spontaneous higher velocity movement. Analyzing data collected prior to the ambush event, we examined if increased velocity, at speeds similar to the fleeing induced by the ambush, was correlated with increased Ca²⁺ dependent GCaMP signal in SuM^VGLUT2+::POA neurons (Figure 3L). We found no correlation (Figure 3K). We further examined the data for a correlation of increased Ca²⁺ dependent signal in SuM^VGLUT2+::POA neurons to movement velocity during open field exploration in a sperate cohort of mice (n=13). Here, we found no evidence for correlation of velocity with Z-score of Ca²⁺ dependent signal including a cross correlation analysis to account for a potential temporal offset (Supplemental Figure 5). In aggregate, the data support recruitment of SuM^VGLUT2+::POA neurons by diverse threatening stressors but not during times of higher velocity spontaneous movement.

SuM^VGLUT2+::POA neurons evoke active coping-like behaviors

To examine how activation of SuM^VGLUT2+::POA neurons contributes to responding to threatening stressors, we assessed behavioral changes evoked by photostimulation of SuM^VGLUT2+::POA neurons. We injected VGLUT2-Cre or WT (Cre-littermates) mice with Retro-AAV5-DIO-ChR2eYFP in the POA, and an optic fiber was placed over SuM (Figure 4A). We employed a paradigm with a 15-minute trial divided into three 5-minute periods: pre-stimulation, stimulation at 10 Hz, and post-stimulation. Based on review of videos obtained during the 15-minute trials, we observed behaviors that could be classified into nine distinct categories: grooming, immobile, walking, chewing of bedding, rearing, rapid locomotion (movement was limited by the size of the arena), digging (moving bedding towards the tail), treading (moving bedding forward with front paws), and jumping (Figure 4B). A blinded observer scored the behaviors in 10-second intervals for the predominant behavior observed during each interval, and each behavior was assigned a color for visualization. Color coded representation of trials from 16 Cre+ (n=16) and WT (n=16) mice is illustrated (Figure 4B). The behavioral pattern in the pre-stimulation period is similar between the Cre+ and WT mice. During the stimulation period, a clear shift in behavior was evident in Cre+ mice. Photostimulation induced rearing, treading, digging, rapid locomotion, and jumping. During the post-stimulation period we observed a new pattern with Cre+ mice spending time immobile and grooming. An example of a Cre+ animal is shown in Supplemental video 2.

Figure 4.

We analyzed behavior, quantifying specific behaviors during each period. Quantification of jumping revealed that Cre+ mice (n=16) engaged in significantly (p=0.012) more jumps during the stimulation period compared to WT (n=16) mice (Figure 4C). During the pre- and post-stimulation periods, there was not a significant difference in the number of jumps (pre-stimulation = p>0.999 and post-stimulation = p>0.999). We also observed that during the stimulation period the Cre+ mice engaged in bouts treading/digging, vigorously moving bedding forwards and backwards, as previously described for defensive burying [43]. Defensive burying, characterized by moving bedding forward or backwards often in alternating pattern, is evoked by threatening and noxious stimuli, and is a described active stress coping strategy in rodents[43–46]. We conservatively quantified together the movement of bedding as treading/digging that may include spontaneous digging (Figure 4D) and found no significant (p=0.95) difference during the pre-stimulation period. In the stimulation period, there was a significant (p=0.004) increase in the time spent treading/digging, and, surprisingly, during the post-stimulation period time treading/digging was significantly (p=0.014) decreased in Cre+ mice. The variability in behaviors can be attributed to exclusion of one behavior by the other, with individual mice having variability in the predominant behavior displayed during the photostimulation period, but all mice shifted to increased active coping behaviors. The behaviors evoked by photostimulation fit with active coping behaviors seen in the context of stressors. Specifically, escape (jumping, rapid locomotion), defensive burying/treading (digging, pushing beading forward).

We also quantified grooming behaviors in each of the three periods (Figure 4E). During the pre-stimulation period, there was not a significant difference (p>0.999). During the stimulation period, the Cre+ mice did not engage in grooming, leading to a significant (p=0.016) decrease in time spent grooming compared to Cre- mice. In the post-stimulation period, Cre+ mice showed a significant (p=0.005) increase in time spent grooming compared to Cre- mice. A reasonable interpretation of the rise in grooming post stimulation is that photostimulation evoked a stressed-like state and cessation of photostimulation led to selfcare grooming, as seen following acute stressors [47, 48]. In summary, the analysis of behaviors elicited by photostimulation of SuM^VGLUT2+::POA neurons without conditioned cues or concomitant stressors demonstrates a dramatic shift in behavior to escape oriented (jumping, rapid locomotion) and threat response behavior, including rearing and defensive burying, during the photostimulation period. In contrast to freezing, the behaviors elicited by activation of SuM^VGLUT2+::POA neurons indicate that they may promote active coping strategies.

Photostimulation of SuM^VGLUT2+::POA neurons drives real-time avoidance

To examine whether activation of SuM^VGLUT2+::POA neurons may contribute to the aversive aspects of threatening stress, we carried out real-time place aversion testing (RTPA) by pairing one side of the chamber with photostimulation of SuM^VGLUT2+::POA (Figure 5A) neurons at multiple frequencies (1,5,10, and 20 Hz). Photostimulation of SuM^VGLUT2+::POA neurons produced significant aversion at all frequencies in Cre+ (n=19) mice compared to Cre- (n=21) littermate control mice, and higher stimulation frequencies evoked greater aversion (p<0.001; Figure 5B and C). Example of Cre+ mice with stimulation at 1, 5, and 10Hz as well as Cre- mice is in Supplemental Video 3. WT and Cre+ mice explored equivalently, with similar number of entries to the stim side (p= 0.41; Figure 5D). Cre+ mice quickly left the stimulation side, and the average time spent on the stimulation side of the area was significantly lower (p<0.001; Figure 5E). These data indicate, surprisingly, that photostimulation, although aversive, did not generate aversive pairing, as found for other brain areas [49]. To examine the relative aversiveness of SuM^VGLUT2+::POA photostimulation, we carried out RTPA experiments using an arena with a dark side and a bright side (Figure 5F inset). We paired photostimulation with the dark side. As expected, Cre- mice show a preference for the dark side of the arena, however, photostimulation of the SuM^VGLUT2+::POA neurons yielded significant aversion of Cre+ (n=13) compared to Cre- (n=12) (p<0.001; Figure 5F). Cre+ mice spent nearly the entire trial in the brightly lit side, demonstrating photostimulation of SuM^VGLUT2+::POA neurons drove avoidance sufficient to overcome a mildly aversive stimulus (bright light).

