Interpeduncular GABAergic neuron function controls threat processing and innate defensive adaptive learning

Interpeduncular GABAergic neuron function controls threat processing and innate defensive adaptive learning | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Interpeduncular GABAergic neuron function controls threat processing and innate defensive adaptive learning Susanna Molas, Elora Williams, Leshia Snively, Benjamin O'Meara, and 5 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4661779/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 16 You are reading this latest preprint version Abstract The selection of appropriate defensive behaviors in the face of potential threat is fundamental to survival. However, after repeated exposures to threatening stimuli that did not signal real danger, an animal must learn to adjust and optimize defensive behaviors. Despite extensive research on innate threat processing, little is known how individuals change their defensive behaviors when presented with recurrent threat exposures without evidence of a real risk. Insight into this process is critical as its dysregulation may contribute to neuropsychiatric conditions, such as anxiety disorders. Here, we used the visual looming stimulus (VLS) paradigm in mice to investigate innate threat processing and adaptive defensive learning. Repeated exposure to VLS over consecutive sessions reduced immediate freezing responses and time spent inside a sheltered area upon VLS events, leading to an increase in foraging behaviors. Fiber photometry recordings and optogenetic manipulations revealed that VLS innate adaptive defensive learning is associated with reduced recruitment of the midbrain interpeduncular nucleus (IPN), a structure associated with fear and anxiety-related behaviors. Functional circuit-mapping identified a role for select IPN projections to the laterodorsal tegmental nucleus in gating defensive learning. Finally, we uncovered a subpopulation of IPN neurons that express the neuropeptide somatostatin and encode safety- and avoidance signals in response to VLS. These results identify critical behavioral signatures of innate defensive responses and a circuit that regulates the essential features of threat processing. Biological sciences/Neuroscience Biological sciences/Psychology Health sciences/Diseases/Psychiatric disorders Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Introduction Individuals select optimal defensive strategies, such as escape or freezing, to avoid danger when threat is present ( 1 , 2 ). Nevertheless, with repeated exposures to a potential threat without evidence of an aversive outcome, defensive behaviors must undergo adaptive learning, thus contributing to efficient action selection. Numerous neuropsychiatric conditions, including anxiety disorders, manifest maladaptation of threat responses ( 3 ), highlighting the importance of understanding these basic neurobehavioral processes. Exposure to an overhead dark visual looming stimulus (VLS) naturally elicits innate defensive responses across multiple species, from fish to humans ( 1 , 4 ). In rodents, exploring an open area supplied with a nest as a shelter, the detection of an expanding VLS to the upper visual field triggers an immediate defensive behavior of freezing and escaping towards a sheltered area, seeking protection ( 5 ). Whether rodents shift their defensive responses to optimal behavioral sequences after repeated VLS and the mechanisms that support this innate defensive adaptive learning remain largely unknown. Processing sensory information and coordinating appropriate motor output for defensive behaviors requires complex neural circuits ( 6 , 7 ). Rodent and human studies have implicated an emerging circuit, arising in the habenula (Hb), that contributes to threat-related behaviors ( 8 ). The habenula nuclei play a crucial role in regulating emotional, motivational, and cognitive behaviors ( 9 – 12 ). Specifically, the medial part of the habenula (mHb) sends descending projections almost exclusively to the interpeduncular nucleus (IPN) of the midbrain ( 13 ), an axis implicated in anxiety ( 14 – 21 ) and fear responses ( 22 – 25 ). The IPN is highly enriched in GABAergic neurons that establish reciprocal connections with regions involved in motivation and affective behaviors, including the raphe, locus coeruleus, and laterodorsal tegmental nucleus (LDTg)( 26 , 27 ). Recent work indicates that the habenulo-interpeduncular axis, and IPN projections to the LDTg, mediate aversive and reward-related behaviors ( 28 , 29 ). Yet, whether the IPN and associated neuronal circuits are recruited by VLS to adjust defensive behaviors has never been explored. Materials and methods Animals All experiments followed the guidelines provided by the National Research Council with approved animal protocols from the Institutional Animal Care and Use Committee of the University of Massachusetts Chan Medical School. C57Bl/6J (Stock #000664, Jackson), GAD2 Cre (Stock #10802, Jackson) and Sst Cre (Stock #013044, Jackson) mice were used. Cre lines were crossed with C57Bl/6J mice and only heterozygous animals used. All experiments included male mice. IPN Sst ablation experiments included males and females. Subject mice were kept under a reverse 12h light/dark cycle (lights ON at 7P.M.) for 3–4 weeks with ad libitum access to food and water, and individually housed for at least 5 days before any behavioral testing. Experiments were performed during the dark cycle phase (8A.M. to 5P.M.). Viral preparations Biosensors, optogenetic and control plasmids packaged into viral particles were purchased from Addgene. For fiber photometry experiments we used pAAV.CAG.Flex.GCaMP6m.WPRE.SV40 (#100839-AAV5, 2.6x10 13 GC/ml), pGP.AAV.CAG.Flex.-jGCaMP7s.WPRE (#104495-AAVrg, 1.1x10 13 GC/ml). For tracing and optogenetic experiments we used pAAV.hSyn.DIO.EGFP (#50457-AAV5, 1.3x10 13 GC/ml and -AAVrg, 1.4x10 13 GC/ml), pAAV.hSyn.DIO.mCherry (#50459-AAV5, 1.8x10 13 GC/ml and -AAVrg, 1.5x10 13 GC/ml), pAAV.Ef1a.DIO.eNpHR3.0.EYFP (#26966-AAV5, 3.8x10 12 GC/ml), pAAV.Ef1a.doublefloxed.hChR2(H134R).mCherry.WPRE.HGHpA (#20297-AAV5 1.2x10 13 GC/ml) and pAAV.flex.taCasp3-TEVp (#45580-AAV5, 2.5x10 13 GC/ml). The viral stock pAAV.Ef1a.DIO.-eNpHR3.0.EYFP (#AV9115-rAAV2, 5.8x10 12 VM/ml) was obtained from UNC GTC Vector Core. Stereotaxic surgeries Surgeries were performed under aseptic conditions as previously described ( 21 ). Mice (6–8 weeks old) were deeply anaesthetized using 100 mg/kg ketamine (VEDCO) and 10 mg/kg xylazine (LLOYD) and placed on a stereotaxic frame (Stoelting Co.). Viral solutions were microinjected at a controlled rate of 50 nl/min using a gas-tight 33G 10-µl neurosyringe (1701RN; Hamilton). Injection coordinates were (in mm, anteroposterior, mediolateral, dorsoventral and angle): IPN (− 3.4, − 0.5, − 4.86, 6°) and LDTg (-5.34, ± 0.4, -3.2, 0°). Viral volumes were 300 nl (IPN) and 300 nl/site (LDTg). For fiber photometry and optogenetic experiments, 3–5 weeks post-viral injection, an optic fiber implant (200-µm core diameter; 0.53N.A., Doric Lenses) held in a magnetic aluminum receptacle (Doric Lenses) was placed above the IPN and secured into the skull using adhesive (C&B Metabond cement, Parkell Inc.) followed by dental cement (Cerebond, PlasticsOne). Mice received IP injections of 1 mg/kg ketoprofen analgesic (Zoetis) and monitored for recovery. Animals showing no viral or off-target site viral expression or incorrect optic fiber placement (< 10%) were excluded from analysis. Behavioral experiments Visual looming stimulus (VLS) paradigm The apparatus consisted of a rectangular Plexiglass maze (40x22x30 cm) with a projector screen (30x20 cm) above the arena and a rectangular shelter (10x12 cm) in one corner. All mice habituated to the apparatus and illumination settings for 8–10 min. After 24h, mice acclimated to the apparatus for 2–5 min before a VLS was randomly displayed from the screen while they actively explored the arena. Each VLS consisted of 15 consecutive dark expansions of 0.5s length and each mouse received 4–7 looming trials per day with a minimum inter-looming trial interval of 60s. The VLS test session was repeated for 3 consecutive days. For side-VLS, the screen was displayed from a wall view. The apparatus was cleaned between animals with 0.1% Micro-90 solution. A video-camera was used to record and track animals’ behavior using Ethovision XT (v15.0). The arena was subdivided in the nest area (12x10 cm), a safety zone adjacent to the nest (10x10 cm), a trigger zone where the VLS were displayed (12x12 cm) and a zone near the walls (5 cm). Early defensive responses included immediate freezing and maximum speed which were reported 2 sec upon VLS initiation, as well as latency to enter the nest and escape run (maximum speed 10 sec upon VLS). Late defensive responses included time spent inside the nest after the first nest entry once the VLS was presented, as well as time spent near the walls. The percent of time in each zone was estimated within 30 upon VLS initiation and averaged per each animal. Latency to and time in nest were manually scored by an experimenter blind to animals’ conditions. Foot shock Mice were habituated to a fear conditioning cage. GCaMP fluorescence from IPN neurons was recorded for 2 min before the first shock. During a 15-min foot-shock period, ten shocks (0.5mA, 1-s duration) were delivered at random intervals and time-stamped into the photometry recording via a transistor-transistor logic (TTL) pulse from the fear conditioning system. Open field The apparatus consisted of an open-field chamber (42x38x30 cm). Each mouse was given 10 min to explore, and the time spent in the center and outer parts of the chamber was tracked from a video recording using Ethovision XT. Elevated plus maze The elevated plus maze (EPM) apparatus consisted of a central junction (5×5 cm) and had four arms elevated 45 cm above the floor with each arm positioned at 90° relative to the adjacent arms. Two closed arms were enclosed by high walls (30×5×15 cm) and the open arms were exposed (30×5×0.25 cm). All mice were given 5 min of free exploration. Tail lift Animals were picked up by their tails by an experimenter while they were actively exploring the home cage. Fiber photometry and data analysis Florescent signals from biosensors were recorded using a Doric Instruments Fiber Photometry System as previously described ( 28 ). A LED driver delivered excitation light at 465 nm and at 405 nm (~ 30-60µW output at the fiber tip). The light was reflected into a 200µm 0.53N.A. optic fiber patch cord via the Dual Fluorescence Minicube. Emissions were detected with a femtowatt photoreceiver (Model 2151, Newport). Sampling (12 kHz) and lock-in demodulation of the fluorescence signals were controlled by Doric Neuroscience Studio software with a decimation factor of 50. A behavioral camera synchronized the photometry recordings with time-locked behavioral tracking systems. Fiber photometry data analysis was performed using custom-written Matlab scripts. The 405 nm channel was scaled to the 465 nm by applying a least mean squares linear regression. Scaled signals were used to calculate the ΔF/F 0 where ΔF/F 0 = (465nm signal – fitted 405nm signal)/fitted 405nm signal. Z-scores were calculated using the average baseline of ΔF/F 0 values from the − 1.0 sec prior to the onset of VLS (considered as time zero, t = 0). The max and mean z-score were estimated between t = 0 and 10 sec upon VLS and averaged per animal. The min z-score for nest entry was estimated between t = 0 (nest entry) and 10 sec upon nest entry and averaged per animal. For IPN Sst + GCaMP recordings, the max z-scores were determined − 2 to 2 sec time-locked to nest entry. Optogenetics Optic fiber implants were connected to a patch cable (Doric Lenses) and a commutator (rotary joint; LEDFRJ-B_FC for blue light and LEDFRJ-A_FC for yellow light, Doric Lenses), by means of an FC/SMC adapter to allow unrestricted movement, as previous reported ( 30 ). Mice habituated for 8–10 min to the VLS apparatus without receiving light photostimulation. On day 1 of VLS test, mice freely explored the apparatus for 2–5 min before VLS were displayed. A high-power LED driver (DC2200, Thorlabs) was used to generate light pulses time locked to VLS events at intensity ~ 2–5 mW at the fiber tip. Light photoinhibition (593nm, constant light) was delivered in time-locked mode by an experimenter blind to animals’ conditions, 2 sec prior, during and 2 sec post each VLS event. On day 2 of the test, mice were subjected to the VLS paradigm with no light delivery. Day 3 followed the same light stimulation protocol as day 1. For optogenetic photostimulation, light pulses (473nm, 20Hz, 12ms pulse, 3s) were delivered at intervals > 90s on days 1 and 3. No VLS was displayed. All sessions were video recorded from above (HDR-CX4440 camera, SONY) and computationally analyzed with Ethovision XT. Immunostaining and microscopy Immunohistochemistry and microscopy were performed as described previously ( 30 ). Mice received sodium pentobarbital (200 mg/kg) and transcardially perfused with ice-cold 0.1 M phosphate buffer saline (PBS, pH7.4) followed by 10 ml of cold 4% (W/V) paraformaldehyde (PFA). Brains were post-fixed in 4% PFA before transferred to 30% sucrose. Coronal sections (25 µm) were obtained using a freezing microtome (HM430; Thermo Fisher Scientific, MA, USA). Brain sections were permeabilized with 0.5% Triton X-100 (Sigma) for 10 min, blocked with 5% donkey serum (DS, Sigma) for 30 min and incubated with the primary antibody (rabbit anti-Sst, 1:700, sc-13099) overnight at 4 ºC. Slices were incubated in secondary antibody for 2 h (1:800; Life Technologies; donkey anti-rabbit 594, R37119). Nuclei were counterstained with DAPI. Viral expression was visualized using the endogenous fluorescence of the virus. All slices were imaged using a fluorescent microscope (Zeiss, Carl Zeiss MicroImmagine, Inc., NY, USA) connected to computer-associated