Somatostatin neurons in the medial amygdala orchestrate flexible, sex-specific social behavior in male mice

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Olucha-Bordonau, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7046653/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Intraspecific social interactions are essential for species survival and require behavioral flexibility to adapt to changing social environments. These behaviors are orchestrated by neural circuits such as those in the medial amygdala (MeA). Within this region, somatostatin-expressing neurons (MeA SST+ ) have been associated with maladaptive social outcomes, particularly following early-life stress. However, whether these neurons contribute to the flexibility of adult male social behavior— modulating responses according to social context and stimulus type—remains unclear. Here, using chemogenetic approaches combined with machine learning-based behavioral tracking, we showed that activation of MeA SST+ neurons reduced sociability, social novelty preference, inter-male aggression, and attention toward males, while enhancing sexual motivation and dominance toward females. Inhibition increased social novelty preference and impaired stress-coping behavior without affecting other social traits. Notably, both manipulations heightened escape-like responses to inanimate stimuli, indicating increased defensive reactivity to non-social cues. These findings identify MeA SST+ neurons as modulators of social context-specific behavior, advancing understanding of circuit-level mechanisms supporting adaptive social responses. Cellular & Molecular Neuroscience Cognitive Neuroscience Neurobiology of Disease sociability aggression mating medial amygdala Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Teaser Neurons that flip the social switch: How the amygdala chooses between fight, flirt, and flight Introduction Social behaviors—including mating, aggression, and dominance—are essential for survival and reproductive success across species, including humans ( 1 , 2 ). Although often considered innate, these behaviors are remarkably flexible, allowing individuals to adapt their responses according to social context and stimulus type ( 3 , 4 ). This behavioral flexibility is especially critical in complex and dynamic social environments, where inappropriate responses can compromise survival or reproductive opportunities ( 5 , 6 ). The medial amygdala (MeA) plays a central role in regulating social behavior ( 7 , 8 ). It integrates direct vomeronasal and indirect main olfactory inputs ( 9 , 10 ) and connects with hypothalamic and limbic structures to orchestrate mating, aggression, and defensive responses ( 11 – 13 ). This amygdaloid nucleus comprises diverse excitatory and inhibitory neuronal populations, which are sexually dimorphic and regulate distinct dimensions of social behavior ( 14 – 19 ). For example, MeA excitatory neurons promote repetitive self-grooming, while GABAergic neurons are involved in aggression and mating motivation ( 14 ). Manipulating ErbB4⁺ GABAergic neurons facilitates mating behavior in adult male mice ( 20 ), and both silencing and activating MeA excitatory neurons delay ejaculation in rats without affecting copulatory patterns or sexual motivation ( 18 ). These results highlight the importance of neuronal subtype in shaping MeA-driven behavior. Somatostatin-expressing (SST⁺) neurons represent a major GABAergic subtype in the MeA ( 21 ) and have been implicated in regulating affective and social behaviors ( 22 ). Alterations in SST signaling have been reported in several neuropsychiatric conditions marked by social dysfunction, including autism ( 23 ), schizophrenia ( 24 ) and major depression ( 25 , 26 ). In aggression-priming paradigms, excitatory neurons in the posterior ventral MeA (MeApv) are preferentially activated, whereas SST⁺ neurons show limited activation ( 27 ). This pattern may be functionally significant, as early-life stress have been shown to produce long-lasting suppression of MeA SST+ neuron activity in males, leading to heightened aggression and reduced sociability. Restoring their activity via chemogenetics reverses these behavioral deficits ( 28 ). Despite these insights, it remains unclear whether MeA SST⁺ neurons contribute to the flexible regulation of social behavior in adulthood— specifically by influencing behavior in response to varying social and non-social cues. We hypothesized that MeA SST⁺ neurons act as modulators of behavioral flexibility, shaping sex-specific social behavior based on contextual and cued stimuli. To investigate this, we used chemogenetic approaches to selectively activate or inhibit MeA SST⁺ neurons in SST-IRES-Cre knock-in male mice, while assessing a range of social and non-social behaviors using automated, machine learning-based tracking. Chemogenetic activation of MeA SST⁺ neurons decreased sociability, intermale aggression, and attention toward males, while enhancing sexual motivation and dominance responses toward females. Conversely, their inhibition increased social novelty preference without altering other social traits. Notably, both manipulations heightened escape-like responses to inanimate objects, suggesting a broader role in regulating defensive reactivity to non-social cues. Together, these findings identify MeA SST⁺ neurons as key contributors to flexible, sex-specific social behavior. By linking cell-type specific function to behavioral modulation, our study advances understanding of the neural basis of adaptive social strategies and may help guide future research into psychiatric conditions characterized by social impairments, including autism, schizophrenia, and major depression. Results To investigate the contribution of MeA SST+ neurons to social behavioral flexibility—particularly in response to different social partners or non-social stimuli—we chronically activated or inhibited these neurons in SST-IRES-Cre male mice using a chemogenetic approach. We assessed their role across a spectrum of behavioral paradigms designed to evaluate social, emotional, aggressive, and defensive responses in both social and non-social contexts. Behavioral parameters were automatically quantified using deep learning-based tracking and multi-pose estimation tools, including DeepLabCut and SimBA, to ensure high-resolution and objective behavioral classification ( Fig. 1A ). Below, we present a structured summary of our findings, beginning with validation of the chemogenetic manipulation and progressing through behavioral domains. Validation of DREADD-based manipulation in MeA SST⁺ neurons We first validated that chronic clozapine-N-oxide (CNO) treatment—used to selectively activate DREADDs—effectively modulated MeA SST⁺ neuron activity in a cell-type–specific manner without adverse physiological effects. Mice injected with Cre-dependent AAVs expressing either excitatory (hM3Dq) or inhibitory (hM4Di) DREADDs fused to mCherry in the MeA showed no significant differences in body weight across groups throughout the experimental timeline (P60, P81, P91, P103, p > 0.05 in all cases; Fig. 1B ), indicating that the treatment did not impair general health. To assess functional DREADD expression, we quantified cFos in mCherry⁺ neurons ( Fig. 1C-F ). A one-way ANOVA revealed a significant group effect (F (2,23) = 16.43; p < 0.001 ). Compared to mCherry controls, hM3Dq mice showed elevated cFos⁺ labeling (58.30 ± 8.29% vs. 26.88 ± 8.32%; p = 0.005 ), while hM4Di mice exhibited reduced labeling (7.91 ± 1.58%; p = 0.017 ), confirming effective bidirectional modulation of MeA SST⁺ neuronal activity ( Fig. 1E, F ). MeA SST+ inhibition impairs stress-coping behavior in the forced swim test without affecting anxiety in the open field Given the role of SST in emotional regulation, we evaluated anxiety-like and stress-coping behaviors in the open field (OF) and the forced swim test (FST), respectively ( Fig. 2A ). In the OF test, no significant differences were observed in locomotion or anxiety-related measures between groups ( Fig. 2B-E ), suggesting that MeA SST+ neurons do not influence baseline anxiety. In contrast, the FST revealed altered stress-coping behavior following MeA SST+ inhibition ( Fig. 2F-K ). hM4Di mice displayed reduced active coping, as indicated by decreased time mobile ( p = 0.021 ; Fig. 2H ), increased total immobility ( p = 0.031; Fig. 2I ), shorter latency to immobility ( p = 0.003; Fig. 2J ), and elevated immobility episode count ( p = 0.027; Fig. 2K ) compared to controls. However, hM3Dq activation had no significant effects. These results indicate that inhibition, but not activation, of MeA SST+ neurons impairs stress-coping strategies without affecting anxiety. Bidirectional control of sociability and social novelty in the tree-chamber test by MeA SST+ neurons We next examined how MeA SST+ neurons regulate social behavior in the three-chamber test (3CH), which includes phases of sociability and social novelty preference ( Fig. 2L ). During the sociability phase ( Fig. 2M-i ), significant main effects of the region of interest (ROI; F (1,40) = 29.83; p < 0.001 ) and Group × ROI interaction (F (2,40) = 7.702; p = 0.001 ) were observed when analyzing the number of entries. A similar pattern was found when analyzing time spent in each ROI, with significant main effects of ROI (F (1,40) = 36,40, p < 0.001 ) and a Group x ROI interaction (F (2,40) = 8.008, p = 0.003 ). Both mCherry control and hM4Di mice showed robust preference for the social target, reflected in a higher number of entries ( p < 0.001; Fig. 2M-ii-iii ) and increased time spent (p < 0.001; Fig. 2M-v-vi ) in the social chamber. Moreover, the discrimination index (DI), calculated as the proportion of entries ( Fig. 2N-iv ) or time spent ( Fig. 2N-vii) in the social ROI relative to the total (social + empty), was significantly reduced in hM3Dq mice ( p = 0.003 and p < 0.001 , respectively). During the social novelty phase ( Fig. 2N-i ), significant main effects were observed for Group (F (2,40) = 14.08; p < 0.001 ), ROI (F (1,40) = 109.8; p < 0.001), and their interaction (F (2,40) = 36.18; p < 0.001 ) when analyzing time spent in the novel vs familiar social ROI. For the number of entries, a significant main effect of ROI was also observed (F (1,40) = 29.86; p < 0.001 ), indicating a general preference for the novel conspecific. Post hoc analyses revealed that mCherry controls showed a strong preference for the novel mouse, with significantly more entries (p < 0.001; Fig. 2N-ii ) and more time spent ( p < 0.001 ; Fig. 2N-v ) in the novel ROI. Similarly, hM4Di mice also preferred the novel conspecific, as shown by a significant increase in time spent ( p < 0.001 ; Fig. 2N-vi ) and a more modest but significant increase in entries ( p = 0.023 ; Fig. 2N-iii ). In contrast, hM3Dq mice failed to show significant differences between the novel and familiar ROIs in either measure. Moreover, the DI, calculated as the proportion of entries or time spent in the novel ROI relative to the total (novel + familiar), was significantly reduced in hM3Dq mice compared to mCherry controls (entries: p = 0.036 , Fig. 2N-iv ; time: p < 0.001 , Fig. 2N-vii ). These findings show that activation of MeA SST⁺ neurons is sufficient to impair sociability and social novelty responses, whereas their inhibition leaves these behaviors unaffected. Activation of MeA SST+ neurons promotes social dominance in novel dyadic interactions in the tube test To assess the role of MeA SST+ neurons in social dominance, we used the tube test (TT) among unfamiliar, single-housed male mice ( Fig. 2O ). This approach, adapted from Fan et al. ( 29 ) has been shown to yield robust results regarding dominance behavior ( 30 , 31 ). Chronic activation of these neurons via hM3Dq significantly increased overall dominance score (t₁₆ = 2.278; p < 0.001 ) ( Fig. 2P-i ) without affecting push frequency ( Fig. 2Q-i-iii ). However, hM4Di inhibition had no effect neither in social dominance ( Fig. 2P-ii-iii ), nor in number of pushes/s ( Fig. 2Q-i-iii ). Upon further analysis of the ethological progression across rounds, a significant Group × Round interaction ( F (2,32) = 3.437; p = 0.044 ) and a main effect of Group ( F (1,16) = 5.532; p = 0.031 ) confirmed that dominance gradually increased over time in the hM3Dq group relative to controls ( Fig. 2P-iii ); this effect became particularly evident in the final round (Round 3: p = 0.0041). In contrast, hM4Di inhibition had no significant effect on dominance score ( Fig. 2P-ii–iii) , and no differences were observed in the number of pushes/s across groups or rounds under any condition ( Fig. 2Q-i–iii ). These findings suggest that MeA SST⁺ neuron activation promotes the progressive acquisition of dominance behavior during novel dyadic encounters. MeA SST+ activation reduces aggression and social attention in male-male resident-intruder interactions Given the proposed role of the MeA in mediating social threat and territoriality ( 13 , 19 ), we next evaluated the contribution of MeA SST⁺ neurons to aggression and dominance in male-male conflict scenarios using the resident-intruder (RI) paradigm. This test allowed us to assess offensive, defensive, and dominance-related behaviors in response to a novel male intruder, as well as attention toward the conspecific ( 32 ) ( Fig. 3A-D ). In the offensive domain, hM3Dq mice showed a significant reduction in the duration of offensive behavior compared to controls ( p = 0.001 ; Fig. 3E ). However, the bout count and latency for these behaviors remained unchanged ( Fig. 3E; fig. S1A-C ). In terms of defensive responses, overall defensive behavior did not differ between groups ( Fig. 3F ), but hM3Dq mice exhibited a significant reduction in escape latency ( p = 0.042 ; fig. S1F ), indicating enhanced defensive reactivity. Dominance-related behaviors were also affected by MeA SST⁺ activation ( Fig. 3G ). hM3Dq mice showed a significant decrease in total dominance duration ( p = 0.049 ; Fig. 3G ), as well as reduced tail rattle bout count ( p = 0.031 ), duration ( p = 0.0063 ), and increased latency to first tail rattle ( p = 0.038 ) ( fig. S1G ). Social attention was also diminished in hM3Dq mice, as reflected by significantly less time spent oriented toward the intruder's nose ( p = 0.046 ) ( Fig. 3K ). Importantly, these changes were not accompanied by alterations in general social interest (approach, allogrooming), self-grooming, or exploratory behaviors ( Fig. 3H-J; fig. S1H-J ), suggesting that the observed reductions in aggression and dominance were not due to general suppression of social motivation ( Fig. 3L ). Activation of MeA SST+ neurons enhances dominance, sexual behavior, and defensive responses during female-directed social encounters We next assessed the role of MeA SST⁺ neurons in regulating social behavior toward female conspecifics using the RI test ( Fig. 4A-B ). This context allowed us to evaluate how neuronal activation influences aggression, sexual behavior, dominance, and social attention in male-female interactions ( 33 ). Among the experimental groups, only hM3Dq mice exhibited notable behavioral alterations. Global offensive behavior remained unaffected ( Fig. 4C ); however, a selective reduction in lateral threat events was observed ( p = 0.013 ; fig. S2C ), with no significant differences in attack-related classifiers ( fig. S2A ) or vigorous allogrooming ( fig. S2B ). While overall defensive behavior remained unchanged ( Fig. 4D ), specific components were significantly modulated in hM3Dq mice. These animals displayed increased duration of defensive attacks ( p = 0.031 ; fig. S2D ), as well as elevated bout count ( p = 0.016 ) and duration ( p = 0.011 ) of upright submission ( fig. S2E ). Escape behavior, by contrast was unaffected ( fig. S2F ). Dominance and sexual behaviors were markedly enhanced in hM3Dq mice. The total duration of this category was significantly elevated compared to controls ( p = 0.036 ; Fig. 4E ), primarily driven by increased durations of tail rattling ( p = 0.045 ; fig. S2G ) and mounting ( p = 0.034; fig. S2H ). Despite a significant increase in the latency to initiate social contact ( p = 0.009 ; Fig. 4F ), approach behavior ( fig. S2I ) and normal allogrooming remained stable in bout count and duration. However, the latency to first normal allogrooming was also significantly delayed ( p = 0.007 ; fig. S2J ), further suggesting a shift in behavioral priorities. In parallel, hM3Dq mice showed a reduced frequency of self-grooming bouts ( p = 0.03 5; Fig. 4G ), while rearing behavior remained unaffected ( Fig. 4H ). Importantly, nose-to-nose orientation toward the female conspecific was not altered in hM3Dq mice ( Fig. 4I ), indicating that social attention was preserved despite the delayed onset of affiliative behaviors. Together, these results indicate that activation of MeA SST⁺ neurons enhances territorial and mating-related behaviors in response to female conspecifics, while preserving general social interest and attention ( Fig. 4J ). Manipulation of MeA SST+ neurons alters behavioral responses to inanimate objects To examine how MeA SST⁺ neurons modulate responses to non-social but potentially threatening stimuli, we exposed mice to three inanimate objects—a mirror, a flipped mirror, and a glove—within the resident’s home cage ( Fig. 5A–C ). The mirror, previously shown to elicit aggression in both fish ( 34 ) and mice ( 35 ), served as a semi-social yet conspecific-free stimulus. The glove, in turn, was used to simulate an experimenter's hand intrusion, providing a physically intrusive but non-social threat ( 35 ). This design allowed us to assess a range of responses from exploratory interest to escape-driven defensive reactions. All groups spent more time facing the object than the opposite corner ( p < 0.001 ), indicating a general attentional bias toward the stimuli ( Fig. 5D, L, T ). However, hM3Dq mice displayed a consistent avoidance pattern, entering the opposite corner more frequently than the object ROI across all conditions (mirror: p < 0.050 ; flipped mirror: p < 0.010 ; glove: p < 0.010 ; Fig. 5F, N, V ). Despite this avoidance, time spent facing the object did not differ between groups, suggesting that attentional salience remained intact ( Fig. 5E, M, U) . During mirror exposure, hM3Dq animals exhibited reduced approach behavior, with fewer approach bouts ( p = 0.035 ) and less time in the ROI ( p = 0.043 ) ( Fig. 5H-i, iii ). Conversely, hM4Di mice exhibited reduced latency to run away ( p = 0.049 ) ( Fig. 5H-ii ) and showed elevated digging behavior ( p = 0.015 ) ( fig. S3A ), indicative of increased avoidance ( 36 ) and anxiety-like behavior ( 37 , 38 ). No significant group differences were observed in tail rattle ( Fig. 5J ). In the flipped mirror test, hM4Di mice again showed reduced latency to run away ( p = 0.045 ) ( Fig. 5Q-ii ) and increased digging behavior ( p = 0.032 ) ( fig. S3E ). By contrast, hM3Dq mice displayed reduced rearing (bout count: p = 0.017 ; duration: p = 0.001; fig. S3H ), suggesting diminished exploratory drive ( 39 ) During glove exposure, hM3Dq mice showed a pronounced “flight response”, with increased number ( p = 0.020 ) and duration ( p = 0.039 ) of run-away events ( Fig. 5Y-i, iii ). hM4Di animals showed increased latency to tail rattle ( p = 0.048 ; Fig. 5Z-ii ), suggesting reduced dominance. Additionally, hM3Dq mice exhibited reduced latency to initiate self-grooming ( p = 0.044 ) ( fig. S3-K ), along with suppressed exploratory behavior (rearing bout count: p = 0.007 ; duration: p = 0.003 ; Fig. 35-L ). These findings demonstrate that activation of MeA SST⁺ neurons enhances escape-like responses to ambiguous or potentially threatening inanimate stimuli, whereas inhibition promotes reactive behaviors such as digging and avoidance. Summary Altogether, our results demonstrate that MeA SST⁺ neurons influence a diverse range of behaviors in adult male mice, including social dominance, mating, coping strategies, and responses to non-social stimuli. The effects were context-dependent and bidirectional, depending on whether MeA SST⁺ neurons were activated or inhibited. Discussion Our findings provide direct functional evidence that MeA SST⁺ neurons contribute to the flexible regulation of social and defensive behaviors in adult male mice. By using bidirectional chemogenetic manipulations, we demonstrated that these neurons are capable of modulating a wide range of social context-dependent behaviors—spanning from sociability and aggression to mating and coping strategies—while also influencing