Figure 5.

Reports have described multiple populations of neurons in supramammillary including populations that project to the dentate gyrus of the hippocampus that release both GABA and glutamate. Our anatomic studies indicated that SuM^VGLUT2+::POA neurons do not project to the dentate gyrus (Figure 2) and are not GABAergic (Supplemental Figure 1G-L). Thus, SuM^VGLUT2+::POA neurons are not part of either a GABAergic or dual transmitter population. To examine if GABAergic neurons in SuM (SuM^VGAT+) can mediate real-time place aversion or preference, we injected VGAT-Cre mice with AAV to express ChR2 (n=26) or control (n=26) in SuM and carried out real-time place aversion testing. Photostimulation at 10 Hz yielded no significant aversion or preference compared to baseline (p=.838) (Supplemental Figure 6A-D). This is in contrast to the robust aversion caused by photostimulation of SuM^VGLUT2+::POA neurons.

Photoactivation of SuM^VGLUT2+::POA neurons does not cause anxiogenic-like behavior

Threat can induce anxiety-like, risk aversion behavioral states [50–52]. To test whether activity of SuM^VGLUT2+::POA neurons contributes to anxiety-like behaviors, we used two established assays for anxiety-like behavior: open field and light-dark exploration [53–56]. During open field testing, 10 Hz photostimulation was applied to SuM^VGLUT2+::POA neurons. We quantified the time spent in the perimeter (outer 50%) vs. the center (inner 50%) of the arena and found VGLUT2-Cre+ (n=18) and WT (n=17) mice did not significantly differ (center p=0.17 and perimeter p=0.19; Figure 5G and H). The total distance traveled was significantly (p<0.001) increased in in Cre+ mice compared to WT mice (Figure 5I). Increased distance traveled in some mice was not surprising, because prior testing (Figure 4) revealed the mice engaged in escape behaviors during photostimulation. We observed behaviors including jumping during open field testing as well. We also carried out open field testing using VGAT-Cre mice to selectively activate SuM^VGAT+ neurons and observed no significant effect on time in center (p=0.254) or perimeter (p=0.287) (Supplemental Figure 6F), but mice expressing ChR2eYFP displayed a small but significant (p=0.035) decrease in distance traveled (Supplemental Figure 6G). We tested if photostimulation of SuM^VGLUT2+::POA neurons would increase preference for the dark area, a potential sign of elevated anxiety (Figures 5J). We found that photostimulation of SuM^VGLUT2+::POA neurons did not significantly alter preference (p=0.68; Figure 5K) in VGLUT2-Cre+ mice (n=8) compared to WT (n=9). The activation of SuM^VGLUT2+::POA neurons evoked escape behaviors in open field testing, as seen in Figure 4; similarly, a significant increase in total distance was detected during light-dark exploration assay in Cre+ compared to WT (p=0.003; Figure 5L). The findings we obtained in open field and light-dark exploration testing do not support a role for SuM^VGLUT2+::POA neurons in driving anxiety-like behaviors.

SuM^VGLUT2+::POA neurons can drive instrumental action-outcome behavior

Current theoretical frameworks for examining threat responses divide behaviors into two broad categories: innate (fixed) and instrumental (action-outcome)[1, 57]. Additionally, stress responses can be divided into active vs passive actions. Behaviors in these separable categories are mediated by distinct neural circuits[1, 58]. For example, areas involved in responding to specific threatening stimuli (looming threat) drive an innate fixed behavior repertoire[10]. Photostimulation of SuM^VGLUT2+::POA neurons evoked multiple active-like behaviors in response to threatening or noxious stimuli (Figure 4). To test if SuM^VGLUT2+::POA neurons can promote flexible repertoires of behavior including instrumental tasks, as opposed to only innate behaviors (ex. fleeing), we used an operant negative reinforcement paradigm (Figure 6A-B). Because our results show photostimulation of SuM^VGLUT2+::POA neurons is aversive, photostimulation of SuM^VGLUT2+::POA neurons could be used as a negative reinforcer in a negative reinforcement paradigm. We employed a paradigm using two nose poke ports, one active and one inactive. SuM^VGLUT2+::POA neurons were photostimulated at 10 Hz during a 10-minute trial. Activation of the active port by a nose poke triggered a 10-second pause in photostimulation of SuM^VGLUT2+::POA neurons and turned on a house light for 10 s (Figure 6A and B). Following this 10-second pause, the house light switched off and photostimulation resumed until the subsequent activation of the active port. We tested animals for four consecutive days with the 10-minute trials without prior training. Importantly, performance of the task had to be completed during photostimulation.

Figure 6.

We hypothesized that if stimulation of SuM^VGLUT2+::POA evokes fixed innate defensive behaviors, or simply promotes high amounts of locomotion, then performance of an operant task (an action-outcome behavior) with active and inactive ports would be impaired because innate defensive behaviors would conflict with performance of the operant task. Importantly, results obtained can help differentiate general elevation in locomotor activity from directed coping behavior. Analyses of port activations during the 10-minute trials revealed that Cre+ mice (n =11) paused the photostimulation (reward) significantly more on all four days of testing compared to Cre- littermate (n=11) mice (Interaction_{Genotype x Day} p<0.001; Day 1 p<0.001; Day 2 p<0.001; Day 3 p<0.001; Day 4 p<0.001; Figure 6C). The number of 10-second pauses approached the maximum, 60 possible in a 10-min period. Cre+ mice engaged the active port significantly more than Cre- mice (p<0.001; Figure 6D) and this effect was consistent for every training session (Day 1 p<0.0001=; Day 2 p<0.0001; Day 3 p<0.0001; Day 4 p=0.0011). There was a slight difference in baseline performance of the task. The time to the first engagement of the active port was significantly different for Cre+ compared to Cre- mice during the first test trial (p=0.046). During the three subsequent tests, Cre+ mice were significantly faster to engage the active port after the start of the trial compared to Cre- mice (Day 2 p=0.009; Day 3 p=0.003; Day 4 p=0.034; Figure 6E). Cre+ mice engaged the active port significantly more than the inactive port on all test days (p<0.001; Supplemental Figure 7A). There was not a significant difference in Cre+ and Cre- mice for engagement of the inactive port on any day (Cre+ p=0.30; Cre- p=0.055; Supplemental Figure 7A-B).Furthermore, Cre+ mice were significantly faster in the second through fourth trials at engaging the active port compared the first trial (Day 1 vs Day 2 p=0.034; vs Day 3 p=0.021; vs Day 4 p=0.027; Figure 6E). Suggesting that mice became more proficient during subsequent trials.