image analyzer software (Axiovision Rel., 4.6.1). Statistical analysis Data were analyzed by means of two-tailed unpaired t -test, one-way and two-way ANOVAs with/without repeated-measures (RM), or the restricted maximum likelihood (REML) mixed model, as indicated. Dunnett’s or Tukey’s post hoc tests were used for multiple comparisons. Two-tailed Pearson r was used for correlation analysis. Comparisons of z-scores photometry signals were made using the calculated average for each animal. Each data set was tested for normal distribution prior to analysis and presented as mean ± standard error of the mean (SEM). All statistical analyses were performed in GraphPad Prism 10.1.0. Software (Graphpad Software Inc.) and statistical significance was established at p < 0.05. Results Innate adaptive defensive learning: mice adjust behavioral response to a potential threat. We implemented the VLS paradigm to investigate an animal’s capacity to adjust innate defensive behaviors in the absence of a real risk (Fig. 1 a). Mice were placed in a plexiglass apparatus that contained a rectangular nest in one corner and a projector screen that displayed a VLS while animals actively explored the arena. The detection of a VLS from above, but not from a side view (Supplementary Fig. 1a-c), triggered an immediate defensive response of freezing followed by running to the nest (here defined as early responses to VLS). Animals also spent a significant amount of time in the confines of the shelter, presumably avoiding potential threat, before engaging in exploratory behaviors (here defined as late responses to VLS). However, with multiple exposures of an overhead VLS for 3 consecutive days, without evidence of aversive outcomes, mice learned to adjust both early and late VLS-evoked innate defensive strategies (Fig. 1 b-j). Across the 3 days, immediate freezing significantly reduced (Fig. 1 c-d), whereas speed increased (Fig. 1 e-f). Mice continued to run with similar maximum speed (10 sec upon VLS) and exhibited similar escape latencies to the nest in response to repeated VLS over the 3 days (Supplementary Fig. 1d-e). To measure late VLS-induced defensive behaviors, we tracked the animal’s position throughout arena zones (Supplementary Fig. 1f and methods). The amount of time spent inside the nest upon VLS exposure decreased over repeated days (Fig. 1 g-h), while animals shifted to more exploratory behavior near the walls (Fig. 1 i-j). We did not observe adaptive changes with time spent in the trigger zone, where the VLS is presented (Supplementary Fig. 1g-h), or time in the safety zone adjacent to the nest (Supplementary Fig. 1i-j). Notably, adjustment of defensive responses was not detected within a single-day trial session (Supplementary Fig. 2), suggesting that optimization of defensive strategies may reflect learning and consolidation processes. We performed linear correlation analysis to further investigate threat-evoked innate adaptive defensive learning with repeated VLS (Supplementary Fig. 3 and Supplementary Tables 1–6). For early responses to VLS, we found that latency to the nest showed a strong negative correlation with the maximum speed the animals reached 10 sec upon VLS, which was maintained across the 3 sessions, indicating the faster the animals ran, the earlier they entered the nest. Interestingly, the early response of freezing upon VLS initiation predicted late defensive responses such as the total amount of time the animals would spend inside the nest. Time spent in the nest was also positively correlated with escape including latency to the nest and maximum speed 10 sec upon VLS only on day 1, but not in later sessions. These results suggest that escape behaviors (i.e. running to the nest) and avoidance behaviors (i.e. time spent inside the nest) are related to each other at initial sessions but may become more dissociable once the animals learn to adjust defensive responses. Exposure to potential threat engages activity of IPN GAD2 neurons that adjusts with defensive learning. To study the neurocircuitry behind threat adaptation and defensive learning we focused on the IPN of the midbrain, an emerging region associated with anxiety and fear ( 31 ). The IPN is an inhibitory nucleus highly enriched in GABAergic neurons that respond to aversive stimuli ( 32 – 34 ). We combined in vivo fiber photometry recordings with mouse behavior to test if IPN GABAergic neuronal activity is engaged by VLS. Specifically, we expressed Cre -dependent GCaMP in the IPN of mice driving Cre recombinase under the control of the glutamic acid decarboxylase 2 enzyme promoter ( GAD2 Cre mice) and recorded IPN activity dynamics time-locked to VLS events (Fig. 2 a). In mice presented with an overhead VLS, we detected a significant increase in IPN GAD2 neuronal activity that was absent in control mice expressing Cre -dependent eGFP in the IPN instead of GCaMP or if the same VLS was presented from a side view (Supplementary Fig. 4a-b). Additional aversive stimuli, including a tail lift or a foot shock, also raised the activity of the IPN GAD2 neuronal population (Supplementary Fig. 4c-d). Remarkably, IPN neuronal responses to the VLS decreased from day 1 to day 3, as mice learned to optimize innate defensive strategies (Fig. 2 b-e). Other behaviors, such as rearing against the wall, also increased IPN activity. However, the IPN GABAergic activity during rearing remained stable across the 3 days (Supplementary Fig. 4e), providing evidence that the decrease in signal was not due to photobleaching. Reductions of VLS-evoked IPN GAD2 neuronal activation across days were not detected within a trial session (Supplementary Fig. 4f-g). Interestingly, IPN GAD2 neuronal activity dynamics inversely mirrored changes in speed during VLS events (Fig. 2 b), as well as across the whole VLS session (Supplementary Fig. 5a). Correlation analysis demonstrated VLS-induced activation levels of IPN GAD2 neurons negatively related with speed 10 sec post-VLS initiation (Supplementary Fig. 5b). Furthermore, VLS-induced engagement of IPN GAD2 neurons positively correlated with latency to nest (Supplementary Fig. 5c) and time spent near the walls (Supplementary Fig. 5d). Notably, we also detected reduced activity of IPN GABAergic neurons when mice entered the nest (Fig. 2 f-h), although these signals did not adjust across repeated sessions. Altogether these results suggested the dynamics of IPN GAD2 neuronal activity reflected different aspects of VLS evoked defensive actions. Silencing activity of IPN GAD2 neurons during VLS presentations reduces innate defensive behaviors. To determine the functional implication of IPN GAD2 neurons in defensive responses, we used optogenetics and selectively silenced these neurons during VLS events. GAD2 Cre mice were injected with Cre -dependent halorhodopsin (NpHR) or eGFP control virus and implanted with an optic fiber in the IPN (Fig. 3 a). Animals then underwent the 3-day VLS paradigm with photoinhibition 2 sec prior to VLS, which remained ON until 2 sec post-VLS, on days 1 and 3 (Fig. 3 b). Photoinhibition of IPN GABAergic neurons reduced both early and late defensive responses. Compared to the controls, IPN NpHR animals exhibited a decrease in VLS-induced immediate freezing response (Fig. 3 c-d) along with increased speed upon VLS display (Fig. 3 e-f). Additionally, silencing IPN GAD2 neurons led to decreased time spent in the nest on day 1 of the looming session (Fig. 3 g-h); instead, animals remained in the vicinity of the safety zone (Supplementary Fig. 6a-b). Other defensive responses including latency to enter the nest (Supplementary Fig. 6c), time spent near the walls (Supplementary Fig. 6d-e), or in the trigger area (Supplementary Fig. 6f-g) were not significantly affected by IPN GAD2 photoinhibition. Overall, these data suggest that IPN GABAergic neurons mediate both early and late VLS-evoked defensive behaviors, particularly on the first exposure session when neurons are highly engaged by VLS. To complement these experiments, we also asked if activating IPN GABAergic neurons is enough to elicit changes in innate defensive strategies. To this aim, we injected the IPN of GAD2 Cr e mice with Cre -dependent channelrhodopsin (ChR2) and implanted an optic fiber in the target site (Supplementary Fig. 7a). In this experiment, no VLS was presented; instead, animals received IPN GAD2 neuronal photostimulation when they entered the trigger zone, on days 1 and 3 (Supplementary Fig. 7b). Optogenetic excitation alone did not produce differences between groups for the defensive responses measured (Supplementary Fig. 7c-k). IPN GAD2 neurons projecting to the LDTg are engaged by VLS and reduce activation with innate defensive adaptive learning. The IPN GAD2 neuronal population consists of projection neurons that innervate brain regions involved in affective and motivational behaviors, including the raphe and the LDTg ( 26 , 27 ). Here, we hypothesize the IPN may use the LDTg, an area previously associated with fear and anxiety ( 35 ), to convey innate defensive behaviors. To test if this circuit responded to the VLS, we recorded activity dynamics in GAD2 Cre mice bilaterally injected with a retrogradely-transported AAVrg Cre -dependent GCaMP into the LDTg and implanted with an optic fiber in the IPN (Fig. 4 a). The presentation of overhead VLS triggered an elevated activation of GAD2 IPN◊LDTg neurons that significantly decreased over consecutive sessions (Fig. 4 b-e). No reductions in VLS-induced GAD2 IPN◊LDTg circuit engagement were detected within a trial session (Supplementary Fig. 8a-b). Similarly to overall IPN GAD2 neurons, activity dynamics of the GAD2 IPN◊LDTg circuit inversely mirrored changes in speed (Fig. 4 b) and negatively correlated with speed levels 10 sec upon VLS initiation (Supplementary Fig. 8c). Additional analysis did not find significant correlations between VLS-induced GAD2 IPN◊LDTg circuit engagement and latency to nest (Supplementary Fig. 8d), although GAD2 IPN◊LDTg activation positively predicted the time mice spent near the walls (Supplementary Fig. 8e). Akin to IPN GAD2 measures, GAD2 IPN◊LDTg neurons showed a decrease in circuit activity when animals entered inside the nest that was sustained across the three sessions (Fig. 4 f-h). This data demonstrates that VLS-related information engages the projection from the IPN to the LDTg area, which may participate in threat processing and adaptive defensive learning. Silencing IPN GAD2 neurons projecting to the LDTg occludes innate defensive adaptive learning. To test if IPN GABAergic IPN neurons projecting to the LDTg are necessary for innate adaptive defensive responses, we used closed-loop optogenetic approaches in a circuit-specific manner. To this aim, we injected a retrograde Cre -dependent AAVrg NpHR or eGFP into the LDTg and placed an optic fiber in the IPN (Fig. 5 a). Animals underwent the same VLS paradigm with circuit photoinhibition time-locked to VLS events. Experimental and control mice demonstrated a significant reduction in the early defensive behavior of freezing across days (Fig. 5 b-c). However, compared to the controls, optogenetic silencing of the IPN◊LDTg circuit slightly increased freezing behavior (Fig. 5 b-c) and significantly reduced max speed 2 sec upon VLS events (Fig. 5 d-e). Noticeably, control mice significantly reduced the time spent in the nest after VLS presentation across sessions, whereas mice with GAD2 IPN◊LDTg circuit photoinhibition continued spending a significant amount of time inside the nest over VLS days (Fig. 5 f-g), suggesting that inhibition of this circuit occludes adaptive learning of innate threat responses. Time spent in other zones, such as the wall, safe, and trigger as well as latency to nest, were not influenced by optogenetic inhibition of the GAD2 IPN◊LDTg circuit (Supplementary Fig. 9). A subpopulation of IPN neurons expressing Sst encodes defensive responses in a threatening environment. Within the IPN, a GABAergic subpopulation expressing somatostatin (Sst)( 36 ) demonstrates a highly selective dorso-rostral to ventro-caudal gradient (Supplementary Fig. 10a). Sst is a neuropeptide typically co-released with GABA and involved in anxiety-like behaviors ( 37 ). To explore whether IPN Sst + responded to VLS, we injected Cre -dependent GCaMP in the IPN of mice expressing Cre recombinase under the control of the Sst promoter (Fig. 6 a and Supplementary Fig. 10b). We found that aversive stimuli such as VLS presentations (Fig. 6 b-e), a tail lift (Supplementary Fig. 10c) or a foot shock (Supplementary Fig. 10d), triggered an increase in IPN Sst + neuronal activity. Remarkably, VLS-induced IPN Sst + activation occurred after reaching max speed upon VLS (Fig. 6 b) and did not reduce from day 1 to day 3 (Fig. 6 c-e). Interestingly, we also detected IPN Sst + activation time-locked to nest entry, although these signals did not adjust across repeated VLS sessions (Fig. 6 f-h). Further analysis demonstrated on day 1 of the looming test, the engagement of IPN Sst + neurons with nest entry positively correlated with speed levels 10 sec upon VLS (Supplementary Fig. 10e) and time spent inside the nest (Supplementary Fig. 10f), while it negatively correlated with nest latency (Supplementary Fig. 10g) or time spent near the walls (Supplementary Fig. 10h). Overall, IPN Sst + activation with nest entry consistently predicted VLS-induced defensive behaviors, suggesting these neurons may encode safety and avoidance aspects of threat processing. Genetic ablation of IPN Sst + neurons reduces anxiety-like behaviors. We next addressed the role of IPN Sst + neurons in threat processing and defensive learning by genetically ablating this neuronal population using a Cre -dependent caspase 3 approach (Fig. 6 i). Removing IPN Sst + neuronal function did not affect VLS-induced freezing (Supplementary Fig. 11a-b) or changes in speed (Supplementary Fig. 11c-d) across the 3-day looming sessions. Nevertheless, we observed that IPN Sst + ablated animals