reactions to non-social stimuli. These effects highlight the role of MeA SST⁺ neurons not simply as modulators of general social engagement, but as key integrators of social behaviors that are sensitive to both stimulus type and social context. Chronic activation of MeA SST⁺ neurons via hM3Dq disrupted social preference and novelty responses in the 3CH test, resulting in reduced sociability and a diminished preference for unfamiliar conspecifics. These results are consistent with our previous observation of a negative correlation between the activity of these neurons and social interaction levels ( 30 ). Importantly, this effect was specific to social novelty and was not attributable to changes in general exploratory behavior, as no significant differences in locomotion were observed in the OF. On the contrary, inhibition of MeA SST⁺ neurons through hM4Di enhanced preference for social novelty, suggesting that their activity gates approach behavior toward novel social partners. These data support the view that MeA SST⁺ neurons help mediate the balance between exploration and social familiarity, possibly through their impact on attentional or motivational components of social interaction. These findings are also aligned with prior work showing that activation of GABAergic neurons in the MeA can shift social exploration toward aggression or sexual behavior depending on context ( 14 ), and that MeA circuits projecting to structures such as the lateral septum or BNST regulate social recognition and memory, often modulated by oxytocin ( 40 , 41 ). In the affective domain, inhibition of MeA SST⁺ neurons promoted a shift toward passive coping strategies in the FST, a widely used assay of behavioral despair and stress-reactivity in rodents ( 42 ). hM4Di-expressing mice exhibited increased immobility and decreased latency to the first immobility episode—behavioral markers that have been linked to depression-related states and reduced stress resilience in humans ( 43 ). This profile is consistent with a large body of literature implicating GABAergic neurons in the regulation of affective homeostasis. Human studies using magnetic resonance spectroscopy and transcranial magnetic stimulation have reported decreased GABA neurotransmission and altered excitation/inhibition balance in patients with major depressive disorder ( 44 – 48 ). Notably, SST-expressing neurons comprise a major subclass of GABAergic cells ( 21 ) and have been found to show reduced expression in the amygdala of major depressive disorder patients ( 22 , 25 ). In rodents, extensive work has confirmed the amygdala’s central role in affective control ( 49 ). Our results extend these findings by showing that targeted inhibition of MeA SST⁺ neurons is sufficient to elicit maladaptive stress-coping behavior, in the absence of changes in anxiety-related exploration in the OF. Interestingly, the role of MeA SST⁺ neurons in dominance and aggression appears highly context-specific. In the TT—a constrained, non-territorial paradigm—activation of MeA SST⁺ neurons increased dominance, as evidenced by a higher proportion of wins and increased dominance scores over successive trials. These effects were not accompanied by heightened aggression (e.g., number of pushes), suggesting that MeA SST⁺ neurons may promote perseverance in competitive interactions without necessarily enhancing offensive behaviors. This aligns with early work in fish, where somatostatin and its receptor sstR3 were associated with dominant male phenotypes ( 50 ). However, this dominance-enhancing effect of MeA SST⁺ activation did not generalize to territorial contexts. In RI test, activation led to a decrease in classical markers of dominance, including tail rattling ( 51 ), as well as a reduction in social attention toward intruder males (nose-to-nose orientation). Instead, hM3Dq mice displayed a shorter latency to escape, suggesting an avoidance-based or submissive strategy. This context-specific suppression of aggression may reflect an adaptive strategy to reduce costly confrontations in socially demanding environments ( 29 , 52 ). Interestingly, although hM4Di mice did not show increased aggression, their shorter latency to tail rattle may suggest an enhanced readiness to assert dominance without escalating to explicit aggression. This suppression of aggression via MeA SST+ neurons is especially notable given the MeA’s well-established role in driving aggressive behavior ( 19 ). One might expect that neurons in this region would promote aggression regardless of context. However, our findings suggest that the presence or absence of a social context, as well as the specific mode of neuromodulation, can override innate circuit outputs. These findings are consistent with anatomical descriptions of the MeA's projections to key aggression-related circuits, including the ventrolateral hypothalamus and olfactory bulb ( 15 , 19 , 53 ). Notably, our previous work also demonstrated that adult male mice subjected to early-life stress show reduced activation of MeA SST⁺ neurons and increased inter-male aggression ( 30 ), highlighting a potential link between SST⁺ dysfunction and aggression across both acute and developmental timescales. Moreover, discrepancies with previous optogenetic findings may reflect differences in spatial precision and activation dynamics. While ChR2-mediated stimulation of GABAergic neurons in the posterodorsal MeA induces aggression ( 14 ), stimulation with faster opsin variants like ChETA led to opposing outcomes, including reduced aggression ( 54 ). These differences suggest that strong depolarization and high-frequency firing favor aggression, whereas milder, sustained activation—such as that induced by DREADDs—may engage inhibitory circuits or dampen excitatory drive. Our chronic, whole-MeA chemogenetic approach likely resembles the subtler activation dynamics of ChETA more than ChR2, offering a mechanistic explanation for the context- and method-dependent suppression of aggression observed here. When male mice encountered a female intruder, activation of MeA SST⁺ neurons increased sexual and dominance-related behaviors (e.g., mounting, tail rattling), but also delayed the onset of affiliative social behaviors such as allogrooming ( 55 , 56 ). These data indicate that MeA SST⁺ activity enhances mating-related responses while suppressing non-reproductive affiliative behaviors, possibly reflecting a shift in behavioral prioritization toward reproductive success. This interpretation is supported by findings that GABAergic MeA populations encode pheromone and sex-specific cues and influence male mating drive ( 20 , 53 , 57 ) and by recent optogenetic work showing that activation of vGAT⁺ neurons within the MeA suppresses self-grooming and other affiliative touch behaviors ( 14 , 56 ). Indeed, Wu et al. ( 56 ) demonstrated that optogenetic activation of Tac1⁺ vGAT⁺ neurons in the MeA robustly induces allogrooming toward stressed conspecifics, identifying a dedicated GABAergic subpopulation that controls affiliative social touch via projections to the MPOA. Furthermore, the delayed initiation of allogrooming in hM3Dq mice suggests a general suppression of prosocial affiliative behavior even in the context of enhanced sexual and dominance signals. These changes may reflect shifts in behavioral prioritization, whereby reproductive and dominance strategies are upregulated while prosocial affiliative circuits are transiently suppressed. We also found that MeA SST⁺ neuron manipulation influenced responses to inanimate stimuli introduced into the home cage, such as a mirror, flipped mirror, or glove. These objects typically elicit exploratory or investigatory behavior ( 35 ), but in our study, both activation and inhibition of MeA SST⁺ neurons led to enhanced avoidance. hM3Dq-expressing mice showed increased entries into the cage corner opposite the stimulus and reduced time facing the object, suggesting a suppression of exploratory drive or a “flight-like” response. Similarly, hM4Di-expressing mice exhibited shorter latencies to escape and increased digging behavior—commonly associated with heightened arousal or anxiety. Notably, inhibition also delayed the onset of tail rattling, a behavior indicative of dominance, pointing to broader shifts in behavioral prioritization ( 35 , 52 ). These results suggest that both hyperactivation and silencing of MeA SST⁺ neurons promote defensive reactivity to non-social, uncertain cues, but potentially through divergent mechanisms—behavioral suppression (hM3Dq) versus hyperarousal (hM4Di). This bidirectional sensitivity aligns with prior studies implicating SST⁺ neurons in affective regulation ( 22 ) and extends their role beyond social contexts to include flexible modulation of threat-related behaviors. Taken together, these findings point to a functional role of MeA SST⁺ neurons in fine-tuning behavioral output according to context. These neurons appear to increase dominance and sexual behavior in competitive or mating contexts while simultaneously suppressing territorial aggression and affiliative social interaction under certain conditions. The same manipulations that enhanced dominance in the tube test suppressed aggression in the home cage, suggesting that MeA SST⁺ neurons prioritize behavioral outputs depending on environmental and social cues. This flexibility may be crucial for balancing reproductive opportunities with social risks. In summary, our study identifies MeA SST⁺ neurons as key regulators of social behavior flexibility in adult males. By demonstrating that both activation and inhibition of these cells shape context-dependent behaviors—from social exploration and dominance to stress coping and threat reactivity—our findings provide new insight into the neural circuitry that supports adaptive responses across social and non-social domains. These results highlight MeA SST⁺ neurons as a potential node of vulnerability in disorders marked by impaired social functioning, such as autism, depression, or schizophrenia. Materials and Methods Animals SST-IRES-Cre knock-in male mice (Sst tm2.1(cre)Zjh /J, strain #: 013044, The Jackson Laboratory) were used in all experiments ( Fig. 1A ). In this strain, the CRE recombinase is driven by the endogenous Sst promoter. Experiments were conducted on adult mice (P60 at surgery, P99 at testing). After weaning (P21), animals were group-housed (2–3 per cage) in standard polycarbonate cages (26.8 x 21.5 x 14.1 cm) under a 12:12 h light/dark cycle (lights on 8:00 am to 8:00 pm), at temperature (21 ± 1°C) and humidity-controlled environment (50 ± 5%). Food and water were provided ad libitum . Mice were socially housed after surgery and singly housed in standard cages one-week prior to testing, except for those assigned to the resident intruder (RI) test, who were housed individually in larger cages (50 x 38 x 18 cm). All procedures complied with EU Directive 2010/63/EU and were approved by the University Jaume I Bioethics Committee (2019/VSC/PEA/0188). Experimental design Adult males were subjected to AAV-DREADD vector surgeries: AAV2-hSyn-DIO-mCherry (control), AAV2-hSyn-DIO-hM3D(Gq)-mCherry (activator), and AAV2-hSyn-DIO-hM4D(Gi)-mCherry (inhibitor). After 3 weeks of recovery, clozapine-N-oxide (CNO) was administered intraperitoneally (1 mg/kg) 30 min before each session for 21 days. Brains were collected 90 min after the last injection for cFos analysis Behavioral tests were conducted in 2 cohorts of animals (experiment 1, experiment 2). Mice from experiment 1 were exposed to open field (OF, P99), forced swim test (FST, P100), three chamber test (3CH, P101) and tube dominance test (TT, day P102) to evaluate anxiety-like behavior, stress-coping strategies, social interest, and aggressive-dominance behavior, respectively. In experiment 2, mice underwent RI test variants: object-directed (mirror: RI mi , P99; flipped mirror: RI flp.mi , P102; glove: RI gl , P102) or social (female: RI fem, P101, male: RI mal, P100). Viral constructs and stereotaxic surgeries Male mice underwent stereotaxic surgery at P60. Anesthesia was induced with isoflurane (2.5% for induction, 1.5% for maintenance; 1.2 L/min O₂, Isofluotek, KARIZOO, Spain) in an induction chamber. The head was shaved and positioned in a stereotaxic frame (David Kopf Instruments). Animals received preoperative analgesia (buprenorphine, 0.02 mg/kg, s.c.; Bupaq, Fatro) and atropine (0.05 mg/kg, s.c.; B. Braun), and ophthalmic gel was applied (Lubrithal, DECHRA) to prevent corneal drying. The skin was incised to expose and clean the skull, and the head was aligned using bregma, lambda, and medial-lateral references. Bilateral craniotomies were performed (0.5 mm drill bit) at the following coordinates targeting the medial amygdala (MeA): AP − 1.4 mm, ML ± 2.6 mm, DV − 5.3 mm ( 58 ). The skull was rinsed with 0.9% saline solution to reduce inflammation. To deliver AAV-encoded DREADDs, a 33-gauge Hamilton syringe (5 µL) was loaded with 900 nL of virus and manually infused at 100 nL/30 s (450 nL per hemisphere). After infusion, the needle remained in place for 10 min to minimize backflow and was withdrawn slowly (1 mm/min). Body temperature was monitored and maintained on a heating pad throughout the procedure. Following surgery, mice recovered in a clean heated cage and were returned to group housing. A 21-day recovery period was allowed before CNO treatment and behavioral testing. AAV vectors were obtained from Addgene and supplied by Bryan Roth’s laboratory. The control virus was pAAV-hSyn-DIO-mCherry (AAV2; RRID: Addgene_50459; titer ≥ 4 × 10¹² vg/mL). The excitatory DREADD construct was pAAV-hSyn-DIO-hM3D(Gq)-mCherry (AAV2; RRID: Addgene_44361; titer ≥ 6 × 10¹² vg/mL), and the inhibitory construct was pAAV-hSyn-DIO-hM4D(Gi)-mCherry (AAV2; RRID: Addgene_44362; titer ≥ 5 × 10¹² vg/mL). Drugs Chemogenetic activation or inhibition of MeA SST+ neurons in Sst-IRES-Cre mice was achieved via DREADD technology, requiring treatment with CNO. Activation of hM3D(Gq) receptors induces neuronal depolarization, whereas stimulation of hM4D(Gi) receptors causes hyperpolarization, effectively silencing neurons. These effects were confirmed by measuring cFos expression as a proxy for neuronal activity (see below). Mice injected with AAV2-hSyn-DIO-hM3D(Gq)-mCherry (hM3Dq), AAV2-hSyn-DIO-hM4D(Gi)-mCherry (hM4Di), or AAV2-hSyn-DIO-mCherry (control) received daily intraperitoneal CNO (1 mg/kg) injections for 21 days. Behavioral testing was conducted during the final four days of treatment, with CNO administered 30 min before testing and animals perfused 90 min after the final injection. CNO (HelloBio, HB6149-25mg, Bristol, UK) was prepared in sterile saline at 1 mg/mL, aliquoted, frozen, and freshly diluted prior to each injection. The final dose for chemogenetic experiments was 0.1 mg/ml, administered intraperitoneally. Weight monitoring To control for potential side effects of chronic CNO treatment or surgery, animal body weight was monitored at four time points ( Fig. 1B ): the day of surgery (P60), the start of CNO administration (P81), 10 days post-CNO onset (P91), and the day of perfusion (P103). Behavioral assays All behavioral tests were conducted between 09:00 and 14:00 under standardized conditions. Testing rooms were illuminated with white light (150 lx), and mice were acclimated to the room for 30 minutes prior to each session to allow for CNO onset. Behavior was recorded using OBS Studio (v27.01) and analyzed with ANY-maze software (v4.98, Stoelting Europe) for the open field, forced swim, and three-chamber tests. The tube test (TT) was scored manually. RI interactions were evaluated using DeepLabCut ( 30 , 59 ) for pose estimation and downstream behavioral classification. Open field test (OFT) To evaluate locomotor and anxiety-like behavior, mice were placed in a 40 × 40 × 40 cm white-floored arena with black walls for 10 min. Total distance traveled was used to assess locomotion, while the ratio of time spent in the center (20 × 20 cm) vs. periphery served as a measure of anxiety-like behavior as described before ( 30 ). Forced Swimming test (FST) Depression-like behavior was assessed in a 15 cm diameter beaker filled with 12 cm of water at 24 ± 1°C. Mice were tested for 5 minutes, and immobility—defined as the absence of movement except to keep the head above water—was manually scored. The procedure was performed as described before ( 30 ). Three-chamber test (3CHT) Sociability and social novelty were measured in a clear acrylic apparatus (60 × 40.2 × 19.5 cm) divided into three chambers. Mice first explored the empty setup (habituation), then encountered a stranger mouse in one side chamber (sociability), followed by exposure to a familiar and a novel mouse (novelty phase). Time spent and number of entries near each stimulus were recorded. Discrimination indexes were computed as in ( 30 ). Dominance Tube Test (TT) Social dominance was assessed using the tube test ( 29 , 60 ), where weight-matched mice from experimental (hM3Dq, hM4Di) and control (mCherry) groups were introduced from opposite ends of a transparent 30 cm PVC tube (3.2 cm inner diameter). Each trial ended when one mouse fully retreated from the tube. Mice underwent three counterbalanced trials with unfamiliar opponents. The number of wins, ties, and losses was used to compute a dominance score and winning ratio, while the number of forward pushes was manually counted as an indicator of aggressive-like behavior, as described previously ( 30 ). Resident Intruder Test and Variants (RI ) All animals from experiment 2 were tested across multiple RI formats: against inanimate stimuli (mirror, flipped mirror, glove), a female conspecific, and a male intruder ( Fig. 1A ). Female intruders were in estrus, as determined by vaginal smear analysis to ensure hormonal receptivity. Tests involving inanimate objects were performed in the home cage and lasted 10 minutes. The mirror (15 × 10 cm) was presented with its reflective side facing the animal, serving as a semi-social stimulus, as prior studies have shown that rodents display aggression toward their own reflections in the absence of a real conspecific ( 35 ). The flipped mirror served as a non-reflective control, while the nitrile glove was partially inflated to resemble a human hand. Encounters with social targets involved either a group-housed female (sociability/sexual behavior) or a group-housed male intruder (sociability/aggression), both lasting 15 minutes. All intruder animals belonged to the same Sst-IRES-Cre strain. The order of testing was fixed over 4 consecutive days as follows ( Fig. 1A ): baseline (RI ba ), mirror (RI mi ), male (RI mal ), female (RI fem ), flipped mirror (RI fl.mir ), and glove (RI gl ). CNO was administered intraperitoneally 30 min prior to each testing session. Behavioral parameters were quantified using machine-learning-based classifiers (see below). Behavioral events were grouped by functional category following prior literature ( 61 ): offensive behaviors (e.g., attack, vigorous allogrooming, lateral threat), defensive behaviors (e.g., defensive attack, upright submissive, escape), dominance and sexual behaviors (tail rattle, mounting), social interest (approach, normal allogrooming), and other behaviors (self-grooming, rearing). Time spent in nose-to-nose orientation was also measured as an indicator of social attention. Machine-learning based analysis of social behavior using DeepLabCut and SimBA Behavioral data from the RI test and variants were acquired using a combination of markerless pose estimation and supervised behavior classification. DeepLabCut (v. 2.3.8; ( 59 , 62 ). was used to track body parts across video recordings. We created and trained one network for each behavior paradigm—resident intruder towards a conspecific and resident intruder towards an inanimate object—to account for visual and postural differences between conditions. Each network was trained on 300 manually labeled frames from 15 videos, identifying eight key body points: nose, center of body, left/right ears, left/right flanks, tail base, and tail end. The RI-social network used the pre-trained dlcrnet_ms5 model (75,000 iterations), while the RI-inanimate network used ResNet_50 (95,000 iterations). Final train/test errors were 3.15/6.55 pixels and 2.72/4.14 pixels, respectively. Tractlet refinement and visual inspection confirmed high tracking accuracy. Behavior classification was performed using Simple Behavioral Analysis (SimBA, v1.85.1). For RI-social videos, 12 validated classifiers were reused from ( 30 ). For RI-inanimate stimuli, seven new classifiers