To examine effort-related motivation, we employed a 30-minute test using a progressive ratio requiring an exponentially increasing number of active port activations to trigger a pause in the photostimulation on the fifth day of operant behavior testing (Figure 6B) [59]. During trials using a progressive ratio, Cre+ mice activated the active port and paused stimulation significantly (p<0.001) more times than Cre- mice (Figure 6F). Examination of individual cumulative active port activation data showed Cre+ mice continued to engage the active port throughout progressive ratio trials despite the increasing work required to generate each pause in photostimulation. No breakpoint was observed during the trial (Figure 6G and H). This finding indicates that activation of SuM^VGLUT2+::POA neurons remains salient and motivating through the tested period and that photostimulation of SuM^VGLUT2+::POA does not evoke behaviors precluding completion of the task. Examination of reward behavioral epochs for representative animals shows that for the lowest total number of rewards earned (green dots), the next pause required 20 activations of the active port, and for the highest (red dots), the next pause required 50 activations of the active port. Taken together, results from the instrumental reinforcement tasks demonstrate that activation of SuM^VGLUT2+::POA neurons does not solely evoke innate stereotyped behaviors and can drive active coping in the form of instrumental behaviors.

SuM^VGLUT2+::POA neurons are recruited during active coping behaviors during forced swim

The forced swim test is increasingly established as an assay of coping strategy with passive and active components, and we used forced swim testing in conjunction with fiber photometry to examine if SuM^VGLUT2+::POA neurons are recruited differentially during active (mobile) or passive (immobile) behaviors [60, 61]. During a first 15-mintute trial of forced swimming, mice shifted from active escape behaviors (wall climbing, robust swimming) to passive (immobile floating) behaviors. We carried out experiments using fiber photometry paired with automated computer-based behavior analysis [62]. The automated behavior scoring enabled high temporal resolution analysis of behaviors (Supplemental Video 4) and each frame of the video was coded for the classified behavior and plotted as a color-coded point. We observed four types of behavior during forced swimming: climbing, swimming, hindpaw swimming, and immobile floating, and we trained a model to identify each. Analysis of behavior showed that after the first minutes of the trial, mice shifted from swimming and climbing to immobile floating with intermittent swinging or hindpaw swimming. We analyzed fiber photometry recordings to examine whether recruitment of SuM^VGLUT2+::POA neurons changed with this shift to more passive behaviors. As shown in the representative trace (Figure 7A), the Ca²⁺-dependent signal, but not the isosbestic signal, markedly decreased at the time the shift in behavior was occurring. To combine behavioral data, we assigned numerical values, one through four, to each behavior: climbing (4), swimming (3), hindpaw swimming (2), and immobile floating (1). The mean was plotted as a heat map (red higher values to blue lower values) to be compared to time locked fiber photometry data (Figure 7B). Examining the timeframe around the behavioral shift in each animal, we found that as behavior shifted from active coping (climbing, swimming) to more passive strategies and greater immobility, the Ca²⁺ signal significantly (95% CI) declined (Figure 7B). Examining the data after this initial transition, we observed that many elevations in the Z-score were accompanied by changes in behavior to swimming or hindpaw swimming (Figure 7A). To further examine this possible correlation, we examined the time frame around transition to hindpaw swimming for changes in Ca²⁺ signal. In a 20-second window centered on the onset of hindpaw swimming, we found that hindpaw swimming was accompanied by a rise in the Ca²⁺ dependent signal across multiple events within a trial (Figure 7C). A similar analysis across events in multiple animals demonstrated a significant (99.9% CI) rise in the Ca²⁺ dependent signal from SuM^VGLUT2+::POA neurons during the shift from immobility to hindpaw swimming or swimming behaviors (Figure 7D). Time series analysis of 20-second epochs centered on random time intervals did not reveal any rise in the Ca²⁺ dependent signal (Figure 7E). These data indicate that recruitment of SuM^VGLUT2+::POA neurons fluctuates with changes in coping strategy during a forced swim assay with decreased engagement of this circuit during times of immobility.

Figure 7.

SuM^VGLUT2+::POA neurons promote active coping during forced swim test

We found that SuM^VGLUT2+::POA neurons are activated by acute stressors (Figure 3) and are recruited during times of greater active coping behaviors (Figure 7). To test if SuM^VGLUT2+::POA neurons can drive a switch in coping strategy in the context of ongoing stressors, we used a two-day forced swim stress test [61, 63]. We tested animals during the second day, when immobile floating is the predominant behavior. As in previous experiments, we used VGLUT2-Cre (Cre+) or WT (Cre-) mice injected in the POA bilaterally with Retro-AVV-DIO-ChR2eYFP. On day one, we subjected mice to 15 mins of forced swim. On the subsequent day, we repeated the forced swim for 6 mins divided into three periods: pre-stimulation, stimulation at 10Hz, and post-stimulation (Figure 7F). Trials on the second day were recorded and scored by a blinded observer for time spent immobile in each 2-minute period. An example of a Cre+ mouse with stimulation 10Hz is shown in Supplemental Video 3. During the pre-stimulation period, there was not a significantly (p>.999) different amount of time immobile between Cre+ (n=9) and Cre- (n=11) mice. During the stimulation period, Cre+ mice began swimming and attempting to climb the wall of the circular swim arena, reflected by significantly (p<0.001) less time immobile (Figure 7H). Interestingly, in the post stimulation phase, the difference in time spent immobile between Cre+ can WT mice decreased but remained significantly (p=0.047) different. These results indicate that in the context of an ongoing stressor, activation of SuM^VGLUT2+::POA neurons is sufficient to trigger a change in coping strategy from largely passive (floating) to active (swimming, climbing). The persistent effect of the acute activation into the poststimulation phase suggests that activation of SuM^VGLUT2+::POA neurons may shift how the stressor is processed or approached.