significantly reduced the time spent inside the nest as compared to mCherry control mice (Fig. 6 j-k). In contrast, animals with IPN Sst ablation spent more time nearby the safety (Supplementary Fig. 11e-f) and wall zones (Supplementary Fig. 11g-h), without affecting nest latency (Supplementary Fig. 11i). The reduction in nest time observed by animals with ablated IPN Sst + neurons support the notation that these neurons play a role in processing avoidance-related aspects of threat-evoked defensive behavior, but not motor function. Along these lines, we also detected that IPN Sst + ablation increased the time spent in the open arms of the elevated plus maze (Supplementary Fig. 11j) but not the number of closed arm entries (Supplementary Fig. 11k). Similarly, IPN Sst ablated animals showed increased time of center exploration in an open field test (Supplementary Fig. 11l) without altering locomotor activity in this assay (Supplementary Fig. 11m). Discussion Defensive behaviors are regulated by adaptive mechanisms contingent on the previous experience of an aversive outcome. Here we define early and late threat-evoked defensive responses that adjust to repeated VLS exposures when there is no evidence of real harm. We identify the GABAergic population in the IPN of the midbrain as a critical node orchestrating adaptive defensive strategies. Inhibitory projections from the IPN to the LDTg control the learning aspect of threat processing. In contrast, a subpopulation of IPN neurons expressing the neuropeptide Sst mediate generalized aspects of avoidance-related behaviors. The present findings have important implications for understanding the neurobiology of innate fear behaviors and how their expression is regulated in the absence of danger, a critical concept in many neuropsychiatric conditions. The selection of ongoing defensive behaviors is constantly updated by recent experiences that contribute to perceptual and value-based decision-making and action selection ( 38 , 39 ). While adaptations of defensive responses to visual looming stimuli have been reported in various animal species ( 38 , 40 – 42 ), the behavioral signatures of threat adaptation and the circuit mechanisms underlying these behavioral changes remain largely unknown. We found that upon repeated VLS sessions, mice reduced immediate freezing, as well as late defensive behaviors including time spent inside the nest. Instead, animals switched behavioral strategies to engage in more foraging behavior. Importantly, changes in defensive strategies were detected over multiple-day sessions but not within a trial session, supporting the view that these may reflect learning and consolidation processes ( 38 ). Although most adaptive defensive behaviors to visual looming have been focused on escape responses ( 38 , 40 ), understanding late emotional components of threat processing has received less attention. Our behavioral analysis revealed a dissociation between time spent inside the nest and escape responses over repeated VLS sessions and learning. Even though multiple defensive behaviors adjust with consecutive threat exposures that have no aversive outcome, they may each require differential circuitry to guide the behavioral output. Escape is a flexible behavior under cognitive control ( 39 ). Integrating variables of escape behavior together with the engagement of foraging strategies upon threat assessment -for instance when animals leave the nest to take new risks-, is necessary to understand how these are coordinated to drive adaptive behaviors. Given that the speed of habituation to VLS depends on the behavioral context ( 42 ) and it is stimulus-specific ( 38 ), future studies could help elucidate early and late defensive components of threat adaptation specific to the context or type of threatening stimuli. The neural basis of threat detection and response to VLS are conserved across species ( 1 , 7 ). Emerging evidence indicates that divergent circuits orchestrate escape and freezing responses to looming stimuli ( 2 , 6 , 43 , 44 ). Yet, most of these studies have focused on a single looming session and thus, the neural circuitry of innate defensive adaptive learning to VLS remains unexplored. Here we provide first-time evidence that IPN GABAergic neurons are recruited by innate visual threats and show neuronal adaptation with defensive learning. These results support that the habenular axis is involved in anxiety ( 14 – 21 ) and fear-related behaviors ( 22 – 25 ), as well as familiarity signaling ( 30 ). Silencing overall IPN GABAergic activity during VLS presentations devalued the stress component of a VLS threat and reduced early freezing responses and late emotional behaviors such as time spent inside the nest. In contrast to the ventral tegmental area (VTA) GABAergic neurons ( 45 ), brief optogenetic stimulation of IPN GABAergic subpopulation was not sufficient to trigger early or late defensive behaviors, indicating specialized roles of VTA and IPN inhibitory networks. Here, we report for the first time that activity dynamics of IPN GABAergic neurons inversely mirrored changes in speed. The IPN is part of the nucleus incertus network that controls locomotor speed, arousal, and hippocampal theta rhythms ( 46 ). Through this circuit connectivity, the IPN may contribute to the animals’ arousal and locomotion. Still, our findings showing reduced IPN GABAergic neuronal activity with nest entries -when animals lower speed-, suggest a role of the IPN beyond controlling locomotion to also processing affective behaviors like avoidance or approach. Freezing is cardinal in stress-coping processes as it corresponds to a state of hypervigilance that enables decision-making ( 2 ). Spending time inside the nest upon VLS could also represent a stress-coping mechanism that prepares for future foraging actions. Altogether, our results support the view that the IPN responds to aversive stimuli to regulate distinctive stress-related coping strategies ( 34 ). IPN GABAergic neurons send strong inhibitory projections to the raphe and tegmentum ( 26 , 27 ). Through the LDTg, the IPN controls nicotine aversion ( 29 ) or social novelty preference ( 28 ). We found IPN◊LDTg circuit engagement with aversive VLS and neuronal adaptation with multiple exposures. Optogenetic silencing the IPN◊LDTg circuit increased freezing and occluded late defensive adaptive learning specifically for time spent in shelter. Recent findings demonstrate LDTg GABAergic neurons inhibit VTA to promote unconditioned freezing responses ( 47 ). Although work from our group and others suggest IPN GABAergic neurons inhibit LDTg cholinergic neurons innervating the VTA ( 28 , 29 ), we cannot exclude the possibility the IPN inhibits LDTg GABAergic function to regulate fear and freezing responses. Considering different LDTg interneurons oppositely regulate innate fear ( 35 ), understanding how the IPN controls LDTg function and network connectivity is necessary to elucidate the critical role of this circuit in innate defensive adaptive learning. Among all IPN GABAergic cell types, we recently showed the neuronal population expressing the neuropeptide Sst is activated by acute stress to drive motivational behaviors ( 34 ). Sst provides retrograde inhibition of excitatory inputs from the mHb to the IPN ( 48 ). Our fiber photometry data revealed engagement of IPN Sst neurons during threatening VLS events but also when animals entered the safety of a protective shelter. In the lateral habenula, heterogeneous neuronal clusters predict the selection of distinct threat-driven defensive behaviors ( 8 ). It could be possible that different IPN Sst populations exist to control various aspects of threat aversion vs. coping mechanisms. We show that genetic ablation of IPN Sst neurons reduced time inside the nest as well as general anxiety-like behaviors. Ablated animals did not recognize the nest as a safe, protective zone. Sst neurons can mediate anxiety by disinhibition of fear-related amygdaloid circuits ( 37 ). Elucidating the precise IPN Sst neuronal connectivity with previously established anxiety networks should be addressed in future investigations. Collectively, our findings implicate the IPN as a critical node of innate threat processing and adaptive defensive learning, suggesting dysregulation of the IPN may contribute to numerous psychiatric disorders. Declarations Acknowledgments We thank Biorender.com for use of graphics. This work was supported by the Brain and Behavior Research Foundation Young Investigator Award 30616 (S.M.), the National Institute of Mental Health award MH129040 (A.R.T.), the National Institute on Drug Abuse awards DA041482 (A.R.T) and DA047678 (A.R.T.). Conflict of interest All authors report no competing financial interests or potential conflicts of interest. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. 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Additional Declarations The authors have declared there is NO conflict of interest to disclose Supplementary Files SupplFigure1.tif SupplFigure2.tif SupplFigure3.tif SupplFigure4.tif SupplFigure5.tif SupplFigure6.tif SupplFigure7.tif SupplFigure8.tif SupplFigure9.tif SupplFigure10.tif SupplFigure11.tif WilliamsEWetal2024Supplementary.docx Supplementary information Supplementary information is available at MP’s website. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4661779","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":343406975,"identity":"ba694a02-37ee-44de-9a48-54d4549b77bf","order_by":0,"name":"Susanna Molas","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAABB0lEQVRIiWNgGAWjYHAD5gYGhgoGBj4QmwdEHCCohRGo5QwDAxtpWhjbiNAiPyP94efCHXYMuu2NjR9/zrOTY2PvffjhTQWDHN+NBKxaDG7kGEvPPJPMYHbmYLM077ZkYzae48aSc84wGEvi0iKRwyDN28bMYHYjsUGacduBxDaJNJAIQ+IGHFqADnv8m7etHqSl+efPOWAtzEARhnpcWhhuJJgBzTwM0tImwdsA1sIGsiXBAJfDzrwxs+ZtO84D9EubNc8xkF+OsVnOOSNhOPPMA+wOa09/fJu3rVrO7Hjz4Zs/auzk+NnbmG+8qbCR5zuOw2FQwIMuIIFX+SgYBaNgFIwC/AAAjWRbe8xlUIsAAAAASUVORK5CYII=","orcid":"https://orcid.org/0000-0002-7985-9373","institution":"University of Colorado Boulder","correspondingAuthor":true,"prefix":"","firstName":"Susanna","middleName":"","lastName":"Molas","suffix":""},{"id":343406976,"identity":"45fa65ac-4b0f-4da5-b748-37010a13a3c1","order_by":1,"name":"Elora Williams","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Elora","middleName":"","lastName":"Williams","suffix":""},{"id":343406977,"identity":"0bf864de-7af7-4c26-961e-9d407d4bb534","order_by":2,"name":"Leshia Snively","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Leshia","middleName":"","lastName":"Snively","suffix":""},{"id":343406978,"identity":"b73d5e7d-91c1-462d-98a7-58b9e79186f8","order_by":3,"name":"Benjamin O'Meara","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Benjamin","middleName":"","lastName":"O'Meara","suffix":""},{"id":343406979,"identity":"6354a6d7-7d34-46a8-889d-68e1a3a63fd9","order_by":4,"name":"Hannah Jacobs","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Hannah","middleName":"","lastName":"Jacobs","suffix":""},{"id":343406980,"identity":"b89ab84b-ff5c-4710-8fed-080c8fc9e4e9","order_by":5,"name":"Miranda Kolb","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Miranda","middleName":"","lastName":"Kolb","suffix":""},{"id":343406981,"identity":"196a54f0-07e2-46f6-acc9-97746ebff52d","order_by":6,"name":"Rubing Zhao-Shea","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Rubing","middleName":"","lastName":"Zhao-Shea","suffix":""},{"id":343406982,"identity":"29c4798c-12e3-49d2-be9c-5e2eaae51009","order_by":7,"name":"Michael Baratta","email":"","orcid":"https://orcid.org/0000-0001-7273-1994","institution":"University of Colorado Boulder","correspondingAuthor":false,"prefix":"","firstName":"Michael","middleName":"","lastName":"Baratta","suffix":""},{"id":343406983,"identity":"a736fa8a-0b98-45d0-bf53-ed7327788c9f","order_by":8,"name":"Andrew Tapper","email":"","orcid":"https://orcid.org/0000-0002-8358-6937","institution":"University of Massachusetts Medical School","correspondingAuthor":false,"prefix":"","firstName":"Andrew","middleName":"","lastName":"Tapper","suffix":""}],"badges":[],"createdAt":"2024-06-30 08:05:29","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4661779/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4661779/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":64908469,"identity":"5bc20302-51b0-4f7c-9339-5133ad0dbf66","added_by":"auto","created_at":"2024-09-20 09:19:24","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":1073134,"visible":true,"origin":"","legend":"\u003cp\u003eMice exhibit innate defensive adaptive learning with repeated VLS exposures.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e|\u003c/strong\u003e (\u003cstrong\u003ea\u003c/strong\u003e) Schematic of VLS paradigm. (\u003cstrong\u003eb\u003c/strong\u003e) Representative example of day 1 and day 3 animals’ tracked behavior 60s upon VLS.\u003cstrong\u003e \u003c/strong\u003e(\u003cstrong\u003ec\u003c/strong\u003e) Trace of freezing time (%) relative to VLS (t = 0), across 3 days. (\u003cstrong\u003ed\u003c/strong\u003e) Quantification of freezing time (%) 2 sec upon VLS in (c). One-way RM ANOVA (day effect: F\u003csub\u003e(2,92)\u003c/sub\u003e = 7.082, \u003cem\u003eP\u003c/em\u003e = 0.0025). Dunnett’s multiple comparisons **p \u0026lt; 0.01. (\u003cstrong\u003ee\u003c/strong\u003e) Trace of speed (cm/s) relative to VLS (t = 0), across 3 days.\u003cstrong\u003e \u003c/strong\u003e(\u003cstrong\u003ef\u003c/strong\u003e) Quantification of max speed (cm/s) 2 sec upon VLS in (e). One-way RM ANOVA (day effect: F\u003csub\u003e(2,92)\u003c/sub\u003e = 10.74, \u003cem\u003eP\u003c/em\u003e = 0.0014). Dunnett’s multiple comparisons *p \u0026lt; 0.05, ***p \u0026lt; 0.001. (\u003cstrong\u003eg\u003c/strong\u003e) Trace of time spent inside the nest (%) relative to VLS (t = 0), across 3 days. (\u003cstrong\u003eh\u003c/strong\u003e) Quantification of time in nest (s) after VLS across days. One-way RM ANOVA (day effect: F\u003csub\u003e(2,92)\u003c/sub\u003e = 6.248, \u003cem\u003eP\u003c/em\u003e = 0.0037). Dunnett’s multiple comparisons **p \u0026lt; 0.01.