were created based on manual annotations of 18,000 frames from 10 videos, covering behaviors such as approach, tail rattle, rearing, self-grooming, biting, runaway, jumping, and digging. In total, 23,184 labeled frames were used for training, with 254 features extracted per frame using linear body part interpolation and 300 ms Gaussian smoothing. Classifier performance was evaluated via precision, recall, and F1 scores (Table 1 ). Thresholds were optimized via interactive probability plots to maximize detection accuracy and minimize false positives, and minimal bout durations were defined for each classifier (Table 2 ). Table 1 Classifiers. Data of the generated classifiers for the Resident Intruder toward inanimate object including labelled frames for training, optimum threshold and minimum bout length used for analysis. Classifier Event Frames Precision Recall F1 Approach Present 394 0.995 0.934 0.963 Absent 3647 0.993 0.999 0.996 Biting Present 451 0.998 0.998 0.998 Absent 3590 1 1 1 Digging Present 646 0.975 0.98 0.978 Absent 3395 0.996 0.995 0.996 Jumping Present 1228 0.999 0.996 0.998 Absent 2813 0.998 1 0.999 Rearing Present 401 0.986 0.893 0.937 Absent 3640 0.988 0.999 0.99 Run away Present 191 0.994 0.88 0.933 Absent 3850 0.994 1 0.997 Self-grooming Present 1129 0.996 0.996 0.996 Absent 3502 0.999 0.999 0.999 Tail rattling Present 131 0.983 0.893 0.936 Absent 4500 0.997 1 0.998 Table 2 Prediction curves. Data of generated prediction curves including present and absent frames, precision, recall, and F1 values for each classifier. Classifier Labeled Frames Labeled Videos Optimum Threshold Minimum bout length Approach 1887 10 0.6 350 Biting 2272 10 0.52 400 Digging 3366 10 0.7 600 Jumping 6042 10 0.6 400 Rearing 2188 10 0.55 500 Run Away 1051 10 0.35 500 Self-Grooming 5712 10 0.75 600 Tail Rattling 666 10 0.8 150 Output measures included event count, event duration, and latency to first occurrence. For RI-inanimate videos, additional spatial metrics were computed, including entries into the region of interest (ROI), time spent facing the stimulus, and time in the opposite cage zone. Tissue Processing and Confocal Microscopy Analysis For histological analysis, animals were anesthetized with an overdose of sodium pentobarbital (1 mg/kg, i.p.; Dolethal, Vetoquinol, Madrid, Spain) and transcardially perfused with 0.9% NaCl for 5 min, followed by 15 min of 4% paraformaldehyde (PFA in 0.1 M PB, pH 7.4; Sigma-Aldrich). Brains were extracted, post-fixed in PFA for 2 h, and cryoprotected for 48 h in 30% sucrose (in 0.01 M PBS). Coronal sections (50 µm thick) were cut using a freezing sliding microtome (LEICA SM2000R), collected in six sequential subseries, and stored at − 20°C in cryoprotective solution (30% ethylene glycol, 30% glycerol, 40% PB 0.1 M; Sigma-Aldrich). Prior to the immunofluorescence assay, injection sites were verified under a fluorescence microscope. Free-floating sections were then processed using the following protocol: (i) washing (3 × 10 min PBS), (ii) blocking (1 h in 10% normal goat serum in PBST), (iii) incubation with primary antibody (guinea pig anti-cFos, 1:500; Synaptic Systems #226008) for 72 h at 4°C, (iv) washing (3 × 10 min PBS), (v) incubation with secondary antibody (Alexa 647 goat anti-guinea pig IgG, 1:200; Biotium #20041) for 2 h at room temperature, (vi) washing (2 × 10 min PBS + 1 × 10 min PB), (vii) counterstaing with DAPI (0.1 µg/mL; ThermoFisher, cat. #D1306), and (viii) mounting using Fluoromount-G (Invitrogen, ThermoFisher). Sections were imaged with a Leica SP8 laser scanning confocal microscope. Confocal images (2048 × 2048 pixels) were acquired at 10× magnification (NA 0.8 air objective) to cover the full MeA region. The expression of cFos, was evaluated as a marker of neuronal activation level ( 63 ) within the AAV infected MeA SST neurons. Quantification was performed using QuPath v0.1.3 ( 64 ). The MeA was manually delineated based on DAPI staining, following ( 58 ). Nuclei were automatically segmented, and a 5 µm cytoplasmic expansion was applied. After background subtraction, mCherry and cFos signals were detected within cell masks. Cells were classified as mCherry + and/or cFos + based on signal thresholds. Data were expressed as the percentage of cFos + cells among mCherry + neurons. Viral infection was further verified by quantifying mCherry fluorescence with ImageJ (FIJI). At least four sections per animal were analyzed and averaged to yield one value per hemisphere per mouse. Statistics Statistical analyses were performed using Prism v8.0 (GraphPad Software). Data were analyzed only from animals that exhibited the behaviors of interest. Normality was assessed using the Shapiro–Wilk test. For comparisons among the three experimental groups (mCherry, hM3Dq, hM4Di), one-way ANOVA was used for normally distributed data. For pairwise comparisons in the tube test (TT), unpaired two-tailed t-tests were applied. Two-way ANOVA with factors Group × ROI was used to analyze three-chamber (3CH) test data across sociability and social novelty phases, followed by post hoc tests where appropriate. All data are presented as mean ± SEM in bar graphs with individual data points represented as dots. The significance threshold (α) was set at 0.05. Statistical significance is indicated in figures as follows: p < 0.05 (* ), p < 0.01 (**), and p < 0.001 (*** ). Declarations Acknowledgments The authors thank “Servei Central d’Instrumentació Científica (SCIC)” and “Servei d’Experimentació Animal (SEA)” at Universitat Jaume I for their technical support. Authors belong to “Red Española de Investigación en Estrés (REIS)” financed by MCIN/AEI /10.13039/501100011033 and FEDER (RED2022-134191-T). Funding: Spanish Ministry of Science, Innovation, and Universities: grant PID2023-153074OB-I00 (ECG, FOB). Generalitat Valenciana: grant CIAICO/2023/244 (ECG) Universitat Jaume I: grant GACUJIMB/2024/30 (ECG, FOB) European Commission grant MSCA-SE101086247 (FOB) Author contributions: Conceptualization: ECG, AOM Methodology: AOM, JHC Investigation: AOM, MAZ Visualization: AOM, MAZ Supervision: ECG, FOB Writing—original draft: AOM, ECG, FOB Writing—review & editing: AOM, MAZ, JHC, ECG, FOB Competing interests: Authors declare that they have no competing interests Data and materials availability: Raw data that support the findings of this study will be deposited at https://repositori.uji.es/ and will be made available from the corresponding author upon reasonable request. References Rubenstein DI, Rubenstein DR (2013) Social Behavior. Encyclopedia of Biodiversity. Elsevier, pp 571–579 Coria-Avila GA, Manzo J, Garcia LI, Carrillo P, Miquel M, Pfaus JG (2014) Neurobiology of social attachments. Neurosci Biobehav Rev 43:173–182 Wei D, Talwar V, Lin D (2021) Neural circuits of social behaviors: Innate yet flexible. Neuron 109:1600–1620 Chen P, Hong W (2018) Neural Circuit Mech Social Behav Neuron 98:16–30 Karigo T, Deutsch D (2022) Flexibility of neural circuits regulating mating behaviors in mice and flies. Front Neural Circuits 16 Mazza V, Šlipogor V (2024) Behavioral flexibility and novel environments: integrating current perspectives for future directions. Curr Zool 70:304 Raam T, Hong W (2021) Organization of neural circuits underlying social behavior: A consideration of the medial amygdala. Curr Opin Neurobiol 68:124–136 Prakash N, Abu Irqeba A, Corbin JG (2025) Development and function of the medial amygdala. Trends Neurosci 48:22–32 Bergan JF, Ben-Shaul Y, Dulac C (2014) Sex-specific processing of social cues in the medial amygdala. Elife 2014 Dulac C, Torello AT (2003) Molecular detection of pheromone signals in mammals: from genes to behaviour. Nature Reviews Neuroscience 2003 4:7 4, 551–562 Dwyer J, Kelly DA, Bergan J (2022) Brain-Wide Synaptic Inputs to Aromatase-Expressing Neurons in the Medial Amygdala Suggest Complex Circuitry for Modulating Social Behavior. eNeuro 9 Canteras NS, Simerly RB, Swanson LW (1995) Organization of projections from the medial nucleus of the amygdala: A PHAL study in the rat. J Comp Neurol 360:213–245 Lischinsky JE, Lin D (2020) Neural mechanisms of aggression across species. Nat Neurosci 23:1317–1328 Hong W, Kim DW, Anderson DJ (2014) Antagonistic control of social versus repetitive self-grooming behaviors by separable amygdala neuronal subsets. Cell 158:1348–1361 Chen PB, Hu RK, Wu YE, Pan L, Huang S, Micevych PE, Hong W (2019) Sexually Dimorphic Control of Parenting Behavior by the Medial Amygdala. Cell 176:1206–1221e18 Newman SW (1999) The medial extended amygdala in male reproductive behavior. A node in the mammalian social behavior network. Ann N Y Acad Sci 877:242–257 Hines M, Allen LS, Gorski RA (1992) Sex differences in subregions of the medial nucleus of the amygdala and the bed nucleus of the stria terminalis of the rat. Brain Res 579:321–326 Huijgens PT, Heijkoop R, Snoeren EMS (2021) Silencing and stimulating the medial amygdala impairs ejaculation but not sexual incentive motivation in male rats. Behav Brain Res 405:113206 Lischinsky JE, Yin L, Shi C, Prakash N, Burke J, Shekaran G, Grba M, Corbin JG, Lin D (2023) Transcriptionally defined amygdala subpopulations play distinct roles in innate social behaviors. Nat Neurosci 26:2131–2146 Huang J, Zhang YY, Qiu YY, Yao S, Qiu WT, Peng JL, Li YQ, You QL, Wu CH, Wu EJ, Wang J, Zhou YL, Ning YP, Wang HS, Chen WB, Hu BJ, Liu Y (2024) Sun, NRG1-ErbB4 signaling in the medial amygdala controls mating motivation in adult male mice. Cell Rep 43:113905 Keshavarzi S, Sullivan RKP, Ianno DJ, Sah P (2014) Functional properties and projections of neurons in the medial amygdala. J Neurosci 34:8699–8715 Lin LC, Sibille E (2015) Somatostatin, neuronal vulnerability and behavioral emotionality. Molecular Psychiatry 2015 20:3 20, 377–387 Sato M, Nakai N, Fujima S, Choe KY, Takumi T (2023) Social circuits and their dysfunction in autism spectrum disorder. Molecular Psychiatry 2023 28:8 28, 3194–3206 Hoftman GD, Volk DW, Bazmi HH, Li S, Sampson AR, Lewis DA (2015) Altered cortical expression of GABA-related genes in schizophrenia: Illness progression vs developmental disturbance. Schizophr Bull 41:180–191 Guilloux JP, Douillard-Guilloux G, Kota R, Wang X, Gardier AM, Martinowich K, Tseng GC, Lewis DA, Sibille E (2012) Molecular evidence for BDNF-and GABA-related dysfunctions in the amygdala of female subjects with major depression. Mol Psychiatry 17:1130–1142 Song YH, Yoon J, Lee SH (2021) The role of neuropeptide somatostatin in the brain and its application in treating neurological disorders. Exp Mol Med 53:328–338 Nordman JC, Ma X, Gu Q, Potegal M, Li H, Kravitz AV, Li Z (2020) Potentiation of Divergent Medial Amygdala Pathways Drives Experience-Dependent Aggression Escalation. J Neurosci 40:4858–4880 Mañas-Ojeda A, Hidalgo-Cortés J, García-Mompó C, Zahran MA, Gil-Miravet I, Olucha-Bordonau FE, Guirado R, Castillo-Gómez E (2024) Activation of somatostatin neurons in the medial amygdala reverses long-term aggression and social deficits associated to early-life stress in male mice. Mol Psychiatry. 10.1038/s41380-024-02829-6 Fan Z, Zhu H, Zhou T, Wang S, Wu Y, Hu H (2019) Using the tube test to measure social hierarchy in mice. Nat Protoc 14:819–831 Mañas-Ojeda A, Hidalgo-Cortés J, García-Mompó C, Zahran MA, Gil-Miravet I, Olucha-Bordonau FE, Guirado R, Castillo-Gómez E (2024) Activation of somatostatin neurons in the medial amygdala reverses long-term aggression and social deficits associated to early-life stress in male mice. Molecular Psychiatry 2024 30:5 30, 2168–2182 Fulenwider HD, Caruso MA, Ryabinin AE (2021) Manifestations of domination: Assessments of social dominance in rodents. Genes Brain Behav 21:e12731 Clipperton-Allen AE, Page DT (2023) Social Behavior Testing in Mice: Social Interest, Recognition, and Aggression. Neuromethods 190:279–307 Morozova MV, Boldyreva LV, Borisova MA, Kozhevnikova EN (2024) Investigating social communication in mice: A two-intruders test approach. J Neurosci Res 102:e25365 Desjardins JK, Fernald RD (2010) What do fish make of mirror images? Biol Lett 6:744–747 Yang T, Yang CF, Chizari MD, Maheswaranathan N, Burke KJ, Borius M, Inoue S, Chiang MC, Bender KJ, Ganguli S, Shah NM (2017) Social Control of Hypothalamus-Mediated Male Aggression. Neuron 95:955–970e4 Powell SB, Geyer MA, Gallagher D, Paulus MP (2004) The balance between approach and avoidance behaviors in a novel object exploration paradigm in mice. Behav Brain Res 152:341–349 Latham N, Mason G (2004) From house mouse to mouse house: the behavioural biology of free-living Mus musculus and its implications in the laboratory. Appl Anim Behav Sci 86:261–289 Dudek BC, Adams N, Boice R, Abbott ME (1983) Genetic influences on digging behaviors in mice (Mus musculus) in laboratory and seminatural settings. J Comp Psychol 97:249–259 Sturman O, Germain PL, Bohacek J (2018) Exploratory rearing: a context- and stress-sensitive behavior recorded in the open-field test. Stress 21:443–452 Ferguson JN, Young LJ, Insel TR (2002) The Neuroendocrine Basis of Social Recognition. Front Neuroendocrinol 23:200–224 Choi GB, Dong H, Murphy AJ, Valenzuela DM, Yancopoulos GD, Swanson LW, Anderson DJ (2005) Lhx6 Delineates a Pathway Mediating Innate Reproductive Behaviors from the Amygdala to the Hypothalamus. Neuron 46:647–660 Armario A (2021) The forced swim test: Historical, conceptual and methodological considerations and its relationship with individual behavioral traits. Neurosci Biobehav Rev 128:74–86 Commons KG, Cholanians AB, Babb JA, Ehlinger DG (2017) The Rodent Forced Swim Test Measures Stress-Coping Strategy, Not Depression-like Behavior. ACS Chem Neurosci 8:955–960 Sanacora G, Mason GF, Rothman DL, Behar KL, Hyder F, Petroff OAC, Berman RM, Charney DS, Krystal JH (1999) Reduced Cortical γ-Aminobutyric Acid Levels in Depressed Patients Determined by Proton Magnetic Resonance Spectroscopy. Arch Gen Psychiatry 56:1043 Sanacora G, Mason GF, Rothman DL, Krystal JH (2002) Increased Occipital Cortex GABA Concentrations in Depressed Patients After Therapy With Selective Serotonin Reuptake Inhibitors. Am J Psychiatry 159:663–665 Hasler G, van der Veen JW, Tumonis T, Meyers N, Shen J, Drevets WC (2007) Reduced Prefrontal Glutamate/Glutamine and γ-Aminobutyric Acid Levels in Major Depression Determined Using Proton Magnetic Resonance Spectroscopy. Arch Gen Psychiatry 64:193 Bajbouj M, Lisanby SH, Lang UE, Danker-Hopfe H, Heuser I (2006) Neu, Evidence for Impaired Cortical Inhibition in Patients with Unipolar Major Depression. Biol Psychiatry 59:395–400 Castricum J, Birkenhager TK, Kushner SA, Elgersma Y, Tulen JHM (2022) Cortical Inhibition and Plasticity in Major Depressive Disorder. Front Psychiatry 13 Lovelock DF, Deak T (2019) Acute stress imposed during adolescence yields heightened anxiety in Sprague Dawley rats that persists into adulthood: Sex differences and potential involvement of the Medial Amygdala. Brain Res 1723:146392 Trainor BC, Hofmann HA (2006) Somatostatin Regulates Aggressive Behavior in an African Cichlid Fish. Endocrinology 147:5119–5125 Yang T, Bayless DW, Wei Y, Landayan D, Marcelo IM, Wang Y, DeNardo LA, Luo L, Druckmann S, Shah NM (2023) Hypothalamic neurons that mirror aggression. Cell 186:1195–1211e19 Shemesh Y, Benjamin A, Shoshani-Haye K, Yizhar O, Chen A (2024) Studying dominance and aggression requires ethologically relevant paradigms. Curr Opin Neurobiol 86:102879 Miller SM, Marcotulli D, Shen A, Zweifel LS (2019) Divergent medial amygdala projections regulate approach–avoidance conflict behavior. Nat Neurosci 22:565–575 Baleisyte A, Schneggenburger R, Kochubey O (2022) Stimulation of medial amygdala GABA neurons with kinetically different channelrhodopsins yields opposite behavioral outcomes. Cell Rep 39:110850 Ranade S (2021) Comforting in mice. Nat Neurosci 24:1640–1640 Wu YE, Dang J, Kingsbury L, Zhang M, Sun F, Hu RK, Hong W (2021) Neural control of affiliative touch in prosocial interaction. Nature 599:262–267 Baum MJ, Everitt BJ (1992) Increased expression of c-fos in the medial preoptic area after mating in male rats: Role of afferent inputs from the medial amygdala and midbrain central tegmental field. Neuroscience 50:627–646 Paxinos G, Franklin KBJ (2012) Paxinos and Franklin’s the Mouse Brain in Stereotaxic Coordinates. Elsevier Science. https://books.google.es/books?id=8RJZLwEACAAJ) Mathis A, Mamidanna P, Cury KM, Abe T, Murthy VN, Mathis MW, Bethge M (2018) DeepLabCut: markerless pose estimation of user-defined body parts with deep learning. Nat Neurosci 21:1281–1289 Deng X, Xu W, Liu Y, Jing H, Zhong J, Sun K, Zhou R, Xu L, Wu X, Zhang B, Chen W, Jiang S, Chen G, Zhu Y (2025) Social rank modulates methamphetamine-seeking in dominant and subordinate male rodents via distinct dopaminergic pathways. Nature Neuroscience 2025 28:6 28, 1268–1279 Walker SE, Wood TC, Cash D, Mesquita M, Williams SCR, Sandi C (2018) Alterations in brain microstructure in rats that develop abnormal aggression following peripubertal stress. Eur J Neurosci 48:1818–1832 Lauer J, Zhou M, Ye S, Menegas W, Schneider S, Nath T, Rahman MM, Di Santo V, Soberanes D, Feng G, Murthy VN, Lauder G, Dulac C, Mathis MW, Mathis A (2022) Multi-animal pose estimation, identification and tracking with DeepLabCut. Nat Methods 19:496–504 Morgan JI, Curran T (1991) Stimulus-transcription coupling in the nervous system: involvement of the inducible proto-oncogenes fos and jun. Annu Rev Neurosci 14:421–451 Bankhead P, Loughrey MB, Fernández JA, Dombrowski Y, McArt DG, Dunne PD, McQuaid S, Gray RT, Murray LJ, Coleman HG, James JA, Salto-Tellez M (2017) Hamilton, QuPath: Open source software for digital pathology image analysis. Sci Rep 7:16878 Additional Declarations The authors declare no competing interests. Supplementary Files SupplResearchSquareDREADDsRIvariantsDEFjun25.docx Supplementary figures S1 to S3 Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. 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-7046653","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":480718266,"identity":"3cdbf406-05c7-456b-8ea6-38d36de866cf","order_by":0,"name":"Aroa Mañas-Ojeda","email":"","orcid":"https://orcid.org/0000-0001-5680-4444","institution":"Universitat Jaume I","correspondingAuthor":false,"prefix":"","firstName":"Aroa","middleName":"","lastName":"Mañas-Ojeda","suffix":""},{"id":480718888,"identity":"46d284d7-d1c8-46f6-a696-5cf86c2f7046","order_by":1,"name":"Mohamed Aly Zahran","email":"","orcid":"https://orcid.org/0000-0002-6537-7496","institution":"Universitat Jaume I","correspondingAuthor":false,"prefix":"","firstName":"Mohamed","middleName":"Aly","lastName":"Zahran","suffix":""},{"id":480718889,"identity":"7856a8df-9919-4413-8df4-ddbeca1c8e50","order_by":2,"name":"José Hidalgo-Cortés","email":"","orcid":"https://orcid.org/0000-0001-7721-8890","institution":"Universitat Jaume I","correspondingAuthor":false,"prefix":"","firstName":"José","middleName":"","lastName":"Hidalgo-Cortés","suffix":""},{"id":480718890,"identity":"28f936eb-ddd7-401e-8ae9-da51f939464e","order_by":3,"name":"Francisco E. Olucha-Bordonau","email":"","orcid":"https://orcid.org/0000-0003-0342-993X","institution":"Universitat Jaume I","correspondingAuthor":false,"prefix":"","firstName":"Francisco","middleName":"E.","lastName":"Olucha-Bordonau","suffix":""},{"id":480718891,"identity":"d75f1a06-9a94-405d-b8ef-989413391f72","order_by":4,"name":"Esther Castillo-Gómez","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAtElEQVRIiWNgGAWjYPACCRl+BoaEAyToSJDgkWwgUQsDjwHR6vn5Dz/8+POHBY/xjYSHBxgq6ghrkZyRZizNA3SY2Y0EoMPOHCasxeAGD4M0A0wLYxsRzrM/f4b55w+gFuMZIC3/iHCYAUMOmwTIYQYSIC0NzIS1SNxIM7PmSZPgkTjzIOFAwjEi/MLff/jxzR82dXL87TnJHz7UEOEwJMCTAIwf0gD7ARI1jIJRMApGwUgBAGPqNxqNPjufAAAAAElFTkSuQmCC","orcid":"https://orcid.org/0000-0002-7566-0340","institution":"Universitat Jaume I","correspondingAuthor":true,"prefix":"","firstName":"Esther","middleName":"","lastName":"Castillo-Gómez","suffix":""}],"badges":[],"createdAt":"2025-07-04 12:12:01","currentVersionCode":1,"declarations":{"humanSubjects":false,"vertebrateSubjects":true,"conflictsOfInterestStatement":false,"humanSubjectEthicalGuidelines":false,"humanSubjectConsent":false,"humanSubjectClinicalTrial":false,"humanSubjectCaseReport":false,"vertebrateSubjectEthicalGuidelines":true},"doi":"10.21203/rs.3.rs-7046653/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-7046653/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":86121047,"identity":"cae9aee6-8c9c-4615-a14f-7741901afad9","added_by":"auto","created_at":"2025-07-07 03:44:25","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":3088482,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eExperimental procedure and cFos analyses in mCherry+ neurons (SST-neurons) expressing DREADDs within the MeA. A\u003c/strong\u003eExperimental design for the AAV injection, CNO treatment, behavioural testing and sample processing for experiment 1 and experiment 2. \u003cstrong\u003eB\u003c/strong\u003e Graph showing weight changes of the three group conditions throughout the experiment at different time points: surgeries day (P60), first day of CNO injection (P81), 10 days after starting CNO treatment (P91) and perfusion day (P103). \u003cstrong\u003eC\u003c/strong\u003eSchematic drawing depicting the viral strategy (AAV-hSyn-DIO-mCherry) used in Sst-Cre mice and the representative slices of bilateral injections in MeA from bregma -1.22 mm to bregma -1.94 mm (MeA highlighted in red). \u003cstrong\u003eD\u003c/strong\u003e Drawings of somatostatin-expressing neurons depicting the three DREADDs receptor associated-neurons driving the specific cFos activity response upon CNO injection. \u003cstrong\u003eE\u003c/strong\u003e Bar graph with dots showing the cFos expression measured by the % of cFos+ cells overall mCherry+ cells. \u0026nbsp;\u003cstrong\u003eF\u003c/strong\u003e Representative confocal microscopy images comparing the three experimental groups. Left to right: confocal images captured at 10X and at 63X, respectively (merge, mCherry in red, and cFos in blue). White head arrows indicate mCherry+cFos+ cells whereas yellow arrow heads indicate mCherry+cFos- cells. Scale bar: 200 μm and 20 μm, respectively. Each data point is an individual animal. Data in graph are presented as mean ± SEM, one-way analyses of variance (ANOVA), post hoc Bonferroni test; *p\u0026lt;0.05; **\u0026lt;p\u0026lt;0.01 (n=6-7/group).