We next examined the effect of 10 Hz photostimulation of SuM^VGAT neurons on coping strategy using the same two-day swim paradigm. VGAT-Cre mice were injected in the SuM to express ChR2eYFP (n=10) or control (eYFP) (n=11), and a fiber optic was placed over SuM (Figure 7F). Quantification of behavior revealed no significant difference in time spent immobile during the pre-stimulation (p=.218) or the stimulation (p>.999) periods. Surprisingly, in the post-stimulation period, we observed a dramatic shift in behavior, marked by a significant (p=0.047) decrease in time spent immobile (Figure 7I). The amplitude of change in behavior was similar to what we observed during the stimulation phase of the experiments on SuM^VGLUT2+::POA neurons (Figure 7H). One interpretation of these data is that release of sustained local inhibition leads to rebound activity of output SuM^VGLUT2+::POA neurons.

Suppression of SuM^VGLUT2+::POA neurons is required for feeding

Feeding and responding to threats are conflicting actions, and SuM^VGLUT2+::POA neurons may play a role in switching between behavioral paradigms (e.g., feeding vs escape). We sought to examine SuM^VGLUT2+::POA neuron activity in relation to feeding using fiber photometry based GCaMP recordings. To promote differential drive for food, mice were given ad lib access to food (fed condition) or food deprivation for 24 hours, and on testing day, were presented with a chow pellet, while Ca²⁺ dependent GCaMP fluorescence and isosbestic signals were recorded. In the fed state, mice spent significantly less time interacting with the food (p<0.001; Figure 8C) and ate significantly less (p<0.001; Figure 8D) compared to food deprived state. We analyzed the Ca²⁺ dependent and isosbestic signals around presentation of food to fed and food deprived mice. In the food deprived state, mice spent more time with the food and ate more food (Figure 8E and F). At the time of food presentation, the Ca²⁺ dependent signal decreased significantly (99.9% CI) but not the isosbestic signal. The change in the Ca²⁺ dependent signal was larger and more sustained when the animals spent longer interacting with the food (Figure 8E and F). Together, the data support suppression of SuM^VGLUT2+::POA neural activity during consummatory behavior with greater consumption associated with enhanced suppression.

Figure 8.

To examine whether activation of SuM^VGLUT2+::POA neurons disrupted consummatory behavior, we tested the impact of photostimulation on feeding behavior in food deprived mice. Using VGLUT2-Cre (Cre+) or WT (Cre- littermates) mice injected with Retro-AAV2-DIO-ChR2eYFP into the POA and implanted with a fiber optic midline over SuM, we examined how feeding behaviors in food deprived mice were altered by 10 Hz photostimulation of SuM^VGLUT2+::POA neurons (Figure 8G-I). We used trials lasting 20 mins with unrestricted access to food added at the start of the trial. Each trial was divided into three periods: a 5-min pre-simulation period, 10-min stimulation period, and a 5-min post-stimulation period. Trials were recorded and scored for time spent interacting with the food pellet. During the pre-stimulation period, Cre+ (n=17) and Cre- (n=16) mice spent interacting with the food was not significantly different (p>0.999). Upon the introduction of photostimulation, the Cre+ mice stopped eating and interacting with the food. During the photostimulation period, Cre+ mice spent significantly less time interacting with the food compared to Cre- mice (p<0.001). Surprisingly, the decrease in time spent interacting with food continued into the post-stimulation phase, where Cre+ mice continued to interact with the food significantly less than Cre- mice (p<0.001; Figure 8H). Reflecting the decrease in time spent with the food, Cre+ mice consumed significantly less food during the 20-min trial than Cre- mice (p=0.001; Figure 8I). These results indicate that SuM^VGLUT2+::POA neurons can redirect behavior away from consumption, and suppression of SuM^VGLUT2+::POA neurons is required for feeding, even in a food deprived state. Ethologically, animals must choose rapidly between responding to threats and feeding when foraging. The findings here implicate this pathway in control of switching between these behaviors.

Discussion

Together, the presented data show SuM^VGLUT2+::POA neurons function as a hub, with connections to many brain areas, to regulate responses to threatening stressors. We report here that SuM^VGLUT2+::POA neurons have broad arborizations to multiple brain regions involved in responding to threat and stress (Figures 1, 2, and Supplemental Figure 2). We identified diverse threatening stressors (dunk, shock, and predator), which elicit differential behavioral responses, swimming, jumping, and fleeing, recruit SuM^VGLUT2+::POA neurons. Using a forced swim test, we found that recruitment of SuM^VGLUT2+::POA neurons is correlated with periods of spontaneous active coping (swimming, climbing) and not with passive (immobile) behaviors. These data collectively indicate that SuM^VGLUT2+::POA neurons are recruited by threatening stressors, preferentially during active coping. We also examined the roles of SuM^VGLUT2+::POA neurons in responding to stressors by stimulating SuM^VGLUT2+::POA neurons, and selective activation of SuM^VGLUT2+::POA neurons evoked active coping-like behaviors in the absence of a stressor (Figure 4). SuM^VGLUT2+::POA neuron activation drove aversion but did not promote anxiety-like behaviors (Figure 5). We also used a negative reinforcement test, requiring selective activation of an active port over the inactive port to show SuM^VGLUT2+::POA neurons can drive flexible, in contrast to fixed or reflexive, behaviors (Figure 6). Activation of SuM^VGLUT2+::POA neurons increased active coping during ongoing stress in a forced swim assay (Figure 7H). Finally, examining the conflicting behaviors required for threat response and feeding, we found SuM^VGLUT2+::POA neurons were suppressed during feeding and that activation of SuM^VGLUT2+::POA neurons blocked feeding activity (Figure 8). Thus, SuM^VGLUT2+::POA neurons contribute to affective component of stress, promote active coping behaviors, and shift behavior to shift threat responsivity away from feeding.