\u003cstrong\u003e \u003c/strong\u003e(\u003cstrong\u003ei\u003c/strong\u003e) Trace of time spent near the wall (%) relative to VLS (t = 0), across 3 days. (\u003cstrong\u003ej\u003c/strong\u003e) Quantification of time spent near the wall (%), 30 sec upon VLS in (i). One-way RM ANOVA (day effect: F\u003csub\u003e(2,92)\u003c/sub\u003e = 4.713, \u003cem\u003eP\u003c/em\u003e = 0.0165). Dunnett’s multiple comparisons *p \u0026lt; 0.05. (n = 31 mice). Data represent mean ± SEM.\u003c/p\u003e","description":"","filename":"OnlineFigure1.png","url":"https://assets-eu.researchsquare.com/files/rs-4661779/v1/e05fdf82ea91053cb3d9a9b0.png"},{"id":64907914,"identity":"29a59b3c-4db6-4813-a436-83a1401596cd","added_by":"auto","created_at":"2024-09-20 09:11:24","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":1358664,"visible":true,"origin":"","legend":"\u003cp\u003eIPN GABAergic neurons respond to VLS and adapt with multiple exposures.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e|\u003c/strong\u003e (\u003cstrong\u003ea\u003c/strong\u003e) \u003cem\u003eLeft\u003c/em\u003e, schematic and representative image of GCaMP injection and fiber placement in the IPN in \u003cem\u003eGAD2\u003c/em\u003e \u003cem\u003eCre \u003c/em\u003emice. Scale bar 100μm. \u003cem\u003eRight\u003c/em\u003e, schematic of VLS paradigm. (\u003cstrong\u003eb\u003c/strong\u003e) Example of speed trace (cm/s) compared to time-locked IPN \u003cem\u003eGAD2\u003c/em\u003e fiber photometry signals (dF/F0) relative to VLS (t = 0), across 3 days. Heatmap representations (\u003cstrong\u003ec\u003c/strong\u003e) and z-score values (\u003cstrong\u003ed\u003c/strong\u003e) of time-locked IPN \u003cem\u003eGAD2\u003c/em\u003e neuronal activity relative to VLS (t = 0), across 3 days. (\u003cstrong\u003ee\u003c/strong\u003e) Quantification of responses in (d) as maximum z-score values detected 10 sec upon VLS. One-way RM ANOVA (day effect: F\u003csub\u003e(2,23) \u003c/sub\u003e= 5.061, \u003cem\u003eP\u003c/em\u003e = 0.0328). # p \u0026lt; 0.05. Heatmap representations (\u003cstrong\u003ef\u003c/strong\u003e) and z-score values (\u003cstrong\u003eg\u003c/strong\u003e) of time-locked IPN \u003cem\u003eGAD2\u003c/em\u003e neuronal activity relative to the time of nest entry (t = 0), across 3 days. (\u003cstrong\u003eh\u003c/strong\u003e) Quantification of activity in (f) as minimum z-score values detected 10 sec upon nest entry. One-way RM ANOVA (day effect: F\u003csub\u003e(2,23) \u003c/sub\u003e= 1.348, \u003cem\u003eP\u003c/em\u003e = 0.2916). (n = 8 mice). Data represent mean ± SEM.\u003c/p\u003e","description":"","filename":"OnlineFigure2.png","url":"https://assets-eu.researchsquare.com/files/rs-4661779/v1/95e0dc9fad17a8670f6d3f7c.png"},{"id":64909465,"identity":"7a2577f8-72c8-4914-b747-19ffa1031b7b","added_by":"auto","created_at":"2024-09-20 09:27:24","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":1601452,"visible":true,"origin":"","legend":"\u003cp\u003ePhotoinhibition of IPN GABAergic neurons reduces early and late defensive behaviors.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e|\u003c/strong\u003e (\u003cstrong\u003ea\u003c/strong\u003e) Schematic and representative image of NpHR3.0 injection and fiber placement in the IPN in \u003cem\u003eGAD2 Cre\u003c/em\u003e mice. Scale bar 100μm. (\u003cstrong\u003eb\u003c/strong\u003e) Schematic representation of 3-day VLS paradigm with NpHR inhibition on days 1 and 3 (yellow). (\u003cstrong\u003ec\u003c/strong\u003e) Traces of freezing time (%) relative to VLS (t = 0), across 3 days in IPN \u003cem\u003eGAD2\u003c/em\u003e eGFP and NpHR animals. (\u003cstrong\u003ed\u003c/strong\u003e) Quantification of freezing time (%) 2 sec upon VLS in (c). Two-way RM ANOVA (day effect: F\u003csub\u003e(2,32)\u003c/sub\u003e = 3.928, \u003cem\u003eP\u003c/em\u003e = 0.0312; treatment effect: F\u003csub\u003e(1,16)\u003c/sub\u003e = 6.679, \u003cem\u003eP\u003c/em\u003e = 0.0200; interaction: F\u003csub\u003e(2,32)\u003c/sub\u003e = 0.2304, \u003cem\u003eP\u003c/em\u003e = 0.7956), treatment effect # p \u0026lt; 0.05, day effect $ p \u0026lt; 0.05. (\u003cstrong\u003ee\u003c/strong\u003e) Traces of speed (cm/s) relative to VLS (t = 0), across 3 days in IPN \u003cem\u003eGAD2\u003c/em\u003e eGFP and NpHR animals.\u003cstrong\u003e \u003c/strong\u003e(\u003cstrong\u003ef\u003c/strong\u003e) Quantification of max speed (cm/s) 2 sec upon VLS in (e). Two-way RM ANOVA (day effect: F\u003csub\u003e(2,32) \u003c/sub\u003e= 2.299, \u003cem\u003eP\u003c/em\u003e = 0.1185; treatment effect: F\u003csub\u003e(1,16) \u003c/sub\u003e= 6.514, \u003cem\u003eP\u003c/em\u003e = 0.0213; interaction: F\u003csub\u003e(2,32)\u003c/sub\u003e = 0.0294, \u003cem\u003eP\u003c/em\u003e = 0.9711), treatment effect # p \u0026lt; 0.05. (\u003cstrong\u003eg\u003c/strong\u003e) Trace of time spent inside the nest (%) relative to VLS (t = 0), across 3 days in IPN \u003cem\u003eGAD2\u003c/em\u003e eGFP and NpHR animals. (\u003cstrong\u003eh\u003c/strong\u003e) Quantification of time in nest (s) after VLS across days. Two-way RM ANOVA (day effect: F\u003csub\u003e(2,32)\u003c/sub\u003e = 17.02, \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.0001; treatment effect: F\u003csub\u003e(1,16)\u003c/sub\u003e = 4.336, \u003cem\u003eP\u003c/em\u003e = 0.0537; interaction: F\u003csub\u003e(2, 32) \u003c/sub\u003e= 11.45, \u003cem\u003eP\u003c/em\u003e = 0.0002), Tukey’s multiple comparisons *p\u0026lt;0.05. (n = 6 eGFP and 12 NpHR3.0 mice). Data represent mean ± SEM.\u003c/p\u003e","description":"","filename":"OnlineFigure3.png","url":"https://assets-eu.researchsquare.com/files/rs-4661779/v1/1ba01fb2fc026464c8ff04a6.png"},{"id":64908471,"identity":"2ca62b47-9c6a-4a90-9800-9357164078ae","added_by":"auto","created_at":"2024-09-20 09:19:24","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":1351965,"visible":true,"origin":"","legend":"\u003cp\u003eIPN GAD2 neurons projecting to the LDTg are engaged by VLS and adapt with multiple exposures.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e| \u003c/strong\u003e(\u003cstrong\u003ea\u003c/strong\u003e) \u003cem\u003eLeft\u003c/em\u003e, schematic of retrograde viral injection and optic fiber strategy used. \u003cem\u003eMiddle\u003c/em\u003e, representative images of retroviral mediated GCaMP injection in the LDTg and fiber placement in the IPN in \u003cem\u003eGAD2 Cre\u003c/em\u003e mice. Scale bar 100μm. \u003cem\u003eRight\u003c/em\u003e, schematic of VLS paradigm. (\u003cstrong\u003eb\u003c/strong\u003e) Example of speed trace (cm/s) compared to time-locked IPNàLDTg \u003cem\u003eGAD2\u003c/em\u003e fiber photometry signals (dF/F0) relative to VLS (t = 0), across 3 days. Heatmap representations (\u003cstrong\u003ec\u003c/strong\u003e) and z-score values (\u003cstrong\u003ed\u003c/strong\u003e) of time-locked IPNàLDTg \u003cem\u003eGAD2\u003c/em\u003e neuronal activity relative to VLS (t = 0), across 3 days. (\u003cstrong\u003ee\u003c/strong\u003e) Quantification of responses in (d) as maximum z-score values detected 10 sec upon VLS. One-way RM ANOVA (day effect: F\u003csub\u003e(2,20) \u003c/sub\u003e= 5.409, \u003cem\u003eP\u003c/em\u003e = 0.050),\u003cu\u003e \u003c/u\u003eDunnett’s multiple comparisons. ** p \u0026lt; 0.01. Heatmap representations (\u003cstrong\u003ef\u003c/strong\u003e) and z-score values (\u003cstrong\u003eg\u003c/strong\u003e) of time-locked IPNàLDTg \u003cem\u003eGAD2\u003c/em\u003e neuronal activity relative to the time of nest entry (t = 0), across 3 days. (\u003cstrong\u003eh\u003c/strong\u003e) Quantification of activity in (f) as minimum z-score values detected 10 sec upon nest entry. One-way RM ANOVA (day effect: F\u003csub\u003e(2,20) \u003c/sub\u003e= 1.856, \u003cem\u003eP\u003c/em\u003e = 0.2059). (n = 7 mice). Data represent mean ± SEM.\u003c/p\u003e","description":"","filename":"OnlineFigure4.png","url":"https://assets-eu.researchsquare.com/files/rs-4661779/v1/0ed96133412b470106c1f910.png"},{"id":64907930,"identity":"1f6b3b72-f517-426b-bb79-898366669b56","added_by":"auto","created_at":"2024-09-20 09:11:25","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":1774672,"visible":true,"origin":"","legend":"\u003cp\u003eIPN\u003cstrong\u003e→\u003c/strong\u003eLDTg GAD2 neuronal circuit controls defensive adaptive learning.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e|\u003c/strong\u003e (\u003cstrong\u003ea\u003c/strong\u003e) \u003cem\u003eLeft\u003c/em\u003e,\u003cstrong\u003e \u003c/strong\u003eschematic and representative image of retrograde NpHR3.0 injection in the LDTg and fiber placement in the IPN in \u003cem\u003eGAD2 Cre\u003c/em\u003e mice. Scale bar 100μm. \u003cem\u003eRight\u003c/em\u003e, schematic representation of 3-day VLS paradigm with NpHR inhibition on days 1 and 3 (yellow). (\u003cstrong\u003eb\u003c/strong\u003e) Traces of freezing time (%) relative to VLS (t = 0), across 3 days in IPNàLDTg \u003cem\u003eGAD2\u003c/em\u003e eGFP and NpHR animals. (\u003cstrong\u003ec\u003c/strong\u003e) Quantification of freezing time (%) 2 sec upon VLS in (b). Two-way RM ANOVA (day effect: F\u003csub\u003e(2,60)\u003c/sub\u003e = 11.16, \u003cem\u003eP\u003c/em\u003e = 0.0001; treatment effect: F\u003csub\u003e(1,30)\u003c/sub\u003e = 3.338, \u003cem\u003eP\u003c/em\u003e = 0.0777; interaction: F\u003csub\u003e(2,60)\u003c/sub\u003e = 0.7984, \u003cem\u003eP\u003c/em\u003e = 0.4548), day effect $$$ p \u0026lt; 0.001. (\u003cstrong\u003ed\u003c/strong\u003e) Traces of speed (cm/s) relative to VLS (t = 0), across 3 days in IPNàLDTg \u003cem\u003eGAD2\u003c/em\u003e eGFP and NpHR animals.\u003cstrong\u003e \u003c/strong\u003e(\u003cstrong\u003ee\u003c/strong\u003e) Quantification of max speed (cm/s) 2 sec upon VLS in (d). Two-way RM ANOVA (day effect: F\u003csub\u003e(2,60) \u003c/sub\u003e= 0.6815, \u003cem\u003eP\u003c/em\u003e = 0.4984; treatment effect: F\u003csub\u003e(1,30) \u003c/sub\u003e= 9.599, \u003cem\u003eP\u003c/em\u003e = 0.0042; interaction: F\u003csub\u003e(2,60)\u003c/sub\u003e = 0.1906, \u003cem\u003eP\u003c/em\u003e = 0.8270). (\u003cstrong\u003ef\u003c/strong\u003e) Trace of time spent inside the nest (%) relative to VLS (t = 0), across 3 days in IPNàLDTg \u003cem\u003eGAD2\u003c/em\u003e eGFP and NpHR animals. (\u003cstrong\u003eg\u003c/strong\u003e) Quantification of time in nest (s) after VLS across days. Two-way RM ANOVA (day effect: F\u003csub\u003e(2,60)\u003c/sub\u003e = 6.927, \u003cem\u003eP\u003c/em\u003e = 0.0020; treatment effect: F\u003csub\u003e(1,30)\u003c/sub\u003e = 3.496, \u003cem\u003eP\u003c/em\u003e = 0.0713; interaction: F\u003csub\u003e(2,60) \u003c/sub\u003e= 3.192, \u003cem\u003eP\u003c/em\u003e = 0.0481), Tukey’s multiple comparisons *p\u0026lt;0.05. (n = 15 eGFP and 17 NpHR3.0 mice). Data represent mean ± SEM.\u003c/p\u003e","description":"","filename":"OnlineFigure5.png","url":"https://assets-eu.researchsquare.com/files/rs-4661779/v1/8bb7f291646d6ebaf3e7fbe8.png"},{"id":64907918,"identity":"52e5615a-8c17-436c-a588-20628e0a6db2","added_by":"auto","created_at":"2024-09-20 09:11:24","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":1883945,"visible":true,"origin":"","legend":"\u003cp\u003eIPN Sst+ neurons encode threat and safety signals and control late defensive actions.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e|\u003c/strong\u003e (\u003cstrong\u003ea) \u003c/strong\u003e\u003cem\u003eLeft\u003c/em\u003e, schematic of viral injection and fiber placement; representative image of viral-mediated GCaMP expression and fiber placement in IPN of Sst\u003csup\u003eCre \u003c/sup\u003emice. Scale bar 100μm. \u003cem\u003eRight\u003c/em\u003e, VLS paradigm. (\u003cstrong\u003eb\u003c/strong\u003e)\u003cstrong\u003e \u003c/strong\u003eRepresentative traces of IPN Sst\u003csup\u003e+\u003c/sup\u003e activity recordings (dF/F0) compared to speed (cm/s) relative to VLS presentations (t=0), across days 1–3. Heatmap representations (\u003cstrong\u003ec\u003c/strong\u003e) and z-score values (\u003cstrong\u003ed\u003c/strong\u003e) of time-locked IPN Sst\u003csup\u003e+\u003c/sup\u003e neuronal activity relative to VLS (t = 0), across 3 days. (\u003cstrong\u003ee\u003c/strong\u003e) Quantification of responses in (d) as maximum z-score values detected 10 sec upon VLS. One-way RM ANOVA (day effect: F\u003csub\u003e(2,14)\u003c/sub\u003e = 0.4439, \u003cem\u003eP\u003c/em\u003e = 0.6262). Heatmap representations (\u003cstrong\u003ef\u003c/strong\u003e) and z-score values (\u003cstrong\u003eg\u003c/strong\u003e) of time-locked IPN Sst\u003csup\u003e+\u003c/sup\u003e neuronal activity relative to the time of nest entry (t = 0), across 3 days. (\u003cstrong\u003eh\u003c/strong\u003e) Quantification of activity in (g) as maximum z-score values detected during nest entry. One-way RM ANOVA (day effect: F\u003csub\u003e(2,14) \u003c/sub\u003e= 0.5364, \u003cem\u003eP\u003c/em\u003e = 0.5166). (n = 5 mice). (\u003cstrong\u003ei\u003c/strong\u003e) \u003cem\u003eLeft\u003c/em\u003e, Schematic of taCasp3-TEVp injection in the IPN in \u003cem\u003eSst Cre\u003c/em\u003e mice and representative image of Sst immunostaining (green) in the IPN of control and ablation animals. Scale bar 50μm. \u003cem\u003eRight\u003c/em\u003e, Schematic representation of 3-day VLS paradigm. (\u003cstrong\u003ej\u003c/strong\u003e) Trace of time spent inside the nest (%) relative to VLS (t = 0), across 3 days in IPN Sst mCherry and taCasp3-TEVp animals. (\u003cstrong\u003ek\u003c/strong\u003e) Quantification of time in nest (s) after VLS across days. Two-way RM ANOVA (day effect: F\u003csub\u003e(2,38)\u003c/sub\u003e = 0.0254, \u003cem\u003eP\u003c/em\u003e = 0.9713; treatment effect: F\u003csub\u003e(1,19)\u003c/sub\u003e = 8.818, \u003cem\u003eP\u003c/em\u003e = 0.0079; interaction: F\u003csub\u003e(2, 38) \u003c/sub\u003e= 1.858, \u003cem\u003eP\u003c/em\u003e = 0.1698), treatment effect ## p \u0026lt; 0.01. (n = 9 mCherry and 12 taCasp3-TEVp mice). Data represent mean ± SEM.