Ventromedial hypothalamus (VMHvl) nNOS neurons regulate social behaviors in a sex-specific manner

Ventromedial hypothalamus (VMHvl) nNOS neurons regulate social behaviors in a sex-specific manner | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Ventromedial hypothalamus (VMHvl) nNOS neurons regulate social behaviors in a sex-specific manner Vinícius Oliveira, Ioana Bodea, Julie Bakker This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-5070073/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 01 Dec, 2025 Read the published version in Communications Biology → Version 1 posted You are reading this latest preprint version Abstract Neuronal nitric oxide synthase (nNOS) neurons are ubiquitously spread in the rodent brain. Data using knockouts and pharmacology have revealed that nNOS is essential for the display of sexual and aggressive behavior. Yet, the specific neuronal populations regulating those behaviors remain elusive. Here, we aimed to study the role of the ventromedial hypothalamus (VMHvl)-nNOS neurons in social behaviors in both sexes. First, we evaluated whether the expression of nNOS overlaps with the well-characterized estrogen receptor alpha (ERα+)-VMHvl population. Next, we assessed how different social stimuli affected VMHvl-nNOS neurons' activity. Lastly, we used transgenic mice and viral approaches to ablate VMHvl-nNOS neurons and evaluate their impact on behavior. Our findings suggest that nNOS neurons constitute a small cluster within the VMHvl-ERα+ population which regulates social behaviors in a sex-specific manner. In males, those neurons seem to be essential for aggression whereas in females for sexual behavior and social motivation. Biological sciences/Neuroscience/Social behaviour Biological sciences/Neuroscience/Sexual behaviour/Sexual dimorphism Biological sciences/Neuroscience/Emotion/Aggression Biological sciences/Neuroscience/Neural circuits Biological sciences/Neuroscience/Sexual behaviour/Oxytocin Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 1. Introduction Social behaviors such as mating, aggression, and prosocial approach are considered innate behaviors as they are displayed readily without the need for previous experience[1–3]. In general, those behaviors rely on stereotypical motor patterns and are embedded in developmentally hardwired neural circuits[2], which are known to exhibit strong sex differences[2–6] and are shaped by internal and external factors[1,4,6] such as reproductive states[2–5,7] and social experience[1,6,8–10], respectively. Particularly hypothalamic areas have been strongly associated with the display of instinctive behaviors such as mating[3,11,12] and fighting[4,13,14]. Of note estrogen receptor α (ERα) and progesterone receptor (PR)-positive neurons (expressed in a ratio of 1:1[15,16]) in the ventrolateral part of the ventromedial nucleus of the hypothalamus (VMHvl) are crucial for the display of aggressive and sexual behavior in both sexes[15–19]. Although the role of ERα-PR neurons (accounting for 40-50% of total neurons in the VMHvl) on sociosexual behaviors has been extensively studied, the participation of other neuronal populations within the VMHvl, which is a highly heterogeneous and sexually differentiated region [20], remains understudied. Of note, neuronal nitric oxide synthase (nNOS) neurons are found in the VMHvl[21] and are known to co-express PR in female mice [22] . Furthermore, compelling evidence has tied nNOS expression and possibly nitric oxide (NO) synthesis to a sex-specific regulation of social behaviors. Briefly, nNOS knockout (KO) males exhibit abnormal sexual behavior (excessive mounting) and exaggerated aggression compared to wild types (WT)[23,24] whereas females showed reduced lordosis [25] and maternal aggression [26] . Pharmacological evidence further demonstrated the role of nNOS activity and possibly NO signaling in social behaviors. In male mice, pharmacological inhibition of nNOS activity increased and decreased aggression depending on factors such as social experience [24,27] and synaptic plasticity [28] . In contrast to KO studies, inhibition of nNOS activity decreased sexual behavior in naive male rats without affecting sexually experienced rats [29] . On the other hand, in females, KO and pharmacological studies seem to be aligned as inhibition of VMHvl-nNOS neurons [22] or nNOS activity [30,31] in the VMHvl both reduced lordosis. Yet the role of nNOS neurons in virgin female aggression [4] as well as the participation of specific nNOS populations in various social behaviors such as social motivation, mate choice, mating, and fighting in a sex-specific context remains elusive. Here we first assessed whether nNOS expression in the VMHvl overlaps with the well-characterized ERα population in a sex-specific manner. Next, we exposed mice of both sexes to different social stimuli in order to trigger different behavioral responses and measured co-expression of nNOS and neuronal activity markers with different temporal dynamics, i.e. c-Fos (mating) and pERK (fighting), taking into account the effect of reproductive states and social experience. Finally, in the last set of experiments, we performed loss of function experiments using nNOS::cre mice and viral injections to specifically ablate nNOS neurons within the VMHvl and measure its consequences on behavior as well as used NO donors to rescue the behavioral deficits induced by deletion. 2. Material and Methods 2.1. Animals Experiments were carried out in adult (8–12 weeks) male and female mice, which were either purchased from Charles River Laboratories (Les Oncins, France) or bred in the animal facilities of the University of Liège. Viral ablation experiments were conducted in nNOS::Cre mice (Figs. 3 , 4 and Supplementary Table 3) whereas immunohistochemistry experiments were conducted in C57BL6/J mice (Figs. 1 , 2 , and Supplementary Fig. 1). For aggressive behavior testing, adult male (5–6 weeks) and juvenile female (p16-21) CD-1 mice were used as intruders for male and female residents, respectively. For sexual behavior receptive (estrous) females (6–8 weeks) and experienced breeders (8–12 weeks old) C57BL6/J were used as stimuli to test male and