The presented results demonstrate SuM^VGLUT2+::POA neurons act as a distinct subset of neurons projecting to many but not all areas receiving projections from the total SuM^VGLUT2+ population. Notably, mHb and DG projections were present in the SuM^VGLUT2+ population but absent in the SuM^VGLUT2+::POA projections (Figure 3K), consistent with a separation of DG and CA2 projecting SuM neurons [64]. The extensive arborization of SuM^VGLUT2+::POA neurons challenges the often-employed neural circuit concept that projection from a single area to a another area mediates or modifies a behavior. The projections to PVT, Hb, and PAG are of particular interest because these areas regulate stress coping [65–68]. Dopamine receptor 2 expressing neurons in the PVT have been implicated in promoting active coping behaviors and may be a potential target of SuM^VGLUT2+::POA neurons [8, 9]. The habenula also regulates active vs passive coping behaviors, and the lHb functions in aversive processing, making the selective projections of SuM^VGLUT2+::POA neurons to the lHb of similar interest [65, 69–72]. In addition, the PAG, which receives projections from SuM^VGLUT2+::POA neurons, has been implicated in regulating defensive behaviors and coping strategies [66, 67, 73]. The arborization to these and other areas position SuM^VGLUT2+::POA neurons to be a central hub for regulating behavioral responses to threatening stressors by concurrent recruitment of multiple areas. Understanding the roles of individual projections in contributing to the overall behavioral response will require further studies.

Superficially, the aversiveness of SuM^VGLUT2+::POA neuron stimulation we report contrasts with a recent study that found photostimulation of SuM neuron projections could be reinforcing. Photostimulation of projections from SuM to the septum drove place preference (septum), but the same report found photostimulation of SuM projections in the PVT was aversive[19]. We report here that SuM^VGLUT2+::POA neurons also project to the PVT and the septum (Figure 2). A plausible interpretation is that SuM contains separable, molecularly defined populations of neurons which gate a host of distinct behavioral strategies through their connectivity and/or co-transmitter content. Delineating the overarching circuitry will require further in depth studies, but our results together with recent studies establish the SuM as an important and poorly understood node which regulates both appetitive and aversive motivated behavior.

We addressed the challenge of potential confounds from locomotor activity inherent in active coping behaviors, which is of special concern here because a population of SuM neurons have been found to correlate with velocity of movement. To do so we used multiple behavioral assays together with fiber photometry and photostimulation. We found multiple threatening stressors (dunk, shock, ambush) with differing evoked behaviors all recruit SuM^VGLUT2+::POA neurons (Figure 3). We also found increased recruitment of SuM^VGLUT2+::POA neurons during active coping behaviors (Figures 3 and 7). We did not find evidence for recruitment of SuM^VGLUT2+::POA neurons during elevated locomotion in two cohorts in assays of free movement (Figure 3K-M and Supplemental Figure 5). These data are in contrast to recent studies that found activity of some neurons in SuM correlated future velocity [42]. We carefully examined the behaviors elicited by activation of SuM^VGLUT2+::POA neurons both in absence of stressors (Figure 4) and during ongoing stressors (Figure 7) and found that stimulation of SuM^VGLUT2+::POA neurons promoted active coping behaviors, which involved increased physical activity. We examined if SuM^VGLUT2+::POA neurons could also drive performance of an operant task which would be impaired if activation of SuM^VGLUT2+::POA neurons lead to fixed escape behaviors. We found that the mice effectively performed the operant task, extensively activating the active port, instead of the inactivate port. Taken together, the results from the behavioral tests indicate that SuM^VGLUT2+::POA promote flexible stressor appropriate coping behaviors and not generalized locomotion.

Although neurons in SuM can release both GABA and glutamate in the dentate gyrus, our anatomic studies indicate that SuM^VGLUT2+::POA neurons were discrete from GABAergic neurons and do not project the dentate gyrus (Figure 2 and Supplemental Figure 2)[21, 31]. Thus, SuM^VGAT neurons could be a functionally distinct population. We tested the effects of photostimulation of SuM^VGAT neuron and, in contrast to SuM^VGLUT2+::POA neurons, found no effect on place aversion, minor decreases in locomotion, and no significant change in active coping behaviors during stress (FST) during stimulation. Surprisingly, immediately after photostimulation stopped, active coping behavior increased dramatically (Figure 7I). These data demonstrate functional separation of SuM^VGAT and suggest connection between the SuM^VGLUT2+::POA and SUM^VGAT+ populations that require further investigation to elucidate.

In conclusion, SuM^VGLUT2+::POA neurons arborize to multiple areas involved in stress and threat response that promote active coping behaviors. Passive coping strategies have been associated with unescapable stress, anhedonia, and depression[7, 74–76]. Targeting neuromodulation of a circuit able to act across many brain areas may represent a therapeutic avenue for common psychiatric conditions, and SuM^VGLUT2+::POA neurons are a newly identified node in critical approach-avoidance circuitry.

Acknowledgements

This work was supported by the NIH through 5R01MH112355 to MRB and P30DA048736 (MRB) and 5K08MH119538 to AJN. Support was also provided by the Hope Center Viral Vectors Core and the Genome Technology Resource Center at Washington University in St. Louis (NIH P30CA91842 and UL1TR000448). This work was supported by a Pilot Project Award from the Hope Center for Neurological Disorders and by the Hope Center Viral Vectors Core at Washington University School of Medicine.

Materials and Methods

Methods

Key Resources Table

For further information regarding reagents and resources, contact Aaron Norris, norrisa@wustl.edu.