\u003c/p\u003e","description":"","filename":"OnlineFigure6.png","url":"https://assets-eu.researchsquare.com/files/rs-4661779/v1/8d5aa9cb925bcd33df2baaad.png"},{"id":64910754,"identity":"71d77738-e405-433b-9b76-f3ae184fa73d","added_by":"auto","created_at":"2024-09-20 09:43:31","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2071442,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4661779/v1/47c21b32-0869-4f72-96e8-2daecf9edc00.pdf"},{"id":64907917,"identity":"fca2aba5-60e7-4370-bf96-f36ab0442e2a","added_by":"auto","created_at":"2024-09-20 09:11:24","extension":"tif","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":7374952,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cbr\u003e\u003c/p\u003e","description":"","filename":"SupplFigure1.tif","url":"https://assets-eu.researchsquare.com/files/rs-4661779/v1/c63b96b8e120f4428f944f2c.tif"},{"id":64907921,"identity":"9f5e4847-8bd4-4de0-ab1f-91d9604ccc05","added_by":"auto","created_at":"2024-09-20 09:11:24","extension":"tif","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":11178268,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cbr\u003e\u003c/p\u003e","description":"","filename":"SupplFigure2.tif","url":"https://assets-eu.researchsquare.com/files/rs-4661779/v1/6086c03b4ee5219e4c013160.tif"},{"id":64907920,"identity":"cee05b85-316f-45a7-b470-bfe5ce5df00e","added_by":"auto","created_at":"2024-09-20 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09:27:24","extension":"tif","order_by":7,"title":"","display":"","copyAsset":false,"role":"supplement","size":17752240,"visible":true,"origin":"","legend":"","description":"","filename":"SupplFigure7.tif","url":"https://assets-eu.researchsquare.com/files/rs-4661779/v1/29fab00d87b5241c8c7483c1.tif"},{"id":64910753,"identity":"4c0940b1-b345-4fe6-b419-d318c1fb163b","added_by":"auto","created_at":"2024-09-20 09:43:24","extension":"tif","order_by":8,"title":"","display":"","copyAsset":false,"role":"supplement","size":7510496,"visible":true,"origin":"","legend":"","description":"","filename":"SupplFigure8.tif","url":"https://assets-eu.researchsquare.com/files/rs-4661779/v1/0490078b15c5348b21cdb173.tif"},{"id":64910752,"identity":"93aa40ab-1dab-4f42-9fab-73f8ab646d8f","added_by":"auto","created_at":"2024-09-20 09:43:24","extension":"tif","order_by":9,"title":"","display":"","copyAsset":false,"role":"supplement","size":11344248,"visible":true,"origin":"","legend":"","description":"","filename":"SupplFigure9.tif","url":"https://assets-eu.researchsquare.com/files/rs-4661779/v1/e35d34b15f5ad8cc7a67187b.tif"},{"id":64907923,"identity":"d6fe47b0-f3ca-4a59-8ab6-b7b464f0aec0","added_by":"auto","created_at":"2024-09-20 09:11:24","extension":"tif","order_by":10,"title":"","display":"","copyAsset":false,"role":"supplement","size":8265716,"visible":true,"origin":"","legend":"","description":"","filename":"SupplFigure10.tif","url":"https://assets-eu.researchsquare.com/files/rs-4661779/v1/382fe4e4dd898ed6733cd448.tif"},{"id":64907931,"identity":"13fdb6a6-48e4-46d1-8b08-87baada7ba0a","added_by":"auto","created_at":"2024-09-20 09:11:25","extension":"tif","order_by":11,"title":"","display":"","copyAsset":false,"role":"supplement","size":19704496,"visible":true,"origin":"","legend":"","description":"","filename":"SupplFigure11.tif","url":"https://assets-eu.researchsquare.com/files/rs-4661779/v1/953e06105c0ceec8d0dbfd53.tif"},{"id":64910035,"identity":"0d12c78c-cf81-4310-ab20-41cb9f4d3f62","added_by":"auto","created_at":"2024-09-20 09:35:24","extension":"docx","order_by":12,"title":"","display":"","copyAsset":false,"role":"supplement","size":650741,"visible":true,"origin":"","legend":"\u003cp\u003eSupplementary information\u003c/p\u003e\n\u003cp\u003eSupplementary information is available at MP’s website.\u003c/p\u003e","description":"","filename":"WilliamsEWetal2024Supplementary.docx","url":"https://assets-eu.researchsquare.com/files/rs-4661779/v1/56a1ee509e6cca9157734320.docx"}],"financialInterests":"The authors have declared there is \u003cb\u003eNO\u003c/b\u003e conflict of interest to disclose","formattedTitle":"Interpeduncular GABAergic neuron function controls threat processing and innate defensive adaptive learning","fulltext":[{"header":"Introduction","content":"\u003cp\u003eIndividuals select optimal defensive strategies, such as escape or freezing, to avoid danger when threat is present (\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e). Nevertheless, with repeated exposures to a potential threat without evidence of an aversive outcome, defensive behaviors must undergo adaptive learning, thus contributing to efficient action selection. Numerous neuropsychiatric conditions, including anxiety disorders, manifest maladaptation of threat responses (\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e), highlighting the importance of understanding these basic neurobehavioral processes.\u003c/p\u003e \u003cp\u003eExposure to an overhead dark visual looming stimulus (VLS) naturally elicits innate defensive responses across multiple species, from fish to humans (\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e). In rodents, exploring an open area supplied with a nest as a shelter, the detection of an expanding VLS to the upper visual field triggers an immediate defensive behavior of freezing and escaping towards a sheltered area, seeking protection (\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e). Whether rodents shift their defensive responses to optimal behavioral sequences after repeated VLS and the mechanisms that support this innate defensive adaptive learning remain largely unknown.\u003c/p\u003e \u003cp\u003eProcessing sensory information and coordinating appropriate motor output for defensive behaviors requires complex neural circuits (\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e). Rodent and human studies have implicated an emerging circuit, arising in the habenula (Hb), that contributes to threat-related behaviors (\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e). The habenula nuclei play a crucial role in regulating emotional, motivational, and cognitive behaviors (\u003cspan additionalcitationids=\"CR10 CR11\" citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e). Specifically, the medial part of the habenula (mHb) sends descending projections almost exclusively to the interpeduncular nucleus (IPN) of the midbrain (\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e), an axis implicated in anxiety (\u003cspan additionalcitationids=\"CR15 CR16 CR17 CR18 CR19 CR20\" citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e) and fear responses (\u003cspan additionalcitationids=\"CR23 CR24\" citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e). The IPN is highly enriched in GABAergic neurons that establish reciprocal connections with regions involved in motivation and affective behaviors, including the raphe, locus coeruleus, and laterodorsal tegmental nucleus (LDTg)(\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e). Recent work indicates that the habenulo-interpeduncular axis, and IPN projections to the LDTg, mediate aversive and reward-related behaviors (\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e, \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e). Yet, whether the IPN and associated neuronal circuits are recruited by VLS to adjust defensive behaviors has never been explored.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eAnimals\u003c/h2\u003e \u003cp\u003e All experiments followed the guidelines provided by the National Research Council with approved animal protocols from the Institutional Animal Care and Use Committee of the University of Massachusetts Chan Medical School. C57Bl/6J (Stock #000664, Jackson), \u003cem\u003eGAD2\u003c/em\u003e\u003csup\u003e\u003cem\u003eCre\u003c/em\u003e\u003c/sup\u003e (Stock #10802, Jackson) and \u003cem\u003eSst\u003c/em\u003e\u003csup\u003e\u003cem\u003eCre\u003c/em\u003e\u003c/sup\u003e (Stock #013044, Jackson) mice were used. \u003cem\u003eCre\u003c/em\u003e lines were crossed with C57Bl/6J mice and only heterozygous animals used. All experiments included male mice. IPN Sst ablation experiments included males and females. Subject mice were kept under a reverse 12h light/dark cycle (lights ON at 7P.M.) for 3\u0026ndash;4 weeks with \u003cem\u003ead libitum\u003c/em\u003e access to food and water, and individually housed for at least 5 days before any behavioral testing. Experiments were performed during the dark cycle phase (8A.M. to 5P.M.).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eViral preparations\u003c/h2\u003e \u003cp\u003eBiosensors, optogenetic and control plasmids packaged into viral particles were purchased from Addgene. For fiber photometry experiments we used pAAV.CAG.Flex.GCaMP6m.WPRE.SV40 (#100839-AAV5, 2.6x10\u003csup\u003e13\u003c/sup\u003eGC/ml), pGP.AAV.CAG.Flex.-jGCaMP7s.WPRE (#104495-AAVrg, 1.1x10\u003csup\u003e13\u003c/sup\u003eGC/ml). For tracing and optogenetic experiments we used pAAV.hSyn.DIO.EGFP (#50457-AAV5, 1.3x10\u003csup\u003e13\u003c/sup\u003eGC/ml and -AAVrg, 1.4x10\u003csup\u003e13\u003c/sup\u003eGC/ml), pAAV.hSyn.DIO.mCherry (#50459-AAV5, 1.8x10\u003csup\u003e13\u003c/sup\u003eGC/ml and -AAVrg, 1.5x10\u003csup\u003e13\u003c/sup\u003eGC/ml), pAAV.Ef1a.DIO.eNpHR3.0.EYFP (#26966-AAV5, 3.8x10\u003csup\u003e12\u003c/sup\u003eGC/ml), pAAV.Ef1a.doublefloxed.hChR2(H134R).mCherry.WPRE.HGHpA (#20297-AAV5 1.2x10\u003csup\u003e13\u003c/sup\u003eGC/ml) and pAAV.flex.taCasp3-TEVp (#45580-AAV5, 2.5x10\u003csup\u003e13\u003c/sup\u003eGC/ml). The viral stock pAAV.Ef1a.DIO.-eNpHR3.0.EYFP (#AV9115-rAAV2, 5.8x10\u003csup\u003e12\u003c/sup\u003eVM/ml) was obtained from UNC GTC Vector Core.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eStereotaxic surgeries\u003c/h2\u003e \u003cp\u003eSurgeries were performed under aseptic conditions as previously described (\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e). Mice (6\u0026ndash;8 weeks old) were deeply anaesthetized using 100 mg/kg ketamine (VEDCO) and 10 mg/kg xylazine (LLOYD) and placed on a stereotaxic frame (Stoelting Co.). Viral solutions were microinjected at a controlled rate of 50 nl/min using a gas-tight 33G 10-\u0026micro;l neurosyringe (1701RN; Hamilton). Injection coordinates were (in mm, anteroposterior, mediolateral, dorsoventral and angle): IPN (\u0026minus;\u0026thinsp;3.4, \u0026minus;\u0026thinsp;0.5, \u0026minus;\u0026thinsp;4.86, 6\u0026deg;) and LDTg (-5.34, \u0026plusmn;\u0026thinsp;0.4, -3.2, 0\u0026deg;). Viral volumes were 300 nl (IPN) and 300 nl/site (LDTg). For fiber photometry and optogenetic experiments, 3\u0026ndash;5 weeks post-viral injection, an optic fiber implant (200-\u0026micro;m core diameter; 0.53N.A., Doric Lenses) held in a magnetic aluminum receptacle (Doric Lenses) was placed above the IPN and secured into the skull using adhesive (C\u0026amp;B Metabond cement, Parkell Inc.) followed by dental cement (Cerebond, PlasticsOne). Mice received IP injections of 1 mg/kg ketoprofen analgesic (Zoetis) and monitored for recovery. Animals showing no viral or off-target site viral expression or incorrect optic fiber placement (\u0026lt;\u0026thinsp;10%) were excluded from analysis.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eBehavioral experiments\u003c/h2\u003e \u003cdiv id=\"Sec7\" class=\"Section3\"\u003e \u003ch2\u003eVisual looming stimulus (VLS) paradigm\u003c/h2\u003e \u003cp\u003eThe apparatus consisted of a rectangular Plexiglass maze (40x22x30 cm) with a projector screen (30x20 cm) above the arena and a rectangular shelter (10x12 cm) in one corner. All mice habituated to the apparatus and illumination settings for 8\u0026ndash;10 min. After 24h, mice acclimated to the apparatus for 2\u0026ndash;5 min before a VLS was randomly displayed from the screen while they actively explored the arena. Each VLS consisted of 15 consecutive dark expansions of 0.5s length and each mouse received 4\u0026ndash;7 looming trials per day with a minimum inter-looming trial interval of 60s. The VLS test session was repeated for 3 consecutive days. For side-VLS, the screen was displayed from a wall view. The apparatus was cleaned between animals with 0.1% Micro-90 solution. A video-camera was used to record and track animals\u0026rsquo; behavior using Ethovision XT (v15.0). The arena was subdivided in the nest area (12x10 cm), a safety zone adjacent to the nest (10x10 cm), a trigger zone where the VLS were displayed (12x12 cm) and a zone near the walls (5 cm). Early defensive responses included immediate freezing and maximum speed which were reported 2 sec upon VLS initiation, as well as latency to enter the nest and escape run (maximum speed 10 sec upon VLS). Late defensive responses included time spent inside the nest after the first nest entry once the VLS was presented, as well as time spent near the walls. The percent of time in each zone was estimated within 30 upon VLS initiation and averaged per each animal. Latency to and time in nest were manually scored by an experimenter blind to animals\u0026rsquo; conditions.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eFoot shock\u003c/h2\u003e \u003cp\u003eMice were habituated to a fear conditioning cage. GCaMP fluorescence from IPN neurons was recorded for 2 min before the first shock. During a 15-min foot-shock period, ten shocks (0.5mA, 1-s duration) were delivered at random intervals and time-stamped into the photometry recording via a transistor-transistor logic (TTL) pulse from the fear conditioning system.\u003c/p\u003e \u003cdiv id=\"Sec9\" class=\"Section3\"\u003e \u003ch2\u003eOpen field\u003c/h2\u003e \u003cp\u003eThe apparatus consisted of an open-field chamber (42x38x30 cm). Each mouse was given 10 min to explore, and the time spent in the center and outer parts of the chamber was tracked from a video recording using Ethovision XT.