\u003c/p\u003e","description":"","filename":"Figure1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7046653/v1/90d1d60b79fdceeaa261fdb5.jpg"},{"id":86121350,"identity":"cc0873dd-9f5d-4c9e-8ec6-4de391e8e3d3","added_by":"auto","created_at":"2025-07-07 03:52:25","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":3284702,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEvaluation of emotional responses and coping strategies upon MeA\u003c/strong\u003e\u003csup\u003e\u003cstrong\u003eSST+\u003c/strong\u003e\u003c/sup\u003e\u003cstrong\u003e neuronal activity manipulation.\u0026nbsp;A \u003c/strong\u003eExperimental design. \u003cstrong\u003eB-E\u003c/strong\u003e OF test analyses. \u003cstrong\u003eB\u003c/strong\u003e Bar graphs showing the locomotor activity measured in the OF; \u003cstrong\u003eC\u003c/strong\u003e (i-iii) Anxiety-like behaviour measured in the total time of the OF test: (i) total time in center zone, (ii) total time in periphery zone, (iii) ratio total time center/time periphery; \u003cstrong\u003eD\u003c/strong\u003e (i-iv) Anxiety-like behaviour measured in different periods of time: (i) time in center zone during the first 5 min, (ii) time in center during the last 5 min, (iii) time in periphery zone during the first 5 min, (iv) time in periphery zone during the last 5 min.\u003cstrong\u003e E\u003c/strong\u003e track plots representative of each experimental group. \u003cstrong\u003eF-K\u003c/strong\u003e Coping strategies evaluated in the FST. \u003cstrong\u003eF\u003c/strong\u003e Drawing depicting the FST; \u003cstrong\u003eG\u003c/strong\u003e Mean speed measured in m/s; \u003cstrong\u003eH\u003c/strong\u003e Time mobile in s; \u003cstrong\u003eI\u003c/strong\u003e time immobile in s; \u003cstrong\u003eJ\u003c/strong\u003e immobility latency in s; \u003cstrong\u003eK\u003c/strong\u003e immobile episodes. \u003cstrong\u003eL-N\u003c/strong\u003e 3CH test analyses. \u003cstrong\u003eL\u003c/strong\u003e Drawing depicting the 3CH paradigm representing the sociability phase (upper) and social novelty preference (lower). \u003cstrong\u003eM\u003c/strong\u003e Sociability phase: (i) drawing showing the zones considered in the 3CH and three heatmaps representative of the results from each group; (ii) entries and (v) time in ROI; (iii) entries and time (vi) in the social target ROI; (iv) discrimination index of the number of entries and (vii) time in social target ROI respect to the empty’s ROI entries. \u003cstrong\u003eN\u003c/strong\u003e Social novelty preference phase: (i) drawing showing the zones considered in the 3CH and three heatmaps representative of the results from each group; (ii) entries and (v) time in ROI; (iii) entries and time (vi) in the novel social target ROI; (iv) discrimination index of the number of entries and (vii) time in novel social target ROI respect to the empty’s ROI entries. \u003cstrong\u003eO-Q\u003c/strong\u003e Tube dominance test results. \u003cstrong\u003eO\u003c/strong\u003e Picture showing the animals in the test and representing the results of each group during the dyadic encounters. \u003cstrong\u003eP\u003c/strong\u003e Social dominance; (i) dominance score of mCherry group and hM3Dq group; (ii) dominance score of mCherry group and hM4Di group; (iii) dominance score per round for each pair of dyadic encounters. \u003cstrong\u003eQ\u003c/strong\u003e Aggressive-like behaviour evaluated in the TT as number of pushes/s (i) of mCherry group and hM3Dq group; (ii) mCherry group and hM4Di group; and (iii) dominance score per round for each pair of dyadic encounters. Each data point is an individual animal. Data in graph are presented as mean ± SEM (mCherry, n=9; hM3Dq, n=8; hM4Di, n=6), one-way analyses of variance (ANOVA), post hoc Bonferroni test; *p\u0026lt;0.05; **\u0026lt;p\u0026lt;0.01, ***p\u0026lt;0.001.\u0026nbsp;\u003c/p\u003e","description":"","filename":"Figure2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7046653/v1/181ccb7119d6716e2e440a7e.jpg"},{"id":86121053,"identity":"96a85ced-b2aa-4a9c-b526-6ac6d7a42d71","added_by":"auto","created_at":"2025-07-07 03:44:25","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":2310094,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eImplication of MeA\u003c/strong\u003e\u003csup\u003e\u003cstrong\u003eSST+ \u003c/strong\u003e\u003c/sup\u003e\u003cstrong\u003eneurons in social and aggressive behaviour toward males via resident intruder.\u0026nbsp;\u003c/strong\u003e\u0026nbsp;\u003cstrong\u003eA\u003c/strong\u003e Schematic drawing representing the classifiers and behaviours considered in this experiment. \u003cstrong\u003eB\u003c/strong\u003e Drawings representing each classifier and grouped based on the type of behaviour. \u003cstrong\u003eC\u003c/strong\u003e Experimental design for the RI toward males, highlighted in blue.\u003cstrong\u003e D\u003c/strong\u003e Schematic drawing depicting mouse surgery and the pose estimation tracking by DeepLabCut during a resident-intruder encounter. \u003cstrong\u003eE- K\u003c/strong\u003e Bar graphs with dots showing the bout counts, the latency to the first behavioural event, and the duration of each behaviour: \u003cstrong\u003eE\u003c/strong\u003e, offensive behavior, \u003cstrong\u003eF\u003c/strong\u003e defensive/submissive behavior, \u003cstrong\u003eG\u003c/strong\u003e dominance behavior, \u003cstrong\u003eH\u003c/strong\u003e social interest behavior, \u003cstrong\u003eI\u003c/strong\u003e self-grooming, \u003cstrong\u003eJ\u003c/strong\u003e rearing and \u003cstrong\u003eK\u003c/strong\u003e social attention measured as the time spent in nose-to-nose directionality. \u003cstrong\u003eL\u003c/strong\u003e Drawing showing a summary of the previous significant results following neuronal activation. Each data point is an individual animal. Data in graph are presented as mean ± SEM (n=7-9/group), one-way analyses of variance (ANOVA), post hoc Bonferroni test; *p\u0026lt;0.05; **\u0026lt;p\u0026lt;0.01.\u003c/p\u003e","description":"","filename":"Figure3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7046653/v1/2f9315b21debaba1aae80162.jpg"},{"id":86121049,"identity":"d7df481d-6754-4e0e-9792-4cce398f6f87","added_by":"auto","created_at":"2025-07-07 03:44:25","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":2216333,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSocial, sexual and aggressive behavior of male mice toward females upon MeASST+ neuronal activity manipulation.\u003c/strong\u003e \u003cstrong\u003eA\u003c/strong\u003e Schematic drawing representing the classifiers and behaviours considered in this experiment. \u003cstrong\u003eB\u003c/strong\u003e Experimental design for the RI toward females, highlighted in red. \u003cstrong\u003eC-I\u003c/strong\u003e Bar graphs with dots showing the bout counts, the latency to the first behavioural event, and the duration of each behaviour: \u003cstrong\u003eC\u003c/strong\u003e offensive behavior, \u003cstrong\u003eD\u003c/strong\u003e defensive/submissive behavior, \u003cstrong\u003eE\u003c/strong\u003e dominance and sexual interest, \u003cstrong\u003eF\u003c/strong\u003e social interest behavior, \u003cstrong\u003eG\u003c/strong\u003e self-grooming, \u003cstrong\u003eH \u003c/strong\u003erearing and \u003cstrong\u003eI\u003c/strong\u003e social attention measured as the time spent in nose-to-nose directionality. \u003cstrong\u003eJ\u003c/strong\u003e Drawing showing a summary of the previous significant results following neuronal activation. Each data point is an individual animal. Data in graph are presented as mean ± SEM (n=7-9/group), one-way analyses of variance (ANOVA), post hoc Bonferroni test; *p\u0026lt;0.05; **\u0026lt;p\u0026lt;0.01.\u003c/p\u003e","description":"","filename":"Figure4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7046653/v1/98a038bed984aa1ef0eefd22.jpg"},{"id":86121352,"identity":"a6c51414-2267-4c70-ae8e-9ad89367e1cd","added_by":"auto","created_at":"2025-07-07 03:52:26","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":2757399,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003e“Fight or flight” responses toward an inanimate object after modulating SST-expressing neurons activity\u003c/strong\u003e. \u0026nbsp;\u003cstrong\u003eA\u003c/strong\u003eExperimental design for the RI toward an inanimate object (highlighted in purple). \u003cstrong\u003eB\u003c/strong\u003e Drawing depicting the pose estimation tracking by DeepLabCut, the two different ROIs considered: the object zone and the opposite corner, the different classifiers evaluated, and drawings showing the RI assay toward the different objects: mirror, flipped mirror and glove. \u003cstrong\u003eC-J \u003c/strong\u003eRI toward mirror. \u003cstrong\u003eC\u003c/strong\u003e Schematic showing the procedure of the RI toward the mirror. For the mirror: \u003cstrong\u003eD\u003c/strong\u003e time facing ROIs; \u003cstrong\u003eE\u003c/strong\u003e time facing the object’s ROI; \u003cstrong\u003eF\u003c/strong\u003e entries in ROIs; \u003cstrong\u003eG\u003c/strong\u003e entries in object’s ROI; \u003cstrong\u003eH\u003c/strong\u003eapproaches; \u003cstrong\u003eI\u003c/strong\u003e run away; \u003cstrong\u003eJ\u003c/strong\u003e tail rattling. \u003cstrong\u003eK-R\u003c/strong\u003e RI toward flipped mirror. \u003cstrong\u003eK\u003c/strong\u003e Schematic showing the procedure of the RI toward the flipped mirror. For the flipped mirror: \u003cstrong\u003eL\u003c/strong\u003e time facing ROIs; \u003cstrong\u003eM\u003c/strong\u003etime facing the object’s ROI; \u003cstrong\u003eN\u003c/strong\u003e entries in ROIs; \u003cstrong\u003eO\u003c/strong\u003e entries in object’s ROI; \u003cstrong\u003eP\u003c/strong\u003e approaches; \u003cstrong\u003eQ\u003c/strong\u003e run away; \u003cstrong\u003eR\u003c/strong\u003e tail rattling. \u003cstrong\u003eS-Z\u003c/strong\u003eRI toward glove. \u003cstrong\u003eS\u003c/strong\u003e Schematic showing the procedure of the RI toward the glove. For the glove: \u003cstrong\u003eT\u003c/strong\u003e time facing ROIs; \u003cstrong\u003eU\u003c/strong\u003e time facing the object’s ROI; \u003cstrong\u003eV\u003c/strong\u003e entries in ROIs; \u003cstrong\u003eW\u003c/strong\u003e entries in object’s ROI; \u003cstrong\u003eX\u003c/strong\u003eapproaches; \u003cstrong\u003eY\u003c/strong\u003e run away; \u003cstrong\u003eZ\u003c/strong\u003e tail rattling. For all the classifiers in all graphs, the bout counts (i), latency to first event (ii) and duration (iii) parameters were considered. Each data point is an individual animal. Data in graph are presented as mean ± SEM (n=7-9/group), one-way analyses of variance (ANOVA), post hoc Bonferroni test; *p\u0026lt;0.05; **\u0026lt;p\u0026lt;0.01.\u003c/p\u003e","description":"","filename":"Figure5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7046653/v1/a621eaa34283aaf4bbe47577.jpg"},{"id":86121834,"identity":"6890f449-bd23-47cf-b262-fcaf54262dc4","added_by":"auto","created_at":"2025-07-07 04:00:30","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":15300574,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7046653/v1/0edb763b-d07d-4a09-ad9a-8fae141120f8.pdf"},{"id":86121058,"identity":"e58f57e8-888b-48c1-84fb-8795c01107cb","added_by":"auto","created_at":"2025-07-07 03:44:26","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":10017365,"visible":true,"origin":"","legend":"\u003cp\u003eSupplementary figures S1 to S3\u003c/p\u003e","description":"","filename":"SupplResearchSquareDREADDsRIvariantsDEFjun25.docx","url":"https://assets-eu.researchsquare.com/files/rs-7046653/v1/89d81984ff71c98b3d2b726a.docx"}],"financialInterests":"The authors declare no competing interests.","formattedTitle":"\u003cp\u003eSomatostatin neurons in the medial amygdala orchestrate flexible, sex-specific social behavior in male mice\u003c/p\u003e","fulltext":[{"header":"Teaser","content":"\u003cp\u003eNeurons that flip the social switch: How the amygdala chooses between fight, flirt, and flight\u003c/p\u003e"},{"header":"Introduction","content":"\u003cp\u003eSocial behaviors\u0026mdash;including mating, aggression, and dominance\u0026mdash;are essential for survival and reproductive success across species, including humans (\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e). Although often considered innate, these behaviors are remarkably flexible, allowing individuals to adapt their responses according to social context and stimulus type (\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e). This behavioral flexibility is especially critical in complex and dynamic social environments, where inappropriate responses can compromise survival or reproductive opportunities (\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eThe medial amygdala (MeA) plays a central role in regulating social behavior (\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e). It integrates direct vomeronasal and indirect main olfactory inputs (\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e) and connects with hypothalamic and limbic structures to orchestrate mating, aggression, and defensive responses (\u003cspan additionalcitationids=\"CR12\" citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eThis amygdaloid nucleus comprises diverse excitatory and inhibitory neuronal populations, which are sexually dimorphic and regulate distinct dimensions of social behavior (\u003cspan additionalcitationids=\"CR15 CR16 CR17 CR18\" citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e). For example, MeA excitatory neurons promote repetitive self-grooming, while GABAergic neurons are involved in aggression and mating motivation (\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e). Manipulating ErbB4⁺ GABAergic neurons facilitates mating behavior in adult male mice (\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e), and both silencing and activating MeA excitatory neurons delay ejaculation in rats without affecting copulatory patterns or sexual motivation (\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e). These results highlight the importance of neuronal subtype in shaping MeA-driven behavior.\u003cdiv class=\"BlockQuote\"\u003e\u003cp\u003eSomatostatin-expressing (SST⁺) neurons represent a major GABAergic subtype in the MeA (\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e) and have been implicated in regulating affective and social behaviors (\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e). Alterations in SST signaling have been reported in several neuropsychiatric conditions marked by social dysfunction, including autism (\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e), schizophrenia (\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e) and major depression (\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eIn aggression-priming paradigms, excitatory neurons in the posterior ventral MeA (MeApv) are preferentially activated, whereas SST⁺ neurons show limited activation (\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e). This pattern may be functionally significant, as early-life stress have been shown to produce long-lasting suppression of MeA\u003csup\u003eSST+\u003c/sup\u003e neuron activity in males, leading to heightened aggression and reduced sociability. Restoring their activity via chemogenetics reverses these behavioral deficits (\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e).\u003c/p\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003eDespite these insights, it remains unclear whether MeA\u003csup\u003eSST⁺\u003c/sup\u003e neurons contribute to the flexible regulation of social behavior in adulthood\u0026mdash; specifically by influencing behavior in response to varying social and non-social cues. We hypothesized that MeA\u003csup\u003eSST⁺\u003c/sup\u003e neurons act as modulators of behavioral flexibility, shaping sex-specific social behavior based on contextual and cued stimuli. To investigate this, we used chemogenetic approaches to selectively activate or inhibit MeA\u003csup\u003eSST⁺\u003c/sup\u003e neurons in SST-IRES-Cre knock-in male mice, while assessing a range of social and non-social behaviors using automated, machine learning-based tracking. Chemogenetic activation of MeA\u003csup\u003eSST⁺\u003c/sup\u003e neurons decreased sociability, intermale aggression, and attention toward males, while enhancing sexual motivation and dominance responses toward females. Conversely, their inhibition increased social novelty preference without altering other social traits. Notably, both manipulations heightened escape-like responses to inanimate objects, suggesting a broader role in regulating defensive reactivity to non-social cues.\u003c/p\u003e\u003cp\u003eTogether, these findings identify MeA\u003csup\u003eSST⁺\u003c/sup\u003e neurons as key contributors to flexible, sex-specific social behavior. By linking cell-type specific function to behavioral modulation, our study advances understanding of the neural basis of adaptive social strategies and may help guide future research into psychiatric conditions characterized by social impairments, including autism, schizophrenia, and major depression.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cdiv class=\"BlockQuote\"\u003e\u003cp\u003eTo investigate the contribution of MeA\u003csup\u003eSST+\u003c/sup\u003e neurons to social behavioral flexibility\u0026mdash;particularly in response to different social partners or non-social stimuli\u0026mdash;we chronically activated or inhibited these neurons in SST-IRES-Cre male mice using a chemogenetic approach. We assessed their role across a spectrum of behavioral paradigms designed to evaluate social, emotional, aggressive, and defensive responses in both social and non-social contexts. Behavioral parameters were automatically quantified using deep learning-based tracking and multi-pose estimation tools, including DeepLabCut and SimBA, to ensure high-resolution and objective behavioral classification (\u003cb\u003eFig.\u0026nbsp;1A\u003c/b\u003e). Below, we present a structured summary of our findings, beginning with validation of the chemogenetic manipulation and progressing through behavioral domains.\u003c/p\u003e\u003cp\u003e\u003cb\u003eValidation of DREADD-based manipulation in MeA\u003c/b\u003e\u003csup\u003e\u003cb\u003eSST⁺\u003c/b\u003e\u003c/sup\u003e \u003cb\u003eneurons\u003c/b\u003e\u003c/p\u003e\u003cp\u003eWe first validated that chronic clozapine-N-oxide (CNO) treatment\u0026mdash;used to selectively activate DREADDs\u0026mdash;effectively modulated MeA\u003csup\u003eSST⁺\u003c/sup\u003e neuron activity in a cell-type\u0026ndash;specific manner without adverse physiological effects. Mice injected with Cre-dependent AAVs expressing either excitatory (hM3Dq) or inhibitory (hM4Di) DREADDs fused to mCherry in the MeA showed no significant differences in body weight across groups throughout the experimental timeline (P60, P81, P91, P103, \u003cem\u003ep\u0026thinsp;\u0026gt;\u0026thinsp;0.05\u003c/em\u003e in all cases; \u003cb\u003eFig.