Experimental model and subject details

Adult (25-35 g, at least 8 weeks of age upon experimental use) male and female VGLUT-Cre (RRID: IMSR_JAX:016963), VGLUT2-Flp (RRID: IMSR_JAX: 030212), Ai14 (RRID: IMSR_JAX: 007908), VGAT-Cre (RRID: IMSR_ JAX: 028862) and C57BL/6J (RRID: IMSR_JAX: 000664) mice (species Mus musculus) were group housed (no more than five littermates per cage) in a 12 hr:12 hr light:dark cycle room with food and water ad libitum[77]. Cre+ and Cre- littermates were used in the experiments unless otherwise noted. The Washington University Animal Care and Use Committee approved all procedures which adhered to NIH guidelines.

Stereotaxic surgery

Injections and implantations were done as described previously[78, 79]. Briefly, in an induction chamber, mice were anesthetized (4% isoflurane) before being placed in a stereotaxic frame (Kopf Instruments). Anesthesia was maintained with 2% isoflurane. Mice were then injected unilaterally or bilaterally, depending on the combination of virus(es) used and brain regions injected. A blunt needle Neuros Syringe (65457-01, Hamilton Con.) and syringe pump (World Precision Instruments) were used to perform the injection schemes below. After surgery, a warmed recovery chamber housed the animal while it recovered from anesthesia before being returned to its home cage.

Injections were made at a rate of 50 nl/min, with the injection needle being withdrawn 5 min after the end of the infusion. Fiber optics for photostimulation or optical fibers for fiber photometry were implanted after injections for all behavioral experiments.

Fiber optic implants for photostimulation were fabricated as previously described using 200μm glass fibers and implanted midline over SuM [78, 80]. For implantation, the skull was cleaned and etched with OptiBond® (Kerr) and the fiber was cemented to the skull with Tetric N-Flow® (Ivoclar Vivadent). Blue light was used to cure and harden cement. Mice were allowed to recover for at least seven days before the start of behavioral experiments. The same process was used for implantation for fiber photometry fibers, which were purchased from Neurophotometrics and trimmed to length.

Anatomical tracing

For anterograde viral tracing experiments, virus was injected at least six weeks prior to transcardial perfusions with 4% paraformaldehyde to allow for anterograde transport of the fluorophore. AAV5-EF1a-DIO-hChR2eYFP or AAV5-EF1a-DIO-eYFP were used. Alternatively, to label only SuM^VGLUT2+::POA neurons for anterograde tracing, Retro-AAV2-DIO-eYFP or Retro-DIO-ChR21eYFP was injected into the POA with AAV-fDIO-Cre injected in the SuM. A minimum of six weeks was allowed prior to sacrifice, harvesting or brains, and sectioning (30μM). Serial 30 μM sections approximately 60μM apart were examined. For retrograde studies, viruses were injected (see figure legends and text for specific viruses) at the targeted site and table for specific viruses[81–83]. Three weeks were allowed to elapse prior to harvesting brains following injections. Images were obtained on a Leica DM6 B upright microscope and processed using Thunder imaging station (Leica).

Brain clearing and light sheet microscopy

Tissue clearing and imaging was carried out on brains collected and fixed in 4% PFA as described above by LifeCanvas Technologies. Briefly, brains were fixed using SHIELD post ix and cleared for seven days in SmartClear II Pro. Index matched with EASYIndex at room temperature. Samples were mounted ventral side up and imaged at 3.6x with pixel size 1.8 x 1.8 mm, axial resolution < 4 μm, z step 4 μm in 488nm channel. Fos was labeled by Alexa Flour 488. SuM boundaries were defined by -2.6 to -2.95 rostral to Bregma. The medial mammillary nucleus and the mammillary recess of the 3^rd ventricle marked the medial and ventral boundaries, while fornix marked the lateral, and fasciculus retroflexus marked the dorsal boundaries. Images were quantified by a trained laboratory member who was blind to the experimental conditions.

Immunohistochemistry

Mice were intracardially perfused with 4% PFA and then brains were sectioned (30 microns) and placed in 1x PB until immunostaining. Free-floating sections were washed three times in 1x PBS for 10 min intervals. Sections were then placed in blocking buffer (0.5% Triton X-100% and 5% natural goat serum in 1z PBS) for 1 hr at room temperature. After blocking buffer, sections were placed in primary antibody rabbit Phospho-c-Fos (Ser32) antibody (1:500, Cell Signaling Technology) overnight 4 °C temperature. After 3 x 10 min 1x PBS washes, sections were incubated in secondary antibody goat anti-rabbit Alexa Fluor 488 (1:2500, Invitrogen) for 2 hr at room temperature. Sections were washed in 1x PBS (3 x 10 min) followed by 2 x 10 min 1x PB washes. After immunostaining, sections were mounted on Super Frost Plus slides (Fisher) and covered with Vectashield Hard set mounting medium with DAPI (RRID:AB_2336788, Vector Laboratories) and cover glass prior to being imaged.

Imaging and cell quantification

‘The Mouse Brain in Stereotaxic Coordinates’ provided the framework to label brain sections relative to bregma[84]. A Leica DM6 B epifluorescent microscope was used to image all sections. For eYFP visualization, a YFP filter cube (Excitation: 490-510, Dichroic: 515, Emission: 520-550) was used and for tdTomato visualization, a Texas Red Filter Cube (Excitation: BP 560/40, Dichroic: LP 585, Emission: BP 630/75) was used. Fos was labeled by Alexa Flour 488. SuM boundaries were defined by -2.6 to -2.95 rostral to Bregma. The medial mammillary nucleus and the mammillary recess of the 3rd ventricle marked the medial and ventral boundaries while fornix marked the lateral, and fasciculus retroflexus the dorsal boundaries. Images were quantified by a trained laboratory member who was blind to the experimental conditions.

Forced-Swim Test for cFos examination

For stress induction via forced swim (Figure S4)[61, 85], mice were individually placed in a cylindrical container (18 cm in diameter) filled with water at 25 +/- 1 °C for 15 min. Prior to stress force swimming, mice were habituated to the arenas for three days prior to the beginning of FST to minimize stress. 90 mins after forced swim, mice were injected with ketamine and xylazine and were intracardially perfused. Control mice were brought to the behavioral testing area but remained in the home cage until perfusion. Water was replaced after every animal. To examine SuM-POA glutamatergic projections, VGlut2-Cre+ littermates were injected with AAV2 retro-Ef1a-DIO-mCherry into the POA.