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section3\"\u003e \u003ch2\u003eElevated plus maze\u003c/h2\u003e \u003cp\u003eThe elevated plus maze (EPM) apparatus consisted of a central junction (5\u0026times;5 cm) and had four arms elevated 45 cm above the floor with each arm positioned at 90\u0026deg; relative to the adjacent arms. Two closed arms were enclosed by high walls (30\u0026times;5\u0026times;15 cm) and the open arms were exposed (30\u0026times;5\u0026times;0.25 cm). All mice were given 5 min of free exploration.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eTail lift\u003c/h2\u003e \u003cp\u003eAnimals were picked up by their tails by an experimenter while they were actively exploring the home cage.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eFiber photometry and data analysis\u003c/h2\u003e \u003cp\u003eFlorescent signals from biosensors were recorded using a Doric Instruments Fiber Photometry System as previously described (\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e). A LED driver delivered excitation light at 465 nm and at 405 nm (~\u0026thinsp;30-60\u0026micro;W output at the fiber tip). The light was reflected into a 200\u0026micro;m 0.53N.A. optic fiber patch cord via the Dual Fluorescence Minicube. Emissions were detected with a femtowatt photoreceiver (Model 2151, Newport). Sampling (12 kHz) and lock-in demodulation of the fluorescence signals were controlled by Doric Neuroscience Studio software with a decimation factor of 50. A behavioral camera synchronized the photometry recordings with time-locked behavioral tracking systems.\u003c/p\u003e \u003cp\u003eFiber photometry data analysis was performed using custom-written Matlab scripts. The 405 nm channel was scaled to the 465 nm by applying a least mean squares linear regression. Scaled signals were used to calculate the ΔF/F\u003csub\u003e0\u003c/sub\u003e where ΔF/F\u003csub\u003e0\u003c/sub\u003e = (465nm signal \u0026ndash; fitted 405nm signal)/fitted 405nm signal. Z-scores were calculated using the average baseline of ΔF/F\u003csub\u003e0\u003c/sub\u003e values from the \u0026minus;\u0026thinsp;1.0 sec prior to the onset of VLS (considered as time zero, t\u0026thinsp;=\u0026thinsp;0). The max and mean z-score were estimated between t\u0026thinsp;=\u0026thinsp;0 and 10 sec upon VLS and averaged per animal. The min z-score for nest entry was estimated between t\u0026thinsp;=\u0026thinsp;0 (nest entry) and 10 sec upon nest entry and averaged per animal. For IPN Sst\u003csup\u003e+\u003c/sup\u003e GCaMP recordings, the max z-scores were determined \u0026minus;\u0026thinsp;2 to 2 sec time-locked to nest entry.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eOptogenetics\u003c/h2\u003e \u003cp\u003eOptic fiber implants were connected to a patch cable (Doric Lenses) and a commutator (rotary joint; LEDFRJ-B_FC for blue light and LEDFRJ-A_FC for yellow light, Doric Lenses), by means of an FC/SMC adapter to allow unrestricted movement, as previous reported (\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e). Mice habituated for 8\u0026ndash;10 min to the VLS apparatus without receiving light photostimulation. On day 1 of VLS test, mice freely explored the apparatus for 2\u0026ndash;5 min before VLS were displayed. A high-power LED driver (DC2200, Thorlabs) was used to generate light pulses time locked to VLS events at intensity\u0026thinsp;~\u0026thinsp;2\u0026ndash;5 mW at the fiber tip. Light photoinhibition (593nm, constant light) was delivered in time-locked mode by an experimenter blind to animals\u0026rsquo; conditions, 2 sec prior, during and 2 sec post each VLS event. On day 2 of the test, mice were subjected to the VLS paradigm with no light delivery. Day 3 followed the same light stimulation protocol as day 1. For optogenetic photostimulation, light pulses (473nm, 20Hz, 12ms pulse, 3s) were delivered at intervals\u0026thinsp;\u0026gt;\u0026thinsp;90s on days 1 and 3. No VLS was displayed. All sessions were video recorded from above (HDR-CX4440 camera, SONY) and computationally analyzed with Ethovision XT.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eImmunostaining and microscopy\u003c/h2\u003e \u003cp\u003eImmunohistochemistry and microscopy were performed as described previously (\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e). Mice received sodium pentobarbital (200 mg/kg) and transcardially perfused with ice-cold 0.1 M phosphate buffer saline (PBS, pH7.4) followed by 10 ml of cold 4% (W/V) paraformaldehyde (PFA). Brains were post-fixed in 4% PFA before transferred to 30% sucrose. Coronal sections (25 \u0026micro;m) were obtained using a freezing microtome (HM430; Thermo Fisher Scientific, MA, USA). Brain sections were permeabilized with 0.5% Triton X-100 (Sigma) for 10 min, blocked with 5% donkey serum (DS, Sigma) for 30 min and incubated with the primary antibody (rabbit anti-Sst, 1:700, sc-13099) overnight at 4 \u0026ordm;C. Slices were incubated in secondary antibody for 2 h (1:800; Life Technologies; donkey anti-rabbit 594, R37119). Nuclei were counterstained with DAPI. Viral expression was visualized using the endogenous fluorescence of the virus. All slices were imaged using a fluorescent microscope (Zeiss, Carl Zeiss MicroImmagine, Inc., NY, USA) connected to computer-associated image analyzer software (Axiovision Rel., 4.6.1).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eData were analyzed by means of two-tailed unpaired \u003cem\u003et\u003c/em\u003e-test, one-way and two-way ANOVAs with/without repeated-measures (RM), or the restricted maximum likelihood (REML) mixed model, as indicated. Dunnett\u0026rsquo;s or Tukey\u0026rsquo;s \u003cem\u003epost hoc\u003c/em\u003e tests were used for multiple comparisons. Two-tailed Pearson r was used for correlation analysis. Comparisons of z-scores photometry signals were made using the calculated average for each animal. Each data set was tested for normal distribution prior to analysis and presented as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard error of the mean (SEM). All statistical analyses were performed in GraphPad Prism 10.1.0. Software (Graphpad Software Inc.) and statistical significance was established at p\u0026thinsp;\u0026lt;\u0026thinsp;0.05.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cp\u003e \u003cb\u003eInnate adaptive defensive learning: mice adjust behavioral response to a potential threat.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eWe implemented the VLS paradigm to investigate an animal\u0026rsquo;s capacity to adjust innate defensive behaviors in the absence of a real risk (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea). Mice were placed in a plexiglass apparatus that contained a rectangular nest in one corner and a projector screen that displayed a VLS while animals actively explored the arena. The detection of a VLS from above, but not from a side view (Supplementary Fig.\u0026nbsp;1a-c), triggered an immediate defensive response of freezing followed by running to the nest (here defined as early responses to VLS). Animals also spent a significant amount of time in the confines of the shelter, presumably avoiding potential threat, before engaging in exploratory behaviors (here defined as late responses to VLS). However, with multiple exposures of an overhead VLS for 3 consecutive days, without evidence of aversive outcomes, mice learned to adjust both early and late VLS-evoked innate defensive strategies (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eb-j). Across the 3 days, immediate freezing significantly reduced (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ec-d), whereas speed increased (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ee-f). Mice continued to run with similar maximum speed (10 sec upon VLS) and exhibited similar escape latencies to the nest in response to repeated VLS over the 3 days (Supplementary Fig.\u0026nbsp;1d-e). To measure late VLS-induced defensive behaviors, we tracked the animal\u0026rsquo;s position throughout arena zones (Supplementary Fig.\u0026nbsp;1f and methods). The amount of time spent inside the nest upon VLS exposure decreased over repeated days (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eg-h), while animals shifted to more exploratory behavior near the walls (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ei-j). We did not observe adaptive changes with time spent in the trigger zone, where the VLS is presented (Supplementary Fig.\u0026nbsp;1g-h), or time in the safety zone adjacent to the nest (Supplementary Fig.\u0026nbsp;1i-j). Notably, adjustment of defensive responses was not detected within a single-day trial session (Supplementary Fig.\u0026nbsp;2), suggesting that optimization of defensive strategies may reflect learning and consolidation processes.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eWe performed linear correlation analysis to further investigate threat-evoked innate adaptive defensive learning with repeated VLS (Supplementary Fig.\u0026nbsp;3 and Supplementary Tables\u0026nbsp;1\u0026ndash;6). For early responses to VLS, we found that latency to the nest showed a strong negative correlation with the maximum speed the animals reached 10 sec upon VLS, which was maintained across the 3 sessions, indicating the faster the animals ran, the earlier they entered the nest. Interestingly, the early response of freezing upon VLS initiation predicted late defensive responses such as the total amount of time the animals would spend inside the nest. Time spent in the nest was also positively correlated with escape including latency to the nest and maximum speed 10 sec upon VLS only on day 1, but not in later sessions. These results suggest that escape behaviors (i.e. running to the nest) and avoidance behaviors (i.e. time spent inside the nest) are related to each other at initial sessions but may become more dissociable once the animals learn to adjust defensive responses.\u003c/p\u003e \u003cp\u003e \u003cb\u003eExposure to potential threat engages activity of IPN\u003c/b\u003e \u003cb\u003eGAD2\u003c/b\u003e \u003cb\u003eneurons that adjusts with defensive learning.\u003c/b\u003e\u003c/p\u003e \u003cp\u003eTo study the neurocircuitry behind threat adaptation and defensive learning we focused on the IPN of the midbrain, an emerging region associated with anxiety and fear (\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e). The IPN is an inhibitory nucleus highly enriched in GABAergic neurons that respond to aversive stimuli (\u003cspan additionalcitationids=\"CR33\" citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e). We combined \u003cem\u003ein vivo\u003c/em\u003e fiber photometry recordings with mouse behavior to test if IPN GABAergic neuronal activity is engaged by VLS. Specifically, we expressed \u003cem\u003eCre\u003c/em\u003e-dependent GCaMP in the IPN of mice driving \u003cem\u003eCre\u003c/em\u003e recombinase under the control of the glutamic acid decarboxylase 2 enzyme promoter (\u003cem\u003eGAD2\u003c/em\u003e\u003csup\u003e\u003cem\u003eCre\u003c/em\u003e\u003c/sup\u003e mice) and recorded IPN activity dynamics time-locked to VLS events (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea). In mice presented with an overhead VLS, we detected a significant increase in IPN \u003cem\u003eGAD2\u003c/em\u003e neuronal activity that was absent in control mice expressing \u003cem\u003eCre\u003c/em\u003e-dependent eGFP in the IPN instead of GCaMP or if the same VLS was presented from a side view (Supplementary Fig.\u0026nbsp;4a-b). Additional aversive stimuli, including a tail lift or a foot shock, also raised the activity of the IPN \u003cem\u003eGAD2\u003c/em\u003e neuronal population (Supplementary Fig.\u0026nbsp;4c-d). Remarkably, IPN neuronal responses to the VLS decreased from day 1 to day 3, as mice learned to optimize innate defensive strategies (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb-e). Other behaviors, such as rearing against the wall, also increased IPN activity. However, the IPN GABAergic activity during rearing remained stable across the 3 days (Supplementary Fig.\u0026nbsp;4e), providing evidence that the decrease in signal was not due to photobleaching. Reductions of VLS-evoked IPN \u003cem\u003eGAD2\u003c/em\u003e neuronal activation across days were not detected within a trial session (Supplementary Fig.\u0026nbsp;4f-g).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eInterestingly, IPN \u003cem\u003eGAD2\u003c/em\u003e neuronal activity dynamics inversely mirrored changes in speed during VLS events (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb), as well as across the whole VLS session (Supplementary Fig.\u0026nbsp;5a). Correlation analysis demonstrated VLS-induced activation levels of IPN \u003cem\u003eGAD2\u003c/em\u003e neurons negatively related with speed 10 sec post-VLS initiation (Supplementary Fig.\u0026nbsp;5b). Furthermore, VLS-induced engagement of IPN \u003cem\u003eGAD2\u003c/em\u003e neurons positively correlated with latency to nest (Supplementary Fig.\u0026nbsp;5c) and time spent near the walls (Supplementary Fig.\u0026nbsp;5d). Notably, we also detected reduced activity of IPN GABAergic neurons when mice entered the nest (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ef-h), although these signals did not adjust across repeated sessions. Altogether these results suggested the dynamics of IPN \u003cem\u003eGAD2\u003c/em\u003e neuronal activity reflected different aspects of VLS evoked defensive actions.\u003c/p\u003e \u003cp\u003e \u003cb\u003eSilencing activity of IPN\u003c/b\u003e \u003cb\u003eGAD2\u003c/b\u003e \u003cb\u003eneurons during VLS presentations reduces innate defensive behaviors.