\u0026nbsp;1B\u003c/b\u003e), indicating that the treatment did not impair general health.\u003c/p\u003e\u003cp\u003eTo assess functional DREADD expression, we quantified cFos in mCherry⁺ neurons (\u003cb\u003eFig.\u0026nbsp;1C-F\u003c/b\u003e). A one-way ANOVA revealed a significant group effect (F\u003csub\u003e(2,23)\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;16.43; \u003cem\u003ep\u0026thinsp;\u0026lt;\u0026thinsp;0.001\u003c/em\u003e). Compared to mCherry controls, hM3Dq mice showed elevated cFos⁺ labeling (58.30\u0026thinsp;\u0026plusmn;\u0026thinsp;8.29% vs. 26.88\u0026thinsp;\u0026plusmn;\u0026thinsp;8.32%; \u003cem\u003ep\u0026thinsp;=\u0026thinsp;0.005\u003c/em\u003e), while hM4Di mice exhibited reduced labeling (7.91\u0026thinsp;\u0026plusmn;\u0026thinsp;1.58%; \u003cem\u003ep\u0026thinsp;=\u0026thinsp;0.017\u003c/em\u003e), confirming effective bidirectional modulation of MeA\u003csup\u003eSST⁺\u003c/sup\u003e neuronal activity (\u003cb\u003eFig.\u0026nbsp;1E, F\u003c/b\u003e).\u003c/p\u003e\u003cp\u003e\u003cb\u003eMeA\u003c/b\u003e\u003csup\u003e\u003cb\u003eSST+\u003c/b\u003e\u003c/sup\u003e \u003cb\u003einhibition impairs stress-coping behavior in the forced swim test without affecting anxiety in the open field\u003c/b\u003e\u003c/p\u003e\u003cp\u003eGiven the role of SST in emotional regulation, we evaluated anxiety-like and stress-coping behaviors in the open field (OF) and the forced swim test (FST), respectively (\u003cb\u003eFig.\u0026nbsp;2A\u003c/b\u003e). In the OF test, no significant differences were observed in locomotion or anxiety-related measures between groups (\u003cb\u003eFig.\u0026nbsp;2B-E\u003c/b\u003e), suggesting that MeA\u003csup\u003eSST+\u003c/sup\u003e neurons do not influence baseline anxiety.\u003c/p\u003e\u003cp\u003eIn contrast, the FST revealed altered stress-coping behavior following MeA\u003csup\u003eSST+\u003c/sup\u003e inhibition (\u003cb\u003eFig.\u0026nbsp;2F-K\u003c/b\u003e). hM4Di mice displayed reduced active coping, as indicated by decreased time mobile (\u003cem\u003ep\u0026thinsp;=\u0026thinsp;0.021\u003c/em\u003e; \u003cb\u003eFig.\u0026nbsp;2H\u003c/b\u003e), increased total immobility (\u003cem\u003ep\u0026thinsp;=\u0026thinsp;0.031;\u003c/em\u003e \u003cb\u003eFig.\u0026nbsp;2I\u003c/b\u003e), shorter latency to immobility (\u003cem\u003ep\u0026thinsp;=\u0026thinsp;0.003;\u003c/em\u003e \u003cb\u003eFig.\u0026nbsp;2J\u003c/b\u003e), and elevated immobility episode count (\u003cem\u003ep\u0026thinsp;=\u0026thinsp;0.027;\u003c/em\u003e \u003cb\u003eFig.\u0026nbsp;2K\u003c/b\u003e) compared to controls. However, hM3Dq activation had no significant effects. These results indicate that inhibition, but not activation, of MeA\u003csup\u003eSST+\u003c/sup\u003e neurons impairs stress-coping strategies without affecting anxiety.\u003c/p\u003e\u003cp\u003e\u003cb\u003eBidirectional control of sociability and social novelty in the tree-chamber test by MeA\u003c/b\u003e\u003csup\u003e\u003cb\u003eSST+\u003c/b\u003e\u003c/sup\u003e \u003cb\u003eneurons\u003c/b\u003e\u003c/p\u003e\u003cp\u003eWe next examined how MeA\u003csup\u003eSST+\u003c/sup\u003e neurons regulate social behavior in the three-chamber test (3CH), which includes phases of sociability and social novelty preference (\u003cb\u003eFig.\u0026nbsp;2L\u003c/b\u003e).\u003c/p\u003e\u003cp\u003eDuring the sociability phase (\u003cb\u003eFig.\u0026nbsp;2M-i\u003c/b\u003e), significant main effects of the region of interest (ROI; F \u003csub\u003e(1,40)\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;29.83; \u003cem\u003ep\u0026thinsp;\u0026lt;\u0026thinsp;0.001\u003c/em\u003e) and Group \u0026times; ROI interaction (F\u003csub\u003e(2,40)\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;7.702; \u003cem\u003ep\u0026thinsp;=\u0026thinsp;0.001\u003c/em\u003e) were observed when analyzing the number of entries. A similar pattern was found when analyzing time spent in each ROI, with significant main effects of ROI (F\u003csub\u003e(1,40)\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;36,40, \u003cem\u003ep\u0026thinsp;\u0026lt;\u0026thinsp;0.001\u003c/em\u003e) and a Group x ROI interaction (F\u003csub\u003e(2,40)\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;8.008, \u003cem\u003ep\u0026thinsp;=\u0026thinsp;0.003\u003c/em\u003e). Both mCherry control and hM4Di mice showed robust preference for the social target, reflected in a higher number of entries (\u003cem\u003ep\u0026thinsp;\u0026lt;\u0026thinsp;0.001;\u003c/em\u003e \u003cb\u003eFig.\u0026nbsp;2M-ii-iii\u003c/b\u003e) and increased time spent (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001; \u003cb\u003eFig.\u0026nbsp;2M-v-vi\u003c/b\u003e) in the social chamber. Moreover, the discrimination index (DI), calculated as the proportion of entries (\u003cb\u003eFig.\u0026nbsp;2N-iv\u003c/b\u003e) or time spent (\u003cb\u003eFig.\u0026nbsp;2N-vii)\u003c/b\u003e in the social ROI relative to the total (social\u0026thinsp;+\u0026thinsp;empty), was significantly reduced in hM3Dq mice (\u003cem\u003ep\u0026thinsp;=\u0026thinsp;0.003\u003c/em\u003e and \u003cem\u003ep\u0026thinsp;\u0026lt;\u0026thinsp;0.001\u003c/em\u003e, respectively).\u003c/p\u003e\u003cp\u003eDuring the social novelty phase (\u003cb\u003eFig.\u0026nbsp;2N-i\u003c/b\u003e), significant main effects were observed for Group (F\u003csub\u003e(2,40)\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;14.08; \u003cem\u003ep\u0026thinsp;\u0026lt;\u0026thinsp;0.001\u003c/em\u003e), ROI (F\u003csub\u003e(1,40)\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;109.8; p\u0026thinsp;\u0026lt;\u0026thinsp;0.001), and their interaction (F\u003csub\u003e(2,40)\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;36.18; \u003cem\u003ep\u0026thinsp;\u0026lt;\u0026thinsp;0.001\u003c/em\u003e) when analyzing time spent in the novel vs familiar social ROI. For the number of entries, a significant main effect of ROI was also observed (F\u003csub\u003e(1,40)\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;29.86; \u003cem\u003ep\u0026thinsp;\u0026lt;\u0026thinsp;0.001\u003c/em\u003e), indicating a general preference for the novel conspecific. Post hoc analyses revealed that mCherry controls showed a strong preference for the novel mouse, with significantly more entries (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001; \u003cb\u003eFig.\u0026nbsp;2N-ii\u003c/b\u003e) and more time spent (\u003cem\u003ep\u0026thinsp;\u0026lt;\u0026thinsp;0.001\u003c/em\u003e; \u003cb\u003eFig.\u0026nbsp;2N-v\u003c/b\u003e) in the novel ROI. Similarly, hM4Di mice also preferred the novel conspecific, as shown by a significant increase in time spent (\u003cem\u003ep\u0026thinsp;\u0026lt;\u0026thinsp;0.001\u003c/em\u003e; \u003cb\u003eFig.\u0026nbsp;2N-vi\u003c/b\u003e) and a more modest but significant increase in entries (\u003cem\u003ep\u0026thinsp;=\u0026thinsp;0.023\u003c/em\u003e; \u003cb\u003eFig.\u0026nbsp;2N-iii\u003c/b\u003e). In contrast, hM3Dq mice failed to show significant differences between the novel and familiar ROIs in either measure. Moreover, the DI, calculated as the proportion of entries or time spent in the novel ROI relative to the total (novel\u0026thinsp;+\u0026thinsp;familiar), was significantly reduced in hM3Dq mice compared to mCherry controls (entries: \u003cem\u003ep\u0026thinsp;=\u0026thinsp;0.036\u003c/em\u003e, \u003cb\u003eFig.\u0026nbsp;2N-iv\u003c/b\u003e; time: \u003cem\u003ep\u0026thinsp;\u0026lt;\u0026thinsp;0.001\u003c/em\u003e, \u003cb\u003eFig.\u0026nbsp;2N-vii\u003c/b\u003e).\u003c/p\u003e\u003cp\u003eThese findings show that activation of MeA\u003csup\u003eSST⁺\u003c/sup\u003e neurons is sufficient to impair sociability and social novelty responses, whereas their inhibition leaves these behaviors unaffected.\u003c/p\u003e\u003cp\u003e\u003cb\u003eActivation of MeA\u003c/b\u003e\u003csup\u003e\u003cb\u003eSST+\u003c/b\u003e\u003c/sup\u003e \u003cb\u003eneurons promotes social dominance in novel dyadic interactions in the tube test\u003c/b\u003e\u003c/p\u003e\u003cp\u003eTo assess the role of MeA\u003csup\u003eSST+\u003c/sup\u003e neurons in social dominance, we used the tube test (TT) among unfamiliar, single-housed male mice (\u003cb\u003eFig.\u0026nbsp;2O\u003c/b\u003e). This approach, adapted from Fan et al. (\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e) has been shown to yield robust results regarding dominance behavior (\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e, \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e). Chronic activation of these neurons via hM3Dq significantly increased overall dominance score \u003cem\u003e(t₁₆ = 2.278; p\u0026thinsp;\u0026lt;\u0026thinsp;0.001\u003c/em\u003e) (\u003cb\u003eFig.\u0026nbsp;2P-i\u003c/b\u003e) without affecting push frequency (\u003cb\u003eFig.\u0026nbsp;2Q-i-iii\u003c/b\u003e). However, hM4Di inhibition had no effect neither in social dominance (\u003cb\u003eFig.\u0026nbsp;2P-ii-iii\u003c/b\u003e), nor in number of pushes/s (\u003cb\u003eFig.\u0026nbsp;2Q-i-iii\u003c/b\u003e).\u003c/p\u003e\u003cp\u003eUpon further analysis of the ethological progression across rounds, a significant Group \u0026times; Round interaction (\u003cem\u003eF\u003c/em\u003e\u003csub\u003e\u003cem\u003e(2,32)\u003c/em\u003e\u003c/sub\u003e\u0026thinsp;\u003cem\u003e=\u0026thinsp;3.437; p\u0026thinsp;=\u0026thinsp;0.044\u003c/em\u003e) and a main effect of Group (\u003cem\u003eF\u003c/em\u003e\u003csub\u003e\u003cem\u003e(1,16)\u003c/em\u003e\u003c/sub\u003e\u0026thinsp;\u003cem\u003e=\u0026thinsp;5.532; p\u0026thinsp;=\u0026thinsp;0.031\u003c/em\u003e) confirmed that dominance gradually increased over time in the hM3Dq group relative to controls (\u003cb\u003eFig.\u0026nbsp;2P-iii\u003c/b\u003e); this effect became particularly evident in the final round (Round 3: p\u0026thinsp;=\u0026thinsp;0.0041). In contrast, hM4Di inhibition had no significant effect on dominance score (\u003cb\u003eFig.\u0026nbsp;2P-ii\u0026ndash;iii)\u003c/b\u003e, and no differences were observed in the number of pushes/s across groups or rounds under any condition (\u003cb\u003eFig.\u0026nbsp;2Q-i\u0026ndash;iii\u003c/b\u003e).\u003c/p\u003e\u003cp\u003eThese findings suggest that MeA\u003csup\u003eSST⁺\u003c/sup\u003e neuron activation promotes the progressive acquisition of dominance behavior during novel dyadic encounters.\u003c/p\u003e\u003cp\u003e\u003cb\u003eMeA\u003c/b\u003e\u003csup\u003e\u003cb\u003eSST+\u003c/b\u003e\u003c/sup\u003e \u003cb\u003eactivation reduces aggression and social attention in male-male resident-intruder interactions\u003c/b\u003e\u003c/p\u003e\u003cp\u003eGiven the proposed role of the MeA in mediating social threat and territoriality (\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e), we next evaluated the contribution of MeA\u003csup\u003eSST⁺\u003c/sup\u003e neurons to aggression and dominance in male-male conflict scenarios using the resident-intruder (RI) paradigm. This test allowed us to assess offensive, defensive, and dominance-related behaviors in response to a novel male intruder, as well as attention toward the conspecific (\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e) (\u003cb\u003eFig.\u0026nbsp;3A-D\u003c/b\u003e).\u003c/p\u003e\u003cp\u003eIn the offensive domain, hM3Dq mice showed a significant reduction in the duration of offensive behavior compared to controls (\u003cem\u003ep\u0026thinsp;=\u0026thinsp;0.001\u003c/em\u003e; \u003cb\u003eFig.\u0026nbsp;3E\u003c/b\u003e). However, the bout count and latency for these behaviors remained unchanged (\u003cb\u003eFig.\u0026nbsp;3E; fig. S1A-C\u003c/b\u003e).\u003c/p\u003e\u003cp\u003eIn terms of defensive responses, overall defensive behavior did not differ between groups (\u003cb\u003eFig.\u0026nbsp;3F\u003c/b\u003e), but hM3Dq mice exhibited a significant reduction in escape latency \u003cb\u003e(\u003c/b\u003e\u003cem\u003ep\u0026thinsp;=\u0026thinsp;0.042\u003c/em\u003e; \u003cb\u003efig. S1F\u003c/b\u003e), indicating enhanced defensive reactivity.\u003c/p\u003e\u003cp\u003eDominance-related behaviors were also affected by MeA\u003csup\u003eSST⁺\u003c/sup\u003e activation (\u003cb\u003eFig.\u0026nbsp;3G\u003c/b\u003e). hM3Dq mice showed a significant decrease in total dominance duration (\u003cem\u003ep\u0026thinsp;=\u0026thinsp;0.049\u003c/em\u003e; \u003cb\u003eFig.\u0026nbsp;3G\u003c/b\u003e), as well as reduced tail rattle bout count \u003cb\u003e(\u003c/b\u003e\u003cem\u003ep\u0026thinsp;=\u0026thinsp;0.031\u003c/em\u003e), duration \u003cb\u003e(\u003c/b\u003e\u003cem\u003ep\u0026thinsp;=\u0026thinsp;0.0063\u003c/em\u003e), and increased latency to first tail rattle (\u003cem\u003ep\u0026thinsp;=\u0026thinsp;0.038\u003c/em\u003e) (\u003cb\u003efig. S1G\u003c/b\u003e).\u003c/p\u003e\u003cp\u003eSocial attention was also diminished in hM3Dq mice, as reflected by significantly less time spent oriented toward the intruder's nose (\u003cem\u003ep\u0026thinsp;=\u0026thinsp;0.046\u003c/em\u003e) (\u003cb\u003eFig.\u0026nbsp;3K\u003c/b\u003e).\u003c/p\u003e\u003cp\u003eImportantly, these changes were not accompanied by alterations in general social interest (approach, allogrooming), self-grooming, or exploratory behaviors (\u003cb\u003eFig.\u0026nbsp;3H-J; fig. S1H-J\u003c/b\u003e), suggesting that the observed reductions in aggression and dominance were not due to general suppression of social motivation (\u003cb\u003eFig.\u0026nbsp;3L\u003c/b\u003e).\u003c/p\u003e\u003cp\u003e\u003cb\u003eActivation of MeA\u003c/b\u003e\u003csup\u003e\u003cb\u003eSST+\u003c/b\u003e\u003c/sup\u003e \u003cb\u003eneurons enhances dominance, sexual behavior, and defensive responses during female-directed social encounters\u003c/b\u003e\u003c/p\u003e\u003cp\u003eWe next assessed the role of MeA\u003csup\u003eSST⁺\u003c/sup\u003e neurons in regulating social behavior toward female conspecifics using the RI test (\u003cb\u003eFig.\u0026nbsp;4A-B\u003c/b\u003e). This context allowed us to evaluate how neuronal activation influences aggression, sexual behavior, dominance, and social attention in male-female interactions (\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eAmong the experimental groups, only hM3Dq mice exhibited notable behavioral alterations. Global offensive behavior remained unaffected (\u003cb\u003eFig.\u0026nbsp;4C\u003c/b\u003e); however, a selective reduction in lateral threat events was observed (\u003cem\u003ep\u0026thinsp;=\u0026thinsp;0.013\u003c/em\u003e; \u003cb\u003efig. S2C\u003c/b\u003e), with no significant differences in attack-related classifiers (\u003cb\u003efig. S2A\u003c/b\u003e) or vigorous allogrooming (\u003cb\u003efig. S2B\u003c/b\u003e).\u003c/p\u003e\u003cp\u003eWhile overall defensive behavior remained unchanged (\u003cb\u003eFig.\u0026nbsp;4D\u003c/b\u003e), specific components were significantly modulated in hM3Dq mice. These animals displayed increased duration of defensive attacks (\u003cem\u003ep\u0026thinsp;=\u0026thinsp;0.031\u003c/em\u003e; \u003cb\u003efig. S2D\u003c/b\u003e), as well as elevated bout count (\u003cem\u003ep\u0026thinsp;=\u0026thinsp;0.016\u003c/em\u003e) and duration (\u003cem\u003ep\u0026thinsp;=\u0026thinsp;0.011\u003c/em\u003e) of upright submission (\u003cb\u003efig. S2E\u003c/b\u003e). Escape behavior, by contrast was unaffected (\u003cb\u003efig. S2F\u003c/b\u003e).\u003c/p\u003e\u003cp\u003eDominance and sexual behaviors were markedly enhanced in hM3Dq mice. The total duration of this category was significantly elevated compared to controls (\u003cem\u003ep\u0026thinsp;=\u0026thinsp;0.036\u003c/em\u003e; \u003cb\u003eFig.\u0026nbsp;4E\u003c/b\u003e), primarily driven by increased durations of tail rattling (\u003cem\u003ep\u0026thinsp;=\u0026thinsp;0.045\u003c/em\u003e; \u003cb\u003efig. S2G\u003c/b\u003e) and mounting (\u003cem\u003ep\u0026thinsp;=\u0026thinsp;0.034;\u003c/em\u003e \u003cb\u003efig. S2H\u003c/b\u003e).\u003c/p\u003e\u003cp\u003eDespite a significant increase in the latency to initiate social contact (\u003cem\u003ep\u0026thinsp;=\u0026thinsp;0.009\u003c/em\u003e; \u003cb\u003eFig.\u0026nbsp;4F\u003c/b\u003e), approach behavior (\u003cb\u003efig. S2I\u003c/b\u003e) and normal allogrooming remained stable in bout count and duration. However, the latency to first normal allogrooming was also significantly delayed (\u003cem\u003ep\u0026thinsp;=\u0026thinsp;0.007\u003c/em\u003e; \u003cb\u003efig. S2J\u003c/b\u003e), further suggesting a shift in behavioral priorities. In parallel, hM3Dq mice showed a reduced frequency of self-grooming bouts (\u003cem\u003ep\u0026thinsp;=\u0026thinsp;0.03\u003c/em\u003e5; \u003cb\u003eFig.\u0026nbsp;4G\u003c/b\u003e), while rearing behavior remained unaffected (\u003cb\u003eFig.\u0026nbsp;4H\u003c/b\u003e). Importantly, nose-to-nose orientation toward the female conspecific was not altered in hM3Dq mice (\u003cb\u003eFig.\u0026nbsp;4I\u003c/b\u003e), indicating that social attention was preserved despite the delayed onset of affiliative behaviors.\u003c/p\u003e\u003cp\u003eTogether, these results indicate that activation of MeA\u003csup\u003eSST⁺\u003c/sup\u003e neurons enhances territorial and mating-related behaviors in response to female conspecifics, while preserving general social interest and attention (\u003cb\u003eFig.\u0026nbsp;4J\u003c/b\u003e).\u003c/p\u003e\u003cp\u003e\u003cb\u003eManipulation of MeA\u003c/b\u003e\u003csup\u003e\u003cb\u003eSST+\u003c/b\u003e\u003c/sup\u003e \u003cb\u003eneurons alters behavioral responses to inanimate objects\u003c/b\u003e\u003c/p\u003e\u003cp\u003eTo examine how MeA\u003csup\u003eSST⁺\u003c/sup\u003e neurons modulate responses to non-social but potentially threatening stimuli, we exposed mice to three inanimate objects\u0026mdash;a mirror, a flipped mirror, and a glove\u0026mdash;within the resident\u0026rsquo;s home cage (\u003cb\u003eFig.\u0026nbsp;5A\u0026ndash;C\u003c/b\u003e). The mirror, previously shown to elicit aggression in both fish (\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e) and mice (\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e), served as a semi-social yet conspecific-free stimulus. The glove, in turn, was used to simulate an experimenter's hand intrusion, providing a physically intrusive but non-social threat (\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e). This design allowed us to assess a range of responses from exploratory interest to escape-driven defensive reactions.\u003c/p\u003e\u003cp\u003eAll groups spent more time facing the object than the opposite corner (\u003cem\u003ep\u0026thinsp;\u0026lt;\u0026thinsp;0.001\u003c/em\u003e), indicating a general attentional bias toward the stimuli (\u003cb\u003eFig.\u0026nbsp;5D, L, T\u003c/b\u003e). However, hM3Dq mice displayed a consistent avoidance pattern, entering the opposite corner more frequently than the object ROI across all conditions (mirror: \u003cem\u003ep\u0026thinsp;\u0026lt;\u0026thinsp;0.050\u003c/em\u003e; flipped mirror: \u003cem\u003ep\u0026thinsp;\u0026lt;\u0026thinsp;0.010\u003c/em\u003e; glove: \u003cem\u003ep\u0026thinsp;\u0026lt;\u0026thinsp;0.010\u003c/em\u003e; \u003cb\u003eFig.\u0026nbsp;5F, N, V\u003c/b\u003e). Despite this avoidance, time spent facing the object did not differ between groups, suggesting that attentional salience remained intact (\u003cb\u003eFig.\u0026nbsp;5E, M, U)\u003c/b\u003e.\u003c/p\u003e\u003cp\u003eDuring mirror exposure, hM3Dq animals exhibited reduced approach behavior, with fewer approach bouts (\u003cem\u003ep\u0026thinsp;=\u0026thinsp;0.035\u003c/em\u003e) and less time in the ROI (\u003cem\u003ep\u0026thinsp;=\u0026thinsp;0.043\u003c/em\u003e) (\u003cb\u003eFig.\u0026nbsp;5H-i, iii\u003c/b\u003e). Conversely, hM4Di mice exhibited reduced latency to run away (\u003cem\u003ep\u0026thinsp;=\u0026thinsp;0.049\u003c/em\u003e) (\u003cb\u003eFig.\u0026nbsp;5H-ii\u003c/b\u003e) and showed elevated digging behavior (\u003cem\u003ep\u0026thinsp;=\u0026thinsp;0.015\u003c/em\u003e) (\u003cb\u003efig. S3A\u003c/b\u003e), indicative of increased avoidance (\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e) and anxiety-like behavior (\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e, \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e). No significant group differences were observed in tail rattle (\u003cb\u003eFig.\u0026nbsp;5J\u003c/b\u003e).\u003c/p\u003e\u003cp\u003eIn the flipped mirror test, hM4Di mice again showed reduced latency to run away (\u003cem\u003ep\u0026thinsp;=\u0026thinsp;0.045\u003c/em\u003e) (\u003cb\u003eFig.