Fiber Photometry

For all fiber photometry experiments, the same strategy to selectively label SuM^VGLUT2+::POA neurons was used. VGLUT2-Flp mice were injected bilaterally in POA with AAV-Retro-EF1a-fDIO-Cre and with AAV-syn-FLEX-GCaMP7s-WPRE in SuM. After two weeks, mice were implanted with fiber-optic cannulas (200μm) in SuM (D/V -4.3-5)[86]. Mice recovered a minimum of one week prior to behavioral testing. Recording of Ca²⁺ dependent and isosbestic signals were obtained using previously described methods with Bonsai software and FP3002 (Neurophotometrics)[59, 87]. 470 and 415 nm LEDs were used to record interleaved isosbestic and Ca²⁺ dependent signals following the manufacturer directions.

For repeated forced swim experiments (dunk tank), mice were placed on top of a square wire mesh platform inside a custom-made plastic rectangular enclosure (20 cm x 20 cm x 23 cm) filled with water 30 +/- 1 °C. The square wire mesh platform could be raised and lowered without touching the animal. Mice were habituated for 2 days before the test day. Each day, mice were tethered to the fiber optic patch cable and placed on top of the platform for 30mins. On test day, mice were tethered and placed on top of the platform for 30mins (habituation) before the beginning of the 1st trial. After 30mins, for each trial the platform was lowered for 30s and raised after 30s with a rest of 2mins between trials for a total of 10 trials (“dunks”). After 10 trials, an additional 2mins were recorded before removing the animal from the enclosure, patted the animals with paper towels, and placed back in its home cage. During testing, mice were tethered to a fiberoptic patch cable that was attached to a counterbalanced arm that prevented downward force on the animal. Water was replaced after each animal. Time mobile was quantified by a trained observer for the ten 30s trials.

For foot shock stress testing, mice were individually placed inside a custom-made clear plastic box (15.24 L x 13.34 W x 14 H cm) inside a sound-attenuated cabinet. A speaker was placed 4 cm above the chamber for the delivery of auditory cues (75dB). A constant current aversive stimulator (ENV-414S) delivered foot shocks through a grid floor (0.7mA). Five shocks of 2s were delivered after a 30s tone. Intertrial interval ranged from 90 to 180s. After the session, the animal was removed from the chamber and placed back in its home cage. The chamber and grid were wiped down with 70% ethanol between animals.

For open field testing velocity measurements, mice were tethered to a fiberoptic patch cable and placed inside a custom-built square arena (50.8 cm x 50.8 cm x 50.8 cm). Mice were allowed to explore the arena for a 20 min session. Velocity was quantified using scripts in Bonsai 2.4. Bedding in the arena was replaced between animals, and the floors and walls of the arena were wiped down with 70% ethanol.

Similar to the repeated forced swim experiments, forced swim mice were placed on top of a square wire mesh platform that could be raised and lowered inside a custom-made plastic rectangular enclosure (20 cm x 20 cm x 23 cm) filled with water at 25 +/- 1 °C. The platform was lowered at the beginning of the test for 15 min and raised at the conclusion of the test. During testing, mice were tethered to a fiberoptic patch cable that was attached to a counter balanced arm that prevented downward force on the animal. Water was replaced between animals and the enclosure was wiped down with 70% ethanol and rinsed with water. Time immobile, hindpaw swim, swimming, and climbing were quantified using LabGym (Hu et al., 2022).

To measure the activity of SuM^VGLUT2+::POA neurons in response to a stressor that mice may encounter in their natural habitat[88], a remote-controlled spider was used to simulate an ambush of a potential predator. The remote-controlled spider (17 cm x 16 cm; Amazon) was placed inside a polylactic acid (PLA) enclosure (19 cm x 20 cm x 23 cm) that was then placed inside a custom-built square arena (50.8 cm x 50.8 cm x 50.8 cm). Mice were tethered to a fiberoptic patch cable and placed inside the arena for 10 min. Baseline activity was recorded for 5 min before the ambush. After 5 min, the animal was ambushed once it moved in close proximity to the spider’s enclosure (Supplemental video 4). Velocity was measured and analyzed using DeepLabCut (Mathis et al., 2019) within Bonsai. The arena and spider’s enclosure were wiped down with 70% ethanol between animals.

For experiments that examined recruitment of SuM^VGLUT2+::POA neurons during consummatory behavior, mice were food deprived or given ad lib access to food for 24 hours prior to testing. On test day, mice were placed inside an 18 cm diameter round arena and allowed to habituate for 30 min before they were tethered to the fiberoptic path cable. Once tethered, mice were placed inside the arena for 20 min. Baseline activity was recorded for 5 min before the introduction of the chow pellet and for 15 min after the chow pellet was introduced. Chow pellets were weighed before and after the 20-min trial. The difference is reported as food eaten. The same procedure was followed for control mice with the only difference being that these animals were not food deprived for 24 hours.

For fiber photometry data analysis, the interleaved isosbestic and Ca²⁺ dependent signals were recorded at 60 fps (30 fps each). Deinterleaved signals were analyzed using methods as previously reported [87]. Briefly, raw signals were smoothed using a moving average, fitted with an exponential curve using a non-linear least squares function for baseline correction, signals were standardized using the mean value and standard deviation (Z-Score), the standardized isosbestic and Ca2+ signals were scaled a non-negative robust linear regression, and normalized dF/F was calculated. In experiments shown in figures 7A and 8 E-F there was sustained step drop evident in the Ca²⁺ dependent signal reflecting change in population activity because it was not seen in the isosbestic signal. The nature and duration of the change of the signal precluded fitting a curve to the Ca²⁺ dependent signal. In these cases, we show both the Ca²⁺ dependent and isosbestic signals. Z-scores were calculated without baseline correction for both Ca²⁺ dependent and isosbestic signals based on the variability in the baseline state.