\u003c/b\u003e\u003c/p\u003e \u003cp\u003eTo determine the functional implication of IPN \u003cem\u003eGAD2\u003c/em\u003e neurons in defensive responses, we used optogenetics and selectively silenced these neurons during VLS events. \u003cem\u003eGAD2\u003c/em\u003e\u003csup\u003e\u003cem\u003eCre\u003c/em\u003e\u003c/sup\u003e mice were injected with \u003cem\u003eCre\u003c/em\u003e-dependent halorhodopsin (NpHR) or eGFP control virus and implanted with an optic fiber in the IPN (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea). Animals then underwent the 3-day VLS paradigm with photoinhibition 2 sec prior to VLS, which remained ON until 2 sec post-VLS, on days 1 and 3 (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eb). Photoinhibition of IPN GABAergic neurons reduced both early and late defensive responses. Compared to the controls, IPN NpHR animals exhibited a decrease in VLS-induced immediate freezing response (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ec-d) along with increased speed upon VLS display (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ee-f). Additionally, silencing IPN \u003cem\u003eGAD2\u003c/em\u003e neurons led to decreased time spent in the nest on day 1 of the looming session (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eg-h); instead, animals remained in the vicinity of the safety zone (Supplementary Fig.\u0026nbsp;6a-b). Other defensive responses including latency to enter the nest (Supplementary Fig.\u0026nbsp;6c), time spent near the walls (Supplementary Fig.\u0026nbsp;6d-e), or in the trigger area (Supplementary Fig.\u0026nbsp;6f-g) were not significantly affected by IPN \u003cem\u003eGAD2\u003c/em\u003e photoinhibition. Overall, these data suggest that IPN GABAergic neurons mediate both early and late VLS-evoked defensive behaviors, particularly on the first exposure session when neurons are highly engaged by VLS.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTo complement these experiments, we also asked if activating IPN GABAergic neurons is enough to elicit changes in innate defensive strategies. To this aim, we injected the IPN of \u003cem\u003eGAD2\u003c/em\u003e\u003csup\u003e\u003cem\u003eCr\u003c/em\u003ee\u003c/sup\u003e mice with \u003cem\u003eCre\u003c/em\u003e-dependent channelrhodopsin (ChR2) and implanted an optic fiber in the target site (Supplementary Fig.\u0026nbsp;7a). In this experiment, no VLS was presented; instead, animals received IPN \u003cem\u003eGAD2\u003c/em\u003e neuronal photostimulation when they entered the trigger zone, on days 1 and 3 (Supplementary Fig.\u0026nbsp;7b). Optogenetic excitation alone did not produce differences between groups for the defensive responses measured (Supplementary Fig.\u0026nbsp;7c-k).\u003c/p\u003e \u003cp\u003e \u003cb\u003eIPN\u003c/b\u003e \u003cb\u003eGAD2\u003c/b\u003e \u003cb\u003eneurons projecting to the LDTg are engaged by VLS and reduce activation with innate defensive adaptive learning.\u003c/b\u003e\u003c/p\u003e \u003cp\u003eThe IPN \u003cem\u003eGAD2\u003c/em\u003e neuronal population consists of projection neurons that innervate brain regions involved in affective and motivational behaviors, including the raphe and the LDTg (\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e). Here, we hypothesize the IPN may use the LDTg, an area previously associated with fear and anxiety (\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e), to convey innate defensive behaviors. To test if this circuit responded to the VLS, we recorded activity dynamics in \u003cem\u003eGAD2\u003c/em\u003e\u003csup\u003e\u003cem\u003eCre\u003c/em\u003e\u003c/sup\u003e mice bilaterally injected with a retrogradely-transported AAVrg \u003cem\u003eCre\u003c/em\u003e-dependent GCaMP into the LDTg and implanted with an optic fiber in the IPN (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea). The presentation of overhead VLS triggered an elevated activation of \u003cem\u003eGAD2\u003c/em\u003e\u003csup\u003eIPN\u0026loz;LDTg\u003c/sup\u003e neurons that significantly decreased over consecutive sessions (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eb-e). No reductions in VLS-induced \u003cem\u003eGAD2\u003c/em\u003e\u003csup\u003eIPN\u0026loz;LDTg\u003c/sup\u003e circuit engagement were detected within a trial session (Supplementary Fig.\u0026nbsp;8a-b). Similarly to overall IPN \u003cem\u003eGAD2\u003c/em\u003e neurons, activity dynamics of the \u003cem\u003eGAD2\u003c/em\u003e\u003csup\u003eIPN\u0026loz;LDTg\u003c/sup\u003e circuit inversely mirrored changes in speed (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eb) and negatively correlated with speed levels 10 sec upon VLS initiation (Supplementary Fig.\u0026nbsp;8c). Additional analysis did not find significant correlations between VLS-induced \u003cem\u003eGAD2\u003c/em\u003e\u003csup\u003eIPN\u0026loz;LDTg\u003c/sup\u003e circuit engagement and latency to nest (Supplementary Fig.\u0026nbsp;8d), although \u003cem\u003eGAD2\u003c/em\u003e\u003csup\u003eIPN\u0026loz;LDTg\u003c/sup\u003e activation positively predicted the time mice spent near the walls (Supplementary Fig.\u0026nbsp;8e). Akin to IPN \u003cem\u003eGAD2\u003c/em\u003e measures, \u003cem\u003eGAD2\u003c/em\u003e\u003csup\u003eIPN\u0026loz;LDTg\u003c/sup\u003e neurons showed a decrease in circuit activity when animals entered inside the nest that was sustained across the three sessions (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ef-h). This data demonstrates that VLS-related information engages the projection from the IPN to the LDTg area, which may participate in threat processing and adaptive defensive learning.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eSilencing IPN\u003c/b\u003e \u003cb\u003eGAD2\u003c/b\u003e \u003cb\u003eneurons projecting to the LDTg occludes innate defensive adaptive learning.\u003c/b\u003e\u003c/p\u003e \u003cp\u003eTo test if IPN GABAergic IPN neurons projecting to the LDTg are necessary for innate adaptive defensive responses, we used closed-loop optogenetic approaches in a circuit-specific manner. To this aim, we injected a retrograde \u003cem\u003eCre\u003c/em\u003e-dependent AAVrg NpHR or eGFP into the LDTg and placed an optic fiber in the IPN (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ea). Animals underwent the same VLS paradigm with circuit photoinhibition time-locked to VLS events. Experimental and control mice demonstrated a significant reduction in the early defensive behavior of freezing across days (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eb-c). However, compared to the controls, optogenetic silencing of the IPN\u0026loz;LDTg circuit slightly increased freezing behavior (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eb-c) and significantly reduced max speed 2 sec upon VLS events (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ed-e). Noticeably, control mice significantly reduced the time spent in the nest after VLS presentation across sessions, whereas mice with \u003cem\u003eGAD2\u003c/em\u003e\u003csup\u003eIPN\u0026loz;LDTg\u003c/sup\u003e circuit photoinhibition continued spending a significant amount of time inside the nest over VLS days (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ef-g), suggesting that inhibition of this circuit occludes adaptive learning of innate threat responses. Time spent in other zones, such as the wall, safe, and trigger as well as latency to nest, were not influenced by optogenetic inhibition of the \u003cem\u003eGAD2\u003c/em\u003e\u003csup\u003eIPN\u0026loz;LDTg\u003c/sup\u003e circuit (Supplementary Fig.\u0026nbsp;9).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eA subpopulation of IPN neurons expressing Sst encodes defensive responses in a threatening environment.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eWithin the IPN, a GABAergic subpopulation expressing somatostatin (Sst)(\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e) demonstrates a highly selective dorso-rostral to ventro-caudal gradient (Supplementary Fig.\u0026nbsp;10a). Sst is a neuropeptide typically co-released with GABA and involved in anxiety-like behaviors (\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e). To explore whether IPN Sst\u003csup\u003e+\u003c/sup\u003e responded to VLS, we injected \u003cem\u003eCre\u003c/em\u003e-dependent GCaMP in the IPN of mice expressing \u003cem\u003eCre\u003c/em\u003e recombinase under the control of the Sst promoter (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ea and Supplementary Fig.\u0026nbsp;10b). We found that aversive stimuli such as VLS presentations (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eb-e), a tail lift (Supplementary Fig.\u0026nbsp;10c) or a foot shock (Supplementary Fig.\u0026nbsp;10d), triggered an increase in IPN Sst\u003csup\u003e+\u003c/sup\u003e neuronal activity. Remarkably, VLS-induced IPN Sst\u003csup\u003e+\u003c/sup\u003e activation occurred after reaching max speed upon VLS (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eb) and did not reduce from day 1 to day 3 (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ec-e). Interestingly, we also detected IPN Sst\u003csup\u003e+\u003c/sup\u003e activation time-locked to nest entry, although these signals did not adjust across repeated VLS sessions (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ef-h). Further analysis demonstrated on day 1 of the looming test, the engagement of IPN Sst\u003csup\u003e+\u003c/sup\u003e neurons with nest entry positively correlated with speed levels 10 sec upon VLS (Supplementary Fig.\u0026nbsp;10e) and time spent inside the nest (Supplementary Fig.\u0026nbsp;10f), while it negatively correlated with nest latency (Supplementary Fig.\u0026nbsp;10g) or time spent near the walls (Supplementary Fig.\u0026nbsp;10h). Overall, IPN Sst\u003csup\u003e+\u003c/sup\u003e activation with nest entry consistently predicted VLS-induced defensive behaviors, suggesting these neurons may encode safety and avoidance aspects of threat processing.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eGenetic ablation of IPN Sst\u0026thinsp;+\u0026thinsp;neurons reduces anxiety-like behaviors.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eWe next addressed the role of IPN Sst\u003csup\u003e+\u003c/sup\u003e neurons in threat processing and defensive learning by genetically ablating this neuronal population using a \u003cem\u003eCre\u003c/em\u003e-dependent caspase 3 approach (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ei). Removing IPN Sst\u003csup\u003e+\u003c/sup\u003e neuronal function did not affect VLS-induced freezing (Supplementary Fig.\u0026nbsp;11a-b) or changes in speed (Supplementary Fig.\u0026nbsp;11c-d) across the 3-day looming sessions. Nevertheless, we observed that IPN Sst\u003csup\u003e+\u003c/sup\u003e ablated animals significantly reduced the time spent inside the nest as compared to mCherry control mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ej-k). In contrast, animals with IPN Sst ablation spent more time nearby the safety (Supplementary Fig.\u0026nbsp;11e-f) and wall zones (Supplementary Fig.\u0026nbsp;11g-h), without affecting nest latency (Supplementary Fig.\u0026nbsp;11i). The reduction in nest time observed by animals with ablated IPN Sst\u0026thinsp;+\u0026thinsp;neurons support the notation that these neurons play a role in processing avoidance-related aspects of threat-evoked defensive behavior, but not motor function. Along these lines, we also detected that IPN Sst\u003csup\u003e+\u003c/sup\u003e ablation increased the time spent in the open arms of the elevated plus maze (Supplementary Fig.\u0026nbsp;11j) but not the number of closed arm entries (Supplementary Fig.\u0026nbsp;11k). Similarly, IPN Sst ablated animals showed increased time of center exploration in an open field test (Supplementary Fig.\u0026nbsp;11l) without altering locomotor activity in this assay (Supplementary Fig.\u0026nbsp;11m).\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eDefensive behaviors are regulated by adaptive mechanisms contingent on the previous experience of an aversive outcome. Here we define early and late threat-evoked defensive responses that adjust to repeated VLS exposures when there is no evidence of real harm. We identify the GABAergic population in the IPN of the midbrain as a critical node orchestrating adaptive defensive strategies. Inhibitory projections from the IPN to the LDTg control the learning aspect of threat processing. In contrast, a subpopulation of IPN neurons expressing the neuropeptide Sst mediate generalized aspects of avoidance-related behaviors. The present findings have important implications for understanding the neurobiology of innate fear behaviors and how their expression is regulated in the absence of danger, a critical concept in many neuropsychiatric conditions.\u003c/p\u003e \u003cp\u003eThe selection of ongoing defensive behaviors is constantly updated by recent experiences that contribute to perceptual and value-based decision-making and action selection (\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e, \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e). While adaptations of defensive responses to visual looming stimuli have been reported in various animal species (\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e, \u003cspan additionalcitationids=\"CR41\" citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e), the behavioral signatures of threat adaptation and the circuit mechanisms underlying these behavioral changes remain largely unknown. We found that upon repeated VLS sessions, mice reduced immediate freezing, as well as late defensive behaviors including time spent inside the nest. Instead, animals switched behavioral strategies to engage in more foraging behavior. Importantly, changes in defensive strategies were detected over multiple-day sessions but not within a trial session, supporting the view that these may reflect learning and consolidation processes (\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e). Although most adaptive defensive behaviors to visual looming have been focused on escape responses (\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e, \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e), understanding late emotional components of threat processing has received less attention. Our behavioral analysis revealed a dissociation between time spent inside the nest and escape responses over repeated VLS sessions and learning. Even though multiple defensive behaviors adjust with consecutive threat exposures that have no aversive outcome, they may each require differential circuitry to guide the behavioral output. Escape is a flexible behavior under cognitive control (\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e). Integrating variables of escape behavior together with the engagement of foraging strategies upon threat assessment -for instance when animals leave the nest to take new risks-, is necessary to understand how these are coordinated to drive adaptive behaviors. Given that the speed of habituation to VLS depends on the behavioral context (\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e) and it is stimulus-specific (\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e), future studies could help elucidate early and late defensive components of threat adaptation specific to the context or type of threatening stimuli.