\u0026nbsp;5Q-ii\u003c/b\u003e) and increased digging behavior (\u003cem\u003ep\u0026thinsp;=\u0026thinsp;0.032\u003c/em\u003e) (\u003cb\u003efig. S3E\u003c/b\u003e). By contrast, hM3Dq mice displayed reduced rearing (bout count: \u003cem\u003ep\u0026thinsp;=\u0026thinsp;0.017\u003c/em\u003e; duration: \u003cem\u003ep\u0026thinsp;=\u0026thinsp;0.001;\u003c/em\u003e \u003cb\u003efig. S3H\u003c/b\u003e), suggesting diminished exploratory drive (\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e)\u003c/p\u003e\u003cp\u003eDuring glove exposure, hM3Dq mice showed a pronounced \u0026ldquo;flight response\u0026rdquo;, with increased number (\u003cem\u003ep\u0026thinsp;=\u0026thinsp;0.020\u003c/em\u003e) and duration (\u003cem\u003ep\u0026thinsp;=\u0026thinsp;0.039\u003c/em\u003e) of run-away events (\u003cb\u003eFig.\u0026nbsp;5Y-i, iii\u003c/b\u003e). hM4Di animals showed increased latency to tail rattle (\u003cem\u003ep\u0026thinsp;=\u0026thinsp;0.048\u003c/em\u003e; \u003cb\u003eFig.\u0026nbsp;5Z-ii\u003c/b\u003e), suggesting reduced dominance. Additionally, hM3Dq mice exhibited reduced latency to initiate self-grooming (\u003cem\u003ep\u0026thinsp;=\u0026thinsp;0.044\u003c/em\u003e) (\u003cb\u003efig. S3-K\u003c/b\u003e), along with suppressed exploratory behavior (rearing bout count: \u003cem\u003ep\u0026thinsp;=\u0026thinsp;0.007\u003c/em\u003e; duration: \u003cem\u003ep\u0026thinsp;=\u0026thinsp;0.003\u003c/em\u003e; \u003cb\u003eFig.\u0026nbsp;35-L\u003c/b\u003e).\u003c/p\u003e\u003cp\u003eThese findings demonstrate that activation of MeA\u003csup\u003eSST⁺\u003c/sup\u003e neurons enhances escape-like responses to ambiguous or potentially threatening inanimate stimuli, whereas inhibition promotes reactive behaviors such as digging and avoidance.\u003c/p\u003e\u003cp\u003e\u003cb\u003eSummary\u003c/b\u003e\u003c/p\u003e\u003cp\u003eAltogether, our results demonstrate that MeA\u003csup\u003eSST⁺\u003c/sup\u003e neurons influence a diverse range of behaviors in adult male mice, including social dominance, mating, coping strategies, and responses to non-social stimuli. The effects were context-dependent and bidirectional, depending on whether MeA\u003csup\u003eSST⁺\u003c/sup\u003e neurons were activated or inhibited.\u003c/p\u003e\u003c/div\u003e\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eOur findings provide direct functional evidence that MeA\u003csup\u003eSST⁺\u003c/sup\u003e neurons contribute to the flexible regulation of social and defensive behaviors in adult male mice. By using bidirectional chemogenetic manipulations, we demonstrated that these neurons are capable of modulating a wide range of social context-dependent behaviors\u0026mdash;spanning from sociability and aggression to mating and coping strategies\u0026mdash;while also influencing reactions to non-social stimuli. These effects highlight the role of MeA\u003csup\u003eSST⁺\u003c/sup\u003e neurons not simply as modulators of general social engagement, but as key integrators of social behaviors that are sensitive to both stimulus type and social context.\u003c/p\u003e\u003cp\u003eChronic activation of MeA\u003csup\u003eSST⁺\u003c/sup\u003e neurons via hM3Dq disrupted social preference and novelty responses in the 3CH test, resulting in reduced sociability and a diminished preference for unfamiliar conspecifics. These results are consistent with our previous observation of a negative correlation between the activity of these neurons and social interaction levels (\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e). Importantly, this effect was specific to social novelty and was not attributable to changes in general exploratory behavior, as no significant differences in locomotion were observed in the OF. On the contrary, inhibition of MeA\u003csup\u003eSST⁺\u003c/sup\u003e neurons through hM4Di enhanced preference for social novelty, suggesting that their activity gates approach behavior toward novel social partners. These data support the view that MeA\u003csup\u003eSST⁺\u003c/sup\u003e neurons help mediate the balance between exploration and social familiarity, possibly through their impact on attentional or motivational components of social interaction. These findings are also aligned with prior work showing that activation of GABAergic neurons in the MeA can shift social exploration toward aggression or sexual behavior depending on context (\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e), and that MeA circuits projecting to structures such as the lateral septum or BNST regulate social recognition and memory, often modulated by oxytocin (\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e, \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eIn the affective domain, inhibition of MeA\u003csup\u003eSST⁺\u003c/sup\u003e neurons promoted a shift toward passive coping strategies in the FST, a widely used assay of behavioral despair and stress-reactivity in rodents (\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e). hM4Di-expressing mice exhibited increased immobility and decreased latency to the first immobility episode\u0026mdash;behavioral markers that have been linked to depression-related states and reduced stress resilience in humans (\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e). This profile is consistent with a large body of literature implicating GABAergic neurons in the regulation of affective homeostasis. Human studies using magnetic resonance spectroscopy and transcranial magnetic stimulation have reported decreased GABA neurotransmission and altered excitation/inhibition balance in patients with major depressive disorder (\u003cspan additionalcitationids=\"CR45 CR46 CR47\" citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e). Notably, SST-expressing neurons comprise a major subclass of GABAergic cells (\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e) and have been found to show reduced expression in the amygdala of major depressive disorder patients (\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e). In rodents, extensive work has confirmed the amygdala\u0026rsquo;s central role in affective control (\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e). Our results extend these findings by showing that targeted inhibition of MeA\u003csup\u003eSST⁺\u003c/sup\u003e neurons is sufficient to elicit maladaptive stress-coping behavior, in the absence of changes in anxiety-related exploration in the OF.\u003c/p\u003e\u003cp\u003eInterestingly, the role of MeA\u003csup\u003eSST⁺\u003c/sup\u003e neurons in dominance and aggression appears highly context-specific. In the TT\u0026mdash;a constrained, non-territorial paradigm\u0026mdash;activation of MeA\u003csup\u003eSST⁺\u003c/sup\u003e neurons increased dominance, as evidenced by a higher proportion of wins and increased dominance scores over successive trials. These effects were not accompanied by heightened aggression (e.g., number of pushes), suggesting that MeA\u003csup\u003eSST⁺\u003c/sup\u003e neurons may promote perseverance in competitive interactions without necessarily enhancing offensive behaviors. This aligns with early work in fish, where somatostatin and its receptor sstR3 were associated with dominant male phenotypes (\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eHowever, this dominance-enhancing effect of MeA\u003csup\u003eSST⁺\u003c/sup\u003e activation did not generalize to territorial contexts. In RI test, activation led to a decrease in classical markers of dominance, including tail rattling (\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e), as well as a reduction in social attention toward intruder males (nose-to-nose orientation). Instead, hM3Dq mice displayed a shorter latency to escape, suggesting an avoidance-based or submissive strategy. This context-specific suppression of aggression may reflect an adaptive strategy to reduce costly confrontations in socially demanding environments (\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e, \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e). Interestingly, although hM4Di mice did not show increased aggression, their shorter latency to tail rattle may suggest an enhanced readiness to assert dominance without escalating to explicit aggression.\u003c/p\u003e\u003cp\u003eThis suppression of aggression via MeA\u003csup\u003eSST+\u003c/sup\u003e neurons is especially notable given the MeA\u0026rsquo;s well-established role in driving aggressive behavior (\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e). One might expect that neurons in this region would promote aggression regardless of context. However, our findings suggest that the presence or absence of a social context, as well as the specific mode of neuromodulation, can override innate circuit outputs. These findings are consistent with anatomical descriptions of the MeA's projections to key aggression-related circuits, including the ventrolateral hypothalamus and olfactory bulb (\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e). Notably, our previous work also demonstrated that adult male mice subjected to early-life stress show reduced activation of MeA\u003csup\u003eSST⁺\u003c/sup\u003e neurons and increased inter-male aggression (\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e), highlighting a potential link between SST⁺ dysfunction and aggression across both acute and developmental timescales. Moreover, discrepancies with previous optogenetic findings may reflect differences in spatial precision and activation dynamics. While ChR2-mediated stimulation of GABAergic neurons in the posterodorsal MeA induces aggression (\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e), stimulation with faster opsin variants like ChETA led to opposing outcomes, including reduced aggression (\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e). These differences suggest that strong depolarization and high-frequency firing favor aggression, whereas milder, sustained activation\u0026mdash;such as that induced by DREADDs\u0026mdash;may engage inhibitory circuits or dampen excitatory drive. Our chronic, whole-MeA chemogenetic approach likely resembles the subtler activation dynamics of ChETA more than ChR2, offering a mechanistic explanation for the context- and method-dependent suppression of aggression observed here.\u003c/p\u003e\u003cp\u003eWhen male mice encountered a female intruder, activation of MeA\u003csup\u003eSST⁺\u003c/sup\u003e neurons increased sexual and dominance-related behaviors (e.g., mounting, tail rattling), but also delayed the onset of affiliative social behaviors such as allogrooming (\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e, \u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e). These data indicate that MeA\u003csup\u003eSST⁺\u003c/sup\u003e activity enhances mating-related responses while suppressing non-reproductive affiliative behaviors, possibly reflecting a shift in behavioral prioritization toward reproductive success. This interpretation is supported by findings that GABAergic MeA populations encode pheromone and sex-specific cues and influence male mating drive (\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e, \u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e, \u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e) and by recent optogenetic work showing that activation of vGAT⁺ neurons within the MeA suppresses self-grooming and other affiliative touch behaviors (\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e). Indeed, Wu et al. (\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e) demonstrated that optogenetic activation of Tac1⁺ vGAT⁺ neurons in the MeA robustly induces allogrooming toward stressed conspecifics, identifying a dedicated GABAergic subpopulation that controls affiliative social touch via projections to the MPOA. Furthermore, the delayed initiation of allogrooming in hM3Dq mice suggests a general suppression of prosocial affiliative behavior even in the context of enhanced sexual and dominance signals. These changes may reflect shifts in behavioral prioritization, whereby reproductive and dominance strategies are upregulated while prosocial affiliative circuits are transiently suppressed.\u003c/p\u003e\u003cp\u003eWe also found that MeA\u003csup\u003eSST⁺\u003c/sup\u003e neuron manipulation influenced responses to inanimate stimuli introduced into the home cage, such as a mirror, flipped mirror, or glove. These objects typically elicit exploratory or investigatory behavior (\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e), but in our study, both activation and inhibition of MeA\u003csup\u003eSST⁺\u003c/sup\u003e neurons led to enhanced avoidance. hM3Dq-expressing mice showed increased entries into the cage corner opposite the stimulus and reduced time facing the object, suggesting a suppression of exploratory drive or a \u0026ldquo;flight-like\u0026rdquo; response. Similarly, hM4Di-expressing mice exhibited shorter latencies to escape and increased digging behavior\u0026mdash;commonly associated with heightened arousal or anxiety. Notably, inhibition also delayed the onset of tail rattling, a behavior indicative of dominance, pointing to broader shifts in behavioral prioritization (\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e, \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e). These results suggest that both hyperactivation and silencing of MeA\u003csup\u003eSST⁺\u003c/sup\u003e neurons promote defensive reactivity to non-social, uncertain cues, but potentially through divergent mechanisms\u0026mdash;behavioral suppression (hM3Dq) versus hyperarousal (hM4Di). This bidirectional sensitivity aligns with prior studies implicating SST⁺ neurons in affective regulation (\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e) and extends their role beyond social contexts to include flexible modulation of threat-related behaviors.\u003c/p\u003e\u003cp\u003eTaken together, these findings point to a functional role of MeA\u003csup\u003eSST⁺\u003c/sup\u003e neurons in fine-tuning behavioral output according to context. These neurons appear to increase dominance and sexual behavior in competitive or mating contexts while simultaneously suppressing territorial aggression and affiliative social interaction under certain conditions. The same manipulations that enhanced dominance in the tube test suppressed aggression in the home cage, suggesting that MeA\u003csup\u003eSST⁺\u003c/sup\u003e neurons prioritize behavioral outputs depending on environmental and social cues. This flexibility may be crucial for balancing reproductive opportunities with social risks.\u003c/p\u003e\u003cp\u003eIn summary, our study identifies MeA\u003csup\u003eSST⁺\u003c/sup\u003e neurons as key regulators of social behavior flexibility in adult males. By demonstrating that both activation and inhibition of these cells shape context-dependent behaviors\u0026mdash;from social exploration and dominance to stress coping and threat reactivity\u0026mdash;our findings provide new insight into the neural circuitry that supports adaptive responses across social and non-social domains. These results highlight MeA\u003csup\u003eSST⁺\u003c/sup\u003e neurons as a potential node of vulnerability in disorders marked by impaired social functioning, such as autism, depression, or schizophrenia.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cp\u003e\u003cb\u003eAnimals\u003c/b\u003e\u003c/p\u003e\u003cp\u003eSST-IRES-Cre knock-in male mice (Sst\u003csup\u003etm2.1(cre)Zjh\u003c/sup\u003e/J, strain #: 013044, The Jackson Laboratory) were used in all experiments (\u003cb\u003eFig.\u0026nbsp;1A\u003c/b\u003e). In this strain, the CRE recombinase is driven by the endogenous Sst promoter. Experiments were conducted on adult mice (P60 at surgery, P99 at testing). After weaning (P21), animals were group-housed (2\u0026ndash;3 per cage) in standard polycarbonate cages (26.8 x 21.5 x 14.1 cm) under a 12:12 h light/dark cycle (lights on 8:00 am to 8:00 pm), at temperature (21\u0026thinsp;\u0026plusmn;\u0026thinsp;1\u0026deg;C) and humidity-controlled environment (50\u0026thinsp;\u0026plusmn;\u0026thinsp;5%). Food and water were provided \u003cem\u003ead libitum\u003c/em\u003e. Mice were socially housed after surgery and singly housed in standard cages one-week prior to testing, except for those assigned to the resident intruder (RI) test, who were housed individually in larger cages (50 x 38 x 18 cm). All procedures complied with EU Directive 2010/63/EU and were approved by the University Jaume I Bioethics Committee (2019/VSC/PEA/0188).\u003c/p\u003e\u003cp\u003e\u003cb\u003eExperimental design\u003c/b\u003e\u003c/p\u003e\u003cp\u003eAdult males were subjected to AAV-DREADD vector surgeries: AAV2-hSyn-DIO-mCherry (control), AAV2-hSyn-DIO-hM3D(Gq)-mCherry (activator), and AAV2-hSyn-DIO-hM4D(Gi)-mCherry (inhibitor). After 3 weeks of recovery, clozapine-N-oxide (CNO) was administered intraperitoneally (1 mg/kg) 30 min before each session for 21 days. Brains were collected 90 min after the last injection for cFos analysis\u003c/p\u003e\u003cp\u003eBehavioral tests were conducted in 2 cohorts of animals (experiment 1, experiment 2). Mice from experiment 1 were exposed to open field (OF, P99), forced swim test (FST, P100), three chamber test (3CH, P101) and tube dominance test (TT, day P102) to evaluate anxiety-like behavior, stress-coping strategies, social interest, and aggressive-dominance behavior, respectively. In experiment 2, mice underwent RI test variants: object-directed (mirror: RI\u003csub\u003emi\u003c/sub\u003e, P99; flipped mirror: RI\u003csub\u003eflp.mi\u003c/sub\u003e, P102; glove: RI\u003csub\u003egl\u003c/sub\u003e, P102) or social (female: RI\u003csub\u003efem,\u003c/sub\u003e P101, male: RI\u003csub\u003emal,\u003c/sub\u003e P100).\u003c/p\u003e\u003cp\u003e\u003cb\u003eViral constructs and stereotaxic surgeries\u003c/b\u003e\u003c/p\u003e\u003cp\u003eMale mice underwent stereotaxic surgery at P60. Anesthesia was induced with isoflurane (2.5% for induction, 1.5% for maintenance; 1.2 L/min O₂, Isofluotek, KARIZOO, Spain) in an induction chamber. The head was shaved and positioned in a stereotaxic frame (David Kopf Instruments). Animals received preoperative analgesia (buprenorphine, 0.02 mg/kg, s.c.; Bupaq, Fatro) and atropine (0.05 mg/kg, s.c.; B. Braun), and ophthalmic gel was applied (Lubrithal, DECHRA) to prevent corneal drying.\u003c/p\u003e\u003cp\u003eThe skin was incised to expose and clean the skull, and the head was aligned using bregma, lambda, and medial-lateral references. Bilateral craniotomies were performed (0.5 mm drill bit) at the following coordinates targeting the medial amygdala (MeA): AP \u0026minus;\u0026thinsp;1.4 mm, ML\u0026thinsp;\u0026plusmn;\u0026thinsp;2.6 mm, DV \u0026minus;\u0026thinsp;5.3 mm (\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e). The skull was rinsed with 0.9% saline solution to reduce inflammation.\u003c/p\u003e\u003cp\u003eTo deliver AAV-encoded DREADDs, a 33-gauge Hamilton syringe (5 \u0026micro;L) was loaded with 900 nL of virus and manually infused at 100 nL/30 s (450 nL per hemisphere). After infusion, the needle remained in place for 10 min to minimize backflow and was withdrawn slowly (1 mm/min). Body temperature was monitored and maintained on a heating pad throughout the procedure. Following surgery, mice recovered in a clean heated cage and were returned to group housing. A 21-day recovery period was allowed before CNO treatment and behavioral testing.\u003c/p\u003e\u003cp\u003eAAV vectors were obtained from Addgene and supplied by Bryan Roth\u0026rsquo;s laboratory. The control virus was pAAV-hSyn-DIO-mCherry (AAV2; RRID: Addgene_50459; titer\u0026thinsp;\u0026ge;\u0026thinsp;4 \u0026times; 10\u0026sup1;\u0026sup2; vg/mL). The excitatory DREADD construct was pAAV-hSyn-DIO-hM3D(Gq)-mCherry (AAV2; RRID: Addgene_44361; titer\u0026thinsp;\u0026ge;\u0026thinsp;6 \u0026times; 10\u0026sup1;\u0026sup2; vg/mL), and the inhibitory construct was pAAV-hSyn-DIO-hM4D(Gi)-mCherry (AAV2; RRID: Addgene_44362; titer\u0026thinsp;\u0026ge;\u0026thinsp;5 \u0026times; 10\u0026sup1;\u0026sup2; vg/mL).