Real-time place aversion testing

For real-time place preference testing with optogenetic photostimulation, we used custom-made, unbiased, balanced two-compartment conditioning apparatus (52.5 x 25.5 x 25.5 cm) as described previously[78, 79, 89]. Mice were tethered to a patch cable that allowed free access to the entire arena for 30 min. Entry into one compartment triggered photostimulation, 1 Hz, 5 Hz, 10 Hz, or 20 Hz (473 nm laser, 10 ms pulse width), that persisted while the mouse remained in the light paired side. The side paired with photostimulation was counterbalanced across mice. Ordering was counterbalanced with respect to stimulation frequency and placement. Bedding in the behavior apparatus was replaced between every trial, and the floors and walls of the apparatus were wiped down with 70% ethanol. Time spent in each chamber and total distance traveled for the entire 30-min trial was measured using Ethovision 10 (Noldus Information Technologies). For optogenetic stimulation of VGAT neurons during RTTA, Cre+ littermates were injected either with AAV5-EF1a-DIO-hChR2(H134R)-EYFP or with AAV5-EF1a-DIO-eYFP as control, and stimulation was provided at 10 Hz.

Light/Dark choice

For light/dark choice, the same arenas as used for real-time preference testing were modified and used as previously described [90]. On the light side, a small sport light was placed overhead, and the walls were covered with white laminated paper. Light levels on this side measure 580-590 lux. For the dark side, an infrared spotlight was placed over head, to allow for video tracking of the mice. The walls were covered with matte black plastic. Light levels in the center of the dark side measure 100-110 lux. Animals were recorded using a USB web cam without an infrared filter. For real-time aversion testing, photostimulation was paired with the dark side of the arena as described above. For anxiety-like behavior testing, stimulation was provided uniformly during the trials. Time spent in each chamber and total distance traveled for the entire 30-min trial was measured using Ethovision 10.

Observational behavioral assay

To observe behaviors evoked by photostimulation of SuM^VGLUT2+::POA neurons, mice were habituated for at least three days prior to testing to a round (18 cm diameter), clear arena with counterbalanced optical commutators to minimize the impact of the head tether on movement. Testing occurred after habitation, and approximately 2 cm of bedding material was placed in the arena. Behavior was recorded from the side and scored by a blinded observer.

Negative reinforcer two nose port operant behavior testing

For operant behavior testing, we used Med Associates mouse operant conditioning chamber with dual nose ports and house light as previously described[59]. Briefly, mice were tethered via cantilevered counterweighted optical commutator to a laser light source. A five-day protocol call was used. Day one through four were 10-min trials. Photostimulation was provided at 10 Hz and activation of the active port resulted in a 10-second pause in the photostimulation and activation of the house light inside the arena. On the first four days, each activation of the active port outside of a 10-second pause resulted in a new pause. On day five was a 30-min trial using a progressive ratio protocol the number activation of the active port to generate a pause increased with each activation based on the number of activations (j) =[5e^(0.2j) – 5] round to the nearest integer generating the schedule 1, 2, 4, 6, 9, 12, 15, 20, 25, 32, 40, 50….[91]. The trial was limited to 30-mins due to concern for animal welfare due to the head tether and confined space. Photostimulation terminated at the conclusion of the trial.

Forced Swim with photostimulation

Forced swim trials with optogenetic stimulation were done as a two-day test. On the first day, all mice were subjected to a 15-min forced swim, dried, and returned to the home cage. On the second day, they were tethered and subjected to a 6-min forced swim, divided into three periods, each two mins in length: pre stimulation, stimulation, and post stimulation. 10 Hz photostimulation was provided during the trial. Mice were closely monitored during each trial of swimming. Trials were recorded and scored by a blinded observer for time spent immobile on the second day of the test. For forced swim trials with optogenetic stimulation of VGAT neurons, Cre+ littermates were injected either with AAV5-EF1a-DIO-hChR2(H134R)-EYFP or with AAV5-EF1a-DIO-eYFP as control.

Feeding after food deprivation

For tests involving brief access to food after deprivation, the same 18 cm diameter round arenas with counterbalanced optical commutators were used. Mice were habituated to the arenas for a minimum of three days prior to testing. Mice were food deprived by removal of food from the home cage 24 hours prior to testing. Mice were placed in the arena and allowed to habituate prior to introduction of a chow pellet. The 20-min trial with the foot pellet was recorded and scored by a blinded observer. Chow pellets were weighed before and after the 20-min trial. The difference is reported as food eaten.

Open Field Test

For Open Field testing, we used a purpose-built 20 in square behavior arena. Mice were tethered to a patch cable and placed into the behavioral arena. The laser frequency was set to 10 Hz and was left on for 20 mins. Distance moved for the 20-min trial was quantified using Ethovision 10. Bedding in the arena was replaced between every trial, and the floors and walls of the arena were wiped down with 70% ethanol. Similarly, for optogenetic stimulation of VGAT neurons, Cre+ littermates were injected either with AAV5-EF1a-DIO-hChR2(H134R)-EYFP or with AAV5-EF1a-DIO-eYFP as control.

Statistical analyses

Statistical analyses were conducted using GraphPad Prism software. Data are shown as mean ±SEM, except for Z-scores which are shown as ± 95% confidence interval, as noted in text. Values for individual p values are given in the text and figure legends. Significance was held at α less than 0.05. Unless noted in the test, nonparametric Mann-Whitney tests were used for statistical comparisons. In cases of multiple comparisons, Holm-Šídák method was used to correct for multiple comparisons. Paired testing was used with comparing within cohorts with repeated measurements and unpaired between cohorts. All “n” values represent the number of animals in a particular group for an experiment. For fiber photometry statistical analysis, the mean signal of the baseline and stimulus windows was used, and comparisons were made using the Wilcoxon ranked-sum test, with α=0.95. To analyze the change in the Ca²⁺ dependent signal, a t-confidence interval method was used, in which 95%, 99%, and 99.9% confidence intervals were calculated for windows preceding and subsequently following the described point of interest. Differences were considered significant if the null hypothesis (zero) was not included in the CI. Because exact p values are not calculated using this method, the highest confidence level at which the difference is significant (95%, 99%, or 99.9% CI) is reported instead.