\u003c/p\u003e \u003cp\u003eThe neural basis of threat detection and response to VLS are conserved across species (\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e). Emerging evidence indicates that divergent circuits orchestrate escape and freezing responses to looming stimuli (\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e, \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e). Yet, most of these studies have focused on a single looming session and thus, the neural circuitry of innate defensive adaptive learning to VLS remains unexplored. Here we provide first-time evidence that IPN GABAergic neurons are recruited by innate visual threats and show neuronal adaptation with defensive learning. These results support that the habenular axis is involved in anxiety (\u003cspan additionalcitationids=\"CR15 CR16 CR17 CR18 CR19 CR20\" citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e) and fear-related behaviors (\u003cspan additionalcitationids=\"CR23 CR24\" citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e), as well as familiarity signaling (\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e). Silencing overall IPN GABAergic activity during VLS presentations devalued the stress component of a VLS threat and reduced early freezing responses and late emotional behaviors such as time spent inside the nest. In contrast to the ventral tegmental area (VTA) GABAergic neurons (\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e), brief optogenetic stimulation of IPN GABAergic subpopulation was not sufficient to trigger early or late defensive behaviors, indicating specialized roles of VTA and IPN inhibitory networks.\u003c/p\u003e \u003cp\u003eHere, we report for the first time that activity dynamics of IPN GABAergic neurons inversely mirrored changes in speed. The IPN is part of the nucleus incertus network that controls locomotor speed, arousal, and hippocampal theta rhythms (\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e). Through this circuit connectivity, the IPN may contribute to the animals\u0026rsquo; arousal and locomotion. Still, our findings showing reduced IPN GABAergic neuronal activity with nest entries -when animals lower speed-, suggest a role of the IPN beyond controlling locomotion to also processing affective behaviors like avoidance or approach. Freezing is cardinal in stress-coping processes as it corresponds to a state of hypervigilance that enables decision-making (\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e). Spending time inside the nest upon VLS could also represent a stress-coping mechanism that prepares for future foraging actions. Altogether, our results support the view that the IPN responds to aversive stimuli to regulate distinctive stress-related coping strategies (\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eIPN GABAergic neurons send strong inhibitory projections to the raphe and tegmentum (\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e). Through the LDTg, the IPN controls nicotine aversion (\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e) or social novelty preference (\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e). We found IPN\u0026loz;LDTg circuit engagement with aversive VLS and neuronal adaptation with multiple exposures. Optogenetic silencing the IPN\u0026loz;LDTg circuit increased freezing and occluded late defensive adaptive learning specifically for time spent in shelter. Recent findings demonstrate LDTg GABAergic neurons inhibit VTA to promote unconditioned freezing responses (\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e). Although work from our group and others suggest IPN GABAergic neurons inhibit LDTg cholinergic neurons innervating the VTA (\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e, \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e), we cannot exclude the possibility the IPN inhibits LDTg GABAergic function to regulate fear and freezing responses. Considering different LDTg interneurons oppositely regulate innate fear (\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e), understanding how the IPN controls LDTg function and network connectivity is necessary to elucidate the critical role of this circuit in innate defensive adaptive learning.\u003c/p\u003e \u003cp\u003eAmong all IPN GABAergic cell types, we recently showed the neuronal population expressing the neuropeptide Sst is activated by acute stress to drive motivational behaviors (\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e). Sst provides retrograde inhibition of excitatory inputs from the mHb to the IPN (\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e). Our fiber photometry data revealed engagement of IPN Sst neurons during threatening VLS events but also when animals entered the safety of a protective shelter. In the lateral habenula, heterogeneous neuronal clusters predict the selection of distinct threat-driven defensive behaviors (\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e). It could be possible that different IPN Sst populations exist to control various aspects of threat aversion vs. coping mechanisms. We show that genetic ablation of IPN Sst neurons reduced time inside the nest as well as general anxiety-like behaviors. Ablated animals did not recognize the nest as a safe, protective zone. Sst neurons can mediate anxiety by disinhibition of fear-related amygdaloid circuits (\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e). Elucidating the precise IPN Sst neuronal connectivity with previously established anxiety networks should be addressed in future investigations. Collectively, our findings implicate the IPN as a critical node of innate threat processing and adaptive defensive learning, suggesting dysregulation of the IPN may contribute to numerous psychiatric disorders.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003eAcknowledgments\u003c/p\u003e\n\u003cp\u003eWe thank Biorender.com for use of graphics. This work was supported by the Brain and Behavior Research Foundation Young Investigator Award 30616 (S.M.), the National Institute of Mental Health award MH129040 (A.R.T.), the National Institute on Drug Abuse awards DA041482 (A.R.T) and DA047678 (A.R.T.).\u003c/p\u003e\n\u003cp\u003eConflict of interest\u003c/p\u003e\n\u003cp\u003eAll authors report no competing financial interests or potential conflicts of interest. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.\u003c/p\u003e\n\u003cp\u003eAuthors contributions\u003c/p\u003e\n\u003cp\u003eS.M., E.W., R.Z., L.S., B.O., M.K. and H.J. performed the experiments and analyzed the data. S.M. and A.R.T. contributed to the study design. S.M. and E.W. prepared the manuscript. All authors contributed to editing and reviewing the manuscript.\u003c/p\u003e\n\u003cp\u003eData availability\u003c/p\u003e\n\u003cp\u003eAll data will be available upon request to the corresponding author.\u003c/p\u003e\n\u003cp\u003eCode availability\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe codes used to process and analyze fiber photometry and Ethovision data will be made available on https://github.com/\u003cem\u003e\u003cbr\u003e \u003c/em\u003e\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003eBranco T, Redgrave P. 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Proc Natl Acad Sci U S A. 2017 Dec 5;114(49):13012\u0026ndash;7.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"molecular-psychiatry","isNatureJournal":false,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"mp","sideBox":"Learn more about [Molecular Psychiatry](http://www.nature.com/mp/)","snPcode":"41380","submissionUrl":"https://mts-mp.nature.com/cgi-bin/main.plex","title":"Molecular Psychiatry","twitterHandle":"@molpsychiatry","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-4661779/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4661779/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThe selection of appropriate defensive behaviors in the face of potential threat is fundamental to survival. However, after repeated exposures to threatening stimuli that did not signal real danger, an animal must learn to adjust and optimize defensive behaviors. Despite extensive research on innate threat processing, little is known how individuals change their defensive behaviors when presented with recurrent threat exposures without evidence of a real risk. Insight into this process is critical as its dysregulation may contribute to neuropsychiatric conditions, such as anxiety disorders. Here, we used the visual looming stimulus (VLS) paradigm in mice to investigate innate threat processing and adaptive defensive learning. Repeated exposure to VLS over consecutive sessions reduced immediate freezing responses and time spent inside a sheltered area upon VLS events, leading to an increase in foraging behaviors. Fiber photometry recordings and optogenetic manipulations revealed that VLS innate adaptive defensive learning is associated with reduced recruitment of the midbrain interpeduncular nucleus (IPN), a structure associated with fear and anxiety-related behaviors. Functional circuit-mapping identified a role for select IPN projections to the laterodorsal tegmental nucleus in gating defensive learning. Finally, we uncovered a subpopulation of IPN neurons that express the neuropeptide somatostatin and encode safety- and avoidance signals in response to VLS. These results identify critical behavioral signatures of innate defensive responses and a circuit that regulates the essential features of threat processing.\u003c/p\u003e","manuscriptTitle":"Interpeduncular GABAergic neuron function controls threat processing and innate defensive adaptive learning","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-09-20 09:11:19","doi":"10.21203/rs.3.rs-4661779/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"revise","date":"2024-09-16T13:14:58+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"This content is not available.","date":"2024-09-10T19:17:53+00:00","index":5,"fulltext":"This content is not available."},{"type":"editorInvitedReview","content":"This content is not available.","date":"2024-09-04T23:08:03+00:00","index":2,"fulltext":"This content is not available."},{"type":"editorInvitedReview","content":"This content is not available.","date":"2024-09-03T07:26:46+00:00","index":3,"fulltext":"This content is not available."},{"type":"editorInvitedReview","content":"This content is not available.","date":"2024-09-02T20:04:10+00:00","index":1,"fulltext":"This content is not available."},{"type":"editorInvitedReview","content":"This content is not available.","date":"2024-08-29T22:05:41+00:00","index":4,"fulltext":"This content is not available."},{"type":"reviewerAgreed","content":"This content is not available.","date":"2024-08-22T22:44:42+00:00","index":5,"fulltext":"This content is not available."},{"type":"reviewerAgreed","content":"This content is not available.","date":"2024-08-22T13:03:26+00:00","index":4,"fulltext":"This content is not available."},{"type":"reviewerAgreed","content":"This content is not available.","date":"2024-08-22T06:18:09+00:00","index":3,"fulltext":"This content is not available."},{"type":"reviewerAgreed","content":"This content is not available.","date":"2024-08-21T22:55:27+00:00","index":2,"fulltext":"This content is not available."},{"type":"reviewerAgreed","content":"This content is not available.","date":"2024-08-21T22:49:26+00:00","index":1,"fulltext":"This content is not available."},{"type":"reviewersInvited","content":"","date":"2024-08-21T22:41:28+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-07-03T10:28:36+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2024-07-03T10:17:34+00:00","index":"","fulltext":""},{"type":"submitted","content":"Molecular Psychiatry","date":"2024-07-01T20:36:32+00:00","index":"","fulltext":""},{"type":"checksFailed","content":"","date":"2024-07-01T10:43:21+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"molecular-psychiatry","isNatureJournal":false,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"mp","sideBox":"Learn more about [Molecular Psychiatry](http://www.nature.com/mp/)","snPcode":"41380","submissionUrl":"https://mts-mp.nature.com/cgi-bin/main.plex","title":"Molecular Psychiatry","twitterHandle":"@molpsychiatry","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"b058536f-0946-4aa1-bc11-f0c96390aabd","owner":[],"postedDate":"September 20th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[{"id":36387523,"name":"Biological sciences/Neuroscience"},{"id":36387524,"name":"Biological sciences/Psychology"},{"id":36387525,"name":"Health sciences/Diseases/Psychiatric disorders"}],"tags":[],"updatedAt":"2025-07-21T15:33:20+00:00","versionOfRecord":[],"versionCreatedAt":"2024-09-20 09:11:19","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-4661779","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4661779","identity":"rs-4661779","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}