\u003c/p\u003e\u003cp\u003e\u003cb\u003eDrugs\u003c/b\u003e\u003c/p\u003e\u003cp\u003eChemogenetic activation or inhibition of MeA\u003csup\u003eSST+\u003c/sup\u003e neurons in Sst-IRES-Cre mice was achieved via DREADD technology, requiring treatment with CNO. Activation of hM3D(Gq) receptors induces neuronal depolarization, whereas stimulation of hM4D(Gi) receptors causes hyperpolarization, effectively silencing neurons. These effects were confirmed by measuring cFos expression as a proxy for neuronal activity (see below). Mice injected with AAV2-hSyn-DIO-hM3D(Gq)-mCherry (hM3Dq), AAV2-hSyn-DIO-hM4D(Gi)-mCherry (hM4Di), or AAV2-hSyn-DIO-mCherry (control) received daily intraperitoneal CNO (1 mg/kg) injections for 21 days. Behavioral testing was conducted during the final four days of treatment, with CNO administered 30 min before testing and animals perfused 90 min after the final injection. CNO (HelloBio, HB6149-25mg, Bristol, UK) was prepared in sterile saline at 1 mg/mL, aliquoted, frozen, and freshly diluted prior to each injection. The final dose for chemogenetic experiments was 0.1 mg/ml, administered intraperitoneally.\u003c/p\u003e\u003cp\u003e\u003cb\u003eWeight monitoring\u003c/b\u003e\u003c/p\u003e\u003cp\u003eTo control for potential side effects of chronic CNO treatment or surgery, animal body weight was monitored at four time points (\u003cb\u003eFig.\u0026nbsp;1B\u003c/b\u003e): the day of surgery (P60), the start of CNO administration (P81), 10 days post-CNO onset (P91), and the day of perfusion (P103).\u003c/p\u003e\u003cp\u003e\u003cb\u003eBehavioral assays\u003c/b\u003e\u003c/p\u003e\u003cp\u003eAll behavioral tests were conducted between 09:00 and 14:00 under standardized conditions. Testing rooms were illuminated with white light (150 lx), and mice were acclimated to the room for 30 minutes prior to each session to allow for CNO onset.\u003c/p\u003e\u003cp\u003eBehavior was recorded using OBS Studio (v27.01) and analyzed with ANY-maze software (v4.98, Stoelting Europe) for the open field, forced swim, and three-chamber tests. The tube test (TT) was scored manually. RI interactions were evaluated using DeepLabCut (\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e, \u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e) for pose estimation and downstream behavioral classification.\u003c/p\u003e\u003cp\u003e\u003cspan type=\"ItalicUnderline\" class=\"ItalicUnderline\" name=\"Emphasis\"\u003eOpen field test (OFT)\u003c/span\u003e\u003c/p\u003e\u003cp\u003eTo evaluate locomotor and anxiety-like behavior, mice were placed in a 40 \u0026times; 40 \u0026times; 40 cm white-floored arena with black walls for 10 min. Total distance traveled was used to assess locomotion, while the ratio of time spent in the center (20 \u0026times; 20 cm) vs. periphery served as a measure of anxiety-like behavior as described before (\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003cspan type=\"ItalicUnderline\" class=\"ItalicUnderline\" name=\"Emphasis\"\u003eForced Swimming test (FST)\u003c/span\u003e\u003c/p\u003e\u003cp\u003eDepression-like behavior was assessed in a 15 cm diameter beaker filled with 12 cm of water at 24\u0026thinsp;\u0026plusmn;\u0026thinsp;1\u0026deg;C. Mice were tested for 5 minutes, and immobility\u0026mdash;defined as the absence of movement except to keep the head above water\u0026mdash;was manually scored. The procedure was performed as described before (\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003cspan type=\"ItalicUnderline\" class=\"ItalicUnderline\" name=\"Emphasis\"\u003eThree-chamber test (3CHT)\u003c/span\u003e\u003c/p\u003e\u003cp\u003eSociability and social novelty were measured in a clear acrylic apparatus (60 \u0026times; 40.2 \u0026times; 19.5 cm) divided into three chambers. Mice first explored the empty setup (habituation), then encountered a stranger mouse in one side chamber (sociability), followed by exposure to a familiar and a novel mouse (novelty phase). Time spent and number of entries near each stimulus were recorded. Discrimination indexes were computed as in (\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003cspan type=\"ItalicUnderline\" class=\"ItalicUnderline\" name=\"Emphasis\"\u003eDominance Tube Test (TT)\u003c/span\u003e\u003c/p\u003e\u003cp\u003eSocial dominance was assessed using the tube test (\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e, \u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e), where weight-matched mice from experimental (hM3Dq, hM4Di) and control (mCherry) groups were introduced from opposite ends of a transparent 30 cm PVC tube (3.2 cm inner diameter). Each trial ended when one mouse fully retreated from the tube. Mice underwent three counterbalanced trials with unfamiliar opponents. The number of wins, ties, and losses was used to compute a dominance score and winning ratio, while the number of forward pushes was manually counted as an indicator of aggressive-like behavior, as described previously (\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003cspan type=\"ItalicUnderline\" class=\"ItalicUnderline\" name=\"Emphasis\"\u003eResident Intruder Test and Variants (RI\u003c/span\u003e\u003cem\u003e)\u003c/em\u003e\u003c/p\u003e\u003cp\u003eAll animals from experiment 2 were tested across multiple RI formats: against inanimate stimuli (mirror, flipped mirror, glove), a female conspecific, and a male intruder (\u003cb\u003eFig.\u0026nbsp;1A\u003c/b\u003e). Female intruders were in estrus, as determined by vaginal smear analysis to ensure hormonal receptivity. Tests involving inanimate objects were performed in the home cage and lasted 10 minutes. The mirror (15 \u0026times; 10 cm) was presented with its reflective side facing the animal, serving as a semi-social stimulus, as prior studies have shown that rodents display aggression toward their own reflections in the absence of a real conspecific (\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e). The flipped mirror served as a non-reflective control, while the nitrile glove was partially inflated to resemble a human hand.\u003c/p\u003e\u003cp\u003eEncounters with social targets involved either a group-housed female (sociability/sexual behavior) or a group-housed male intruder (sociability/aggression), both lasting 15 minutes. All intruder animals belonged to the same Sst-IRES-Cre strain. The order of testing was fixed over 4 consecutive days as follows (\u003cb\u003eFig.\u0026nbsp;1A\u003c/b\u003e): baseline (RI\u003csub\u003eba\u003c/sub\u003e), mirror (RI\u003csub\u003emi\u003c/sub\u003e), male (RI\u003csub\u003emal\u003c/sub\u003e), female (RI\u003csub\u003efem\u003c/sub\u003e), flipped mirror (RI\u003csub\u003efl.mir\u003c/sub\u003e), and glove (RI\u003csub\u003egl\u003c/sub\u003e). CNO was administered intraperitoneally 30 min prior to each testing session.\u003c/p\u003e\u003cp\u003eBehavioral parameters were quantified using machine-learning-based classifiers (see below). Behavioral events were grouped by functional category following prior literature (\u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e61\u003c/span\u003e): offensive behaviors (e.g., attack, vigorous allogrooming, lateral threat), defensive behaviors (e.g., defensive attack, upright submissive, escape), dominance and sexual behaviors (tail rattle, mounting), social interest (approach, normal allogrooming), and other behaviors (self-grooming, rearing). Time spent in nose-to-nose orientation was also measured as an indicator of social attention.\u003c/p\u003e\u003cp\u003e\u003cspan type=\"ItalicUnderline\" class=\"ItalicUnderline\" name=\"Emphasis\"\u003eMachine-learning based analysis of social behavior using DeepLabCut and SimBA\u003c/span\u003e\u003c/p\u003e\u003cp\u003eBehavioral data from the RI test and variants were acquired using a combination of markerless pose estimation and supervised behavior classification. DeepLabCut (v. 2.3.8; (\u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e, \u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e62\u003c/span\u003e). was used to track body parts across video recordings. We created and trained one network for each behavior paradigm\u0026mdash;resident intruder towards a conspecific and resident intruder towards an inanimate object\u0026mdash;to account for visual and postural differences between conditions. Each network was trained on 300 manually labeled frames from 15 videos, identifying eight key body points: nose, center of body, left/right ears, left/right flanks, tail base, and tail end. The RI-social network used the pre-trained dlcrnet_ms5 model (75,000 iterations), while the RI-inanimate network used ResNet_50 (95,000 iterations). Final train/test errors were 3.15/6.55 pixels and 2.72/4.14 pixels, respectively. Tractlet refinement and visual inspection confirmed high tracking accuracy.\u003c/p\u003e\u003cp\u003eBehavior classification was performed using Simple Behavioral Analysis (SimBA, v1.85.1). For RI-social videos, 12 validated classifiers were reused from (\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e). For RI-inanimate stimuli, seven new classifiers were created based on manual annotations of 18,000 frames from 10 videos, covering behaviors such as approach, tail rattle, rearing, self-grooming, biting, runaway, jumping, and digging. In total, 23,184 labeled frames were used for training, with 254 features extracted per frame using linear body part interpolation and 300 ms Gaussian smoothing. Classifier performance was evaluated via precision, recall, and F1 scores (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Thresholds were optimized via interactive probability plots to maximize detection accuracy and minimize false positives, and minimal bout durations were defined for each classifier (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003e\u003cb\u003eClassifiers.\u003c/b\u003e Data of the generated classifiers for the Resident Intruder toward inanimate object including labelled frames for training, optimum threshold and minimum bout length used for analysis.\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"6\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eClassifier\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eEvent\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eFrames\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003ePrecision\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u003cp\u003eRecall\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" 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colname=\"c6\"\u003e\u003cp\u003e0.978\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eAbsent\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e3395\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e0.996\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e0.995\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e0.996\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003eJumping\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003ePresent\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e1228\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e0.999\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e0.996\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e0.998\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eAbsent\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e2813\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e0.998\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e0.999\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003eRearing\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003ePresent\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e401\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e0.986\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e0.893\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e0.937\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eAbsent\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e3640\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e0.988\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e0.999\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e0.99\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003eRun away\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003ePresent\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e191\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e0.994\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e0.88\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e0.933\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eAbsent\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e3850\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e0.994\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e0.997\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003eSelf-grooming\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003ePresent\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e1129\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e0.996\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e0.996\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e0.996\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eAbsent\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e3502\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e0.999\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e0.999\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e0.999\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003eTail rattling\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003ePresent\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e131\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e0.983\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e0.893\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e0.936\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eAbsent\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e4500\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e0.997\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e0.998\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003e\u003cb\u003ePrediction curves.\u003c/b\u003e Data of generated prediction curves including present and absent frames, precision, recall, and F1 values for each classifier.\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"5\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eClassifier\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eLabeled Frames\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eLabeled Videos\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003eOptimum Threshold\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u003cp\u003eMinimum bout length\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eApproach\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e1887\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e10\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e0.6\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e350\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eBiting\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e2272\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e10\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e0.52\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e400\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eDigging\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e3366\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e10\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e0.7\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e600\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eJumping\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e6042\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e10\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e0.6\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e400\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eRearing\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e2188\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e10\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e0.55\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e500\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eRun Away\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e1051\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e10\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e0.35\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e500\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eSelf-Grooming\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e5712\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e10\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e0.75\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e600\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eTail Rattling\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e666\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e10\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e0.8\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e150\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003eOutput measures included event count, event duration, and latency to first occurrence. For RI-inanimate videos, additional spatial metrics were computed, including entries into the region of interest (ROI), time spent facing the stimulus, and time in the opposite cage zone.\u003c/p\u003e\u003cp\u003e\u003cb\u003eTissue Processing and Confocal Microscopy Analysis\u003c/b\u003e\u003c/p\u003e\u003cp\u003eFor histological analysis, animals were anesthetized with an overdose of sodium pentobarbital (1 mg/kg, i.p.; Dolethal, Vetoquinol, Madrid, Spain) and transcardially perfused with 0.9% NaCl for 5 min, followed by 15 min of 4% paraformaldehyde (PFA in 0.1 M PB, pH 7.4; Sigma-Aldrich). Brains were extracted, post-fixed in PFA for 2 h, and cryoprotected for 48 h in 30% sucrose (in 0.01 M PBS). Coronal sections (50 \u0026micro;m thick) were cut using a freezing sliding microtome (LEICA SM2000R), collected in six sequential subseries, and stored at \u0026minus;\u0026thinsp;20\u0026deg;C in cryoprotective solution (30% ethylene glycol, 30% glycerol, 40% PB 0.1 M; Sigma-Aldrich).\u003c/p\u003e\u003cp\u003ePrior to the immunofluorescence assay, injection sites were verified under a fluorescence microscope. Free-floating sections were then processed using the following protocol: (i) washing (3 \u0026times; 10 min PBS), (ii) blocking (1 h in 10% normal goat serum in PBST), (iii) incubation with primary antibody (guinea pig anti-cFos, 1:500; Synaptic Systems #226008) for 72 h at 4\u0026deg;C, (iv) washing (3 \u0026times; 10 min PBS), (v) incubation with secondary antibody (Alexa 647 goat anti-guinea pig IgG, 1:200; Biotium #20041) for 2 h at room temperature, (vi) washing (2 \u0026times; 10 min PBS\u0026thinsp;+\u0026thinsp;1 \u0026times; 10 min PB), (vii) counterstaing with DAPI (0.1 \u0026micro;g/mL; ThermoFisher, cat. #D1306), and (viii) mounting using Fluoromount-G (Invitrogen, ThermoFisher). Sections were imaged with a Leica SP8 laser scanning confocal microscope. Confocal images (2048 \u0026times; 2048 pixels) were acquired at 10\u0026times; magnification (NA 0.8 air objective) to cover the full MeA region. The expression of cFos, was evaluated as a marker of neuronal activation level (\u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e63\u003c/span\u003e) within the AAV infected MeA\u003csup\u003eSST\u003c/sup\u003e neurons. Quantification was performed using QuPath v0.1.3 (\u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e64\u003c/span\u003e). The MeA was manually delineated based on DAPI staining, following (\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e). Nuclei were automatically segmented, and a 5 \u0026micro;m cytoplasmic expansion was applied. After background subtraction, mCherry and cFos signals were detected within cell masks. Cells were classified as mCherry\u0026thinsp;+\u0026thinsp;and/or cFos\u0026thinsp;+\u0026thinsp;based on signal thresholds. Data were expressed as the percentage of cFos\u0026thinsp;+\u0026thinsp;cells among mCherry\u0026thinsp;+\u0026thinsp;neurons. Viral infection was further verified by quantifying mCherry fluorescence with ImageJ (FIJI). At least four sections per animal were analyzed and averaged to yield one value per hemisphere per mouse.\u003c/p\u003e\u003cp\u003e\u003cb\u003eStatistics\u003c/b\u003e\u003c/p\u003e\u003cp\u003eStatistical analyses were performed using Prism v8.0 (GraphPad Software). Data were analyzed only from animals that exhibited the behaviors of interest. Normality was assessed using the Shapiro\u0026ndash;Wilk test.\u003c/p\u003e\u003cp\u003eFor comparisons among the three experimental groups (mCherry, hM3Dq, hM4Di), one-way ANOVA was used for normally distributed data. For pairwise comparisons in the tube test (TT), unpaired two-tailed t-tests were applied. Two-way ANOVA with factors Group \u0026times; ROI was used to analyze three-chamber (3CH) test data across sociability and social novelty phases, followed by post hoc tests where appropriate.\u003c/p\u003e\u003cp\u003eAll data are presented as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SEM in bar graphs with individual data points represented as dots. The significance threshold (α) was set at 0.05. Statistical significance is indicated in figures as follows: \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05 (*\u003cem\u003e), p\u0026thinsp;\u0026lt;\u0026thinsp;0.01 (**), and p\u0026thinsp;\u0026lt;\u0026thinsp;0.001 (***\u003c/em\u003e).\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors thank \u0026ldquo;Servei Central d\u0026rsquo;Instrumentaci\u0026oacute; Cient\u0026iacute;fica (SCIC)\u0026rdquo; and \u0026ldquo;Servei d\u0026rsquo;Experimentaci\u0026oacute; Animal (SEA)\u0026rdquo; at Universitat Jaume I for their technical support. Authors belong to \u0026ldquo;Red Espa\u0026ntilde;ola de Investigaci\u0026oacute;n en Estr\u0026eacute;s (REIS)\u0026rdquo; financed by MCIN/AEI /10.13039/501100011033 and FEDER (RED2022-134191-T).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSpanish Ministry of Science, Innovation, and Universities: grant PID2023-153074OB-I00 (ECG, FOB).\u003c/p\u003e\n\u003cp\u003eGeneralitat Valenciana: grant CIAICO/2023/244 (ECG)\u003c/p\u003e\n\u003cp\u003eUniversitat Jaume I: grant GACUJIMB/2024/30 (ECG, FOB)\u003c/p\u003e\n\u003cp\u003eEuropean Commission grant MSCA-SE101086247 (FOB)\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eConceptualization: ECG, AOM\u003c/p\u003e\n\u003cp\u003eMethodology: AOM, JHC\u003c/p\u003e\n\u003cp\u003eInvestigation: AOM, MAZ\u003c/p\u003e\n\u003cp\u003eVisualization: AOM, MAZ\u003c/p\u003e\n\u003cp\u003eSupervision: ECG, FOB\u003c/p\u003e\n\u003cp\u003eWriting\u0026mdash;original draft: AOM, ECG, FOB\u003c/p\u003e\n\u003cp\u003eWriting\u0026mdash;review \u0026amp; editing: AOM, MAZ, JHC, ECG, FOB\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests:\u003c/strong\u003e Authors declare that they have no competing interests\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData and materials availability:\u003c/strong\u003e Raw data that support the findings of this study will be deposited at https://repositori.uji.es/ and will be made available from the corresponding author upon reasonable request.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eRubenstein DI, Rubenstein DR (2013) Social Behavior. Encyclopedia of Biodiversity. Elsevier, pp 571\u0026ndash;579\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eCoria-Avila GA, Manzo J, Garcia LI, Carrillo P, Miquel M, Pfaus JG (2014) Neurobiology of social attachments. Neurosci Biobehav Rev 43:173\u0026ndash;182\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eWei D, Talwar V, Lin D (2021) Neural circuits of social behaviors: Innate yet flexible. Neuron 109:1600\u0026ndash;1620\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eChen P, Hong W (2018) Neural Circuit Mech Social Behav Neuron 98:16\u0026ndash;30\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eKarigo T, Deutsch D (2022) Flexibility of neural circuits regulating mating behaviors in mice and flies. Front Neural Circuits 16\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eMazza V, Šlipogor V (2024) Behavioral flexibility and novel environments: integrating current perspectives for future directions. Curr Zool 70:304\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eRaam T, Hong W (2021) Organization of neural circuits underlying social behavior: A consideration of the medial amygdala. Curr Opin Neurobiol 68:124\u0026ndash;136\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003ePrakash N, Abu Irqeba A, Corbin JG (2025) Development and function of the medial amygdala. Trends Neurosci 48:22\u0026ndash;32\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eBergan JF, Ben-Shaul Y, Dulac C (2014) Sex-specific processing of social cues in the medial amygdala. Elife 2014\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eDulac C, Torello AT (2003) Molecular detection of pheromone signals in mammals: from genes to behaviour. \u003cem\u003eNature Reviews Neuroscience 2003 4:7\u003c/em\u003e 4, 551\u0026ndash;562\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eDwyer J, Kelly DA, Bergan J (2022) Brain-Wide Synaptic Inputs to Aromatase-Expressing Neurons in the Medial Amygdala Suggest Complex Circuitry for Modulating Social Behavior. \u003cem\u003eeNeuro\u003c/em\u003e 9\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eCanteras NS, Simerly RB, Swanson LW (1995) Organization of projections from the medial nucleus of the amygdala: A PHAL study in the rat. J Comp Neurol 360:213\u0026ndash;245\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eLischinsky JE, Lin D (2020) Neural mechanisms of aggression across species. Nat Neurosci 23:1317\u0026ndash;1328\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eHong W, Kim DW, Anderson DJ (2014) Antagonistic control of social versus repetitive self-grooming behaviors by separable amygdala neuronal subsets. Cell 158:1348\u0026ndash;1361\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eChen PB, Hu RK, Wu YE, Pan L, Huang S, Micevych PE, Hong W (2019) Sexually Dimorphic Control of Parenting Behavior by the Medial Amygdala. Cell 176:1206\u0026ndash;1221e18\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eNewman SW (1999) The medial extended amygdala in male reproductive behavior. A node in the mammalian social behavior network. Ann N Y Acad Sci 877:242\u0026ndash;257\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eHines M, Allen LS, Gorski RA (1992) Sex differences in subregions of the medial nucleus of the amygdala and the bed nucleus of the stria terminalis of the rat. Brain Res 579:321\u0026ndash;326\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eHuijgens PT, Heijkoop R, Snoeren EMS (2021) Silencing and stimulating the medial amygdala impairs ejaculation but not sexual incentive motivation in male rats. Behav Brain Res 405:113206\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eLischinsky JE, Yin L, Shi C, Prakash N, Burke J, Shekaran G, Grba M, Corbin JG, Lin D (2023) Transcriptionally defined amygdala subpopulations play distinct roles in innate social behaviors. Nat Neurosci 26:2131\u0026ndash;2146\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eHuang J, Zhang YY, Qiu YY, Yao S, Qiu WT, Peng JL, Li YQ, You QL, Wu CH, Wu EJ, Wang J, Zhou YL, Ning YP, Wang HS, Chen WB, Hu BJ, Liu Y (2024) Sun, NRG1-ErbB4 signaling in the medial amygdala controls mating motivation in adult male mice. Cell Rep 43:113905\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eKeshavarzi S, Sullivan RKP, Ianno DJ, Sah P (2014) Functional properties and projections of neurons in the medial amygdala. J Neurosci 34:8699\u0026ndash;8715\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eLin LC, Sibille E (2015) Somatostatin, neuronal vulnerability and behavioral emotionality. \u003cem\u003eMolecular Psychiatry 2015 20:3\u003c/em\u003e 20, 377\u0026ndash;387\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eSato M, Nakai N, Fujima S, Choe KY, Takumi T (2023) Social circuits and their dysfunction in autism spectrum disorder. \u003cem\u003eMolecular Psychiatry 2023 28:8\u003c/em\u003e 28, 3194\u0026ndash;3206\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eHoftman GD, Volk DW, Bazmi HH, Li S, Sampson AR, Lewis DA (2015) Altered cortical expression of GABA-related genes in schizophrenia: Illness progression vs developmental disturbance. Schizophr Bull 41:180\u0026ndash;191\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eGuilloux JP, Douillard-Guilloux G, Kota R, Wang X, Gardier AM, Martinowich K, Tseng GC, Lewis DA, Sibille E (2012) Molecular evidence for BDNF-and GABA-related dysfunctions in the amygdala of female subjects with major depression. Mol Psychiatry 17:1130\u0026ndash;1142\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eSong YH, Yoon J, Lee SH (2021) The role of neuropeptide somatostatin in the brain and its application in treating neurological disorders. Exp Mol Med 53:328\u0026ndash;338\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eNordman JC, Ma X, Gu Q, Potegal M, Li H, Kravitz AV, Li Z (2020) Potentiation of Divergent Medial Amygdala Pathways Drives Experience-Dependent Aggression Escalation. J Neurosci 40:4858\u0026ndash;4880\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eMa\u0026ntilde;as-Ojeda A, Hidalgo-Cort\u0026eacute;s J, Garc\u0026iacute;a-Momp\u0026oacute; C, Zahran MA, Gil-Miravet I, Olucha-Bordonau FE, Guirado R, Castillo-G\u0026oacute;mez E (2024) Activation of somatostatin neurons in the medial amygdala reverses long-term aggression and social deficits associated to early-life stress in male mice. Mol Psychiatry. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1038/s41380-024-02829-6\u003c/span\u003e\u003cspan address=\"10.1038/s41380-024-02829-6\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eFan Z, Zhu H, Zhou T, Wang S, Wu Y, Hu H (2019) Using the tube test to measure social hierarchy in mice. Nat Protoc 14:819\u0026ndash;831\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eMa\u0026ntilde;as-Ojeda A, Hidalgo-Cort\u0026eacute;s J, Garc\u0026iacute;a-Momp\u0026oacute; C, Zahran MA, Gil-Miravet I, Olucha-Bordonau FE, Guirado R, Castillo-G\u0026oacute;mez E (2024) Activation of somatostatin neurons in the medial amygdala reverses long-term aggression and social deficits associated to early-life stress in male mice. \u003cem\u003eMolecular Psychiatry 2024 30:5\u003c/em\u003e 30, 2168\u0026ndash;2182\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eFulenwider HD, Caruso MA, Ryabinin AE (2021) Manifestations of domination: Assessments of social dominance in rodents. Genes Brain Behav 21:e12731\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eClipperton-Allen AE, Page DT (2023) Social Behavior Testing in Mice: Social Interest, Recognition, and Aggression. Neuromethods 190:279\u0026ndash;307\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eMorozova MV, Boldyreva LV, Borisova MA, Kozhevnikova EN (2024) Investigating social communication in mice: A two-intruders test approach. J Neurosci Res 102:e25365\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eDesjardins JK, Fernald RD (2010) What do fish make of mirror images? Biol Lett 6:744\u0026ndash;747\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eYang T, Yang CF, Chizari MD, Maheswaranathan N, Burke KJ, Borius M, Inoue S, Chiang MC, Bender KJ, Ganguli S, Shah NM (2017) Social Control of Hypothalamus-Mediated Male Aggression. Neuron 95:955\u0026ndash;970e4\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003ePowell SB, Geyer MA, Gallagher D, Paulus MP (2004) The balance between approach and avoidance behaviors in a novel object exploration paradigm in mice. Behav Brain Res 152:341\u0026ndash;349\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eLatham N, Mason G (2004) From house mouse to mouse house: the behavioural biology of free-living Mus musculus and its implications in the laboratory. Appl Anim Behav Sci 86:261\u0026ndash;289\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eDudek BC, Adams N, Boice R, Abbott ME (1983) Genetic influences on digging behaviors in mice (Mus musculus) in laboratory and seminatural settings. J Comp Psychol 97:249\u0026ndash;259\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eSturman O, Germain PL, Bohacek J (2018) Exploratory rearing: a context- and stress-sensitive behavior recorded in the open-field test. Stress 21:443\u0026ndash;452\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eFerguson JN, Young LJ, Insel TR (2002) The Neuroendocrine Basis of Social Recognition. Front Neuroendocrinol 23:200\u0026ndash;224\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eChoi GB, Dong H, Murphy AJ, Valenzuela DM, Yancopoulos GD, Swanson LW, Anderson DJ (2005) Lhx6 Delineates a Pathway Mediating Innate Reproductive Behaviors from the Amygdala to the Hypothalamus. Neuron 46:647\u0026ndash;660\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eArmario A (2021) The forced swim test: Historical, conceptual and methodological considerations and its relationship with individual behavioral traits. Neurosci Biobehav Rev 128:74\u0026ndash;86\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eCommons KG, Cholanians AB, Babb JA, Ehlinger DG (2017) The Rodent Forced Swim Test Measures Stress-Coping Strategy, Not Depression-like Behavior. ACS Chem Neurosci 8:955\u0026ndash;960\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eSanacora G, Mason GF, Rothman DL, Behar KL, Hyder F, Petroff OAC, Berman RM, Charney DS, Krystal JH (1999) Reduced Cortical γ-Aminobutyric Acid Levels in Depressed Patients Determined by Proton Magnetic Resonance Spectroscopy. Arch Gen Psychiatry 56:1043\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eSanacora G, Mason GF, Rothman DL, Krystal JH (2002) Increased Occipital Cortex GABA Concentrations in Depressed Patients After Therapy With Selective Serotonin Reuptake Inhibitors. Am J Psychiatry 159:663\u0026ndash;665\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eHasler G, van der Veen JW, Tumonis T, Meyers N, Shen J, Drevets WC (2007) Reduced Prefrontal Glutamate/Glutamine and γ-Aminobutyric Acid Levels in Major Depression Determined Using Proton Magnetic Resonance Spectroscopy. Arch Gen Psychiatry 64:193\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eBajbouj M, Lisanby SH, Lang UE, Danker-Hopfe H, Heuser I (2006) Neu, Evidence for Impaired Cortical Inhibition in Patients with Unipolar Major Depression. Biol Psychiatry 59:395\u0026ndash;400\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eCastricum J, Birkenhager TK, Kushner SA, Elgersma Y, Tulen JHM (2022) Cortical Inhibition and Plasticity in Major Depressive Disorder. Front Psychiatry 13\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eLovelock DF, Deak T (2019) Acute stress imposed during adolescence yields heightened anxiety in Sprague Dawley rats that persists into adulthood: Sex differences and potential involvement of the Medial Amygdala. Brain Res 1723:146392\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eTrainor BC, Hofmann HA (2006) Somatostatin Regulates Aggressive Behavior in an African Cichlid Fish. Endocrinology 147:5119\u0026ndash;5125\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eYang T, Bayless DW, Wei Y, Landayan D, Marcelo IM, Wang Y, DeNardo LA, Luo L, Druckmann S, Shah NM (2023) Hypothalamic neurons that mirror aggression. Cell 186:1195\u0026ndash;1211e19\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eShemesh Y, Benjamin A, Shoshani-Haye K, Yizhar O, Chen A (2024) Studying dominance and aggression requires ethologically relevant paradigms. Curr Opin Neurobiol 86:102879\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eMiller SM, Marcotulli D, Shen A, Zweifel LS (2019) Divergent medial amygdala projections regulate approach\u0026ndash;avoidance conflict behavior. Nat Neurosci 22:565\u0026ndash;575\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eBaleisyte A, Schneggenburger R, Kochubey O (2022) Stimulation of medial amygdala GABA neurons with kinetically different channelrhodopsins yields opposite behavioral outcomes. Cell Rep 39:110850\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eRanade S (2021) Comforting in mice. Nat Neurosci 24:1640\u0026ndash;1640\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eWu YE, Dang J, Kingsbury L, Zhang M, Sun F, Hu RK, Hong W (2021) Neural control of affiliative touch in prosocial interaction. Nature 599:262\u0026ndash;267\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eBaum MJ, Everitt BJ (1992) Increased expression of c-fos in the medial preoptic area after mating in male rats: Role of afferent inputs from the medial amygdala and midbrain central tegmental field. Neuroscience 50:627\u0026ndash;646\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003ePaxinos G, Franklin KBJ (2012) Paxinos and Franklin\u0026rsquo;s the Mouse Brain in Stereotaxic Coordinates. Elsevier Science. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://books.google.es/books?id=8RJZLwEACAAJ)\u003c/span\u003e\u003cspan address=\"https://books.google.es/books?id=8RJZLwEACAAJ)\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eMathis A, Mamidanna P, Cury KM, Abe T, Murthy VN, Mathis MW, Bethge M (2018) DeepLabCut: markerless pose estimation of user-defined body parts with deep learning. Nat Neurosci 21:1281\u0026ndash;1289\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eDeng X, Xu W, Liu Y, Jing H, Zhong J, Sun K, Zhou R, Xu L, Wu X, Zhang B, Chen W, Jiang S, Chen G, Zhu Y (2025) Social rank modulates methamphetamine-seeking in dominant and subordinate male rodents via distinct dopaminergic pathways. \u003cem\u003eNature Neuroscience 2025 28:6\u003c/em\u003e 28, 1268\u0026ndash;1279\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eWalker SE, Wood TC, Cash D, Mesquita M, Williams SCR, Sandi C (2018) Alterations in brain microstructure in rats that develop abnormal aggression following peripubertal stress. Eur J Neurosci 48:1818\u0026ndash;1832\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eLauer J, Zhou M, Ye S, Menegas W, Schneider S, Nath T, Rahman MM, Di Santo V, Soberanes D, Feng G, Murthy VN, Lauder G, Dulac C, Mathis MW, Mathis A (2022) Multi-animal pose estimation, identification and tracking with DeepLabCut. Nat Methods 19:496\u0026ndash;504\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eMorgan JI, Curran T (1991) Stimulus-transcription coupling in the nervous system: involvement of the inducible proto-oncogenes fos and jun. Annu Rev Neurosci 14:421\u0026ndash;451\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eBankhead P, Loughrey MB, Fern\u0026aacute;ndez JA, Dombrowski Y, McArt DG, Dunne PD, McQuaid S, Gray RT, Murray LJ, Coleman HG, James JA, Salto-Tellez M (2017) Hamilton, QuPath: Open source software for digital pathology image analysis. Sci Rep 7:16878\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":true,"highlight":"","institution":"Jaume I University","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"sociability, aggression, mating, medial amygdala","lastPublishedDoi":"10.21203/rs.3.rs-7046653/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7046653/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eIntraspecific social interactions are essential for species survival and require behavioral flexibility to adapt to changing social environments. These behaviors are orchestrated by neural circuits such as those in the medial amygdala (MeA). Within this region, somatostatin-expressing neurons (MeA\u003csup\u003eSST+\u003c/sup\u003e) have been associated with maladaptive social outcomes, particularly following early-life stress. However, whether these neurons contribute to the flexibility of adult male social behavior— modulating responses according to social context and stimulus type—remains unclear. Here, using chemogenetic approaches combined with machine learning-based behavioral tracking, we showed that activation of MeA\u003csup\u003eSST+\u003c/sup\u003e neurons reduced sociability, social novelty preference, inter-male aggression, and attention toward males, while enhancing sexual motivation and dominance toward females. Inhibition increased social novelty preference and impaired stress-coping behavior without affecting other social traits. Notably, both manipulations heightened escape-like responses to inanimate stimuli, indicating increased defensive reactivity to non-social cues. These findings identify MeA\u003csup\u003eSST+\u003c/sup\u003e neurons as modulators of social context-specific behavior, advancing understanding of circuit-level mechanisms supporting adaptive social responses.\u003c/p\u003e","manuscriptTitle":"Somatostatin neurons in the medial amygdala orchestrate flexible, sex-specific social behavior in male mice","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-07-07 03:44:21","doi":"10.21203/rs.3.rs-7046653/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"53d9bb97-c207-460e-b0af-24ba7e51e7f9","owner":[],"postedDate":"July 7th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":51053185,"name":"Cellular \u0026 Molecular Neuroscience"},{"id":51053186,"name":"Cognitive Neuroscience"},{"id":51053187,"name":"Neurobiology of Disease"}],"tags":[],"updatedAt":"2025-07-07T03:44:21+00:00","versionOfRecord":[],"versionCreatedAt":"2025-07-07 03:44:21","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7046653","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7046653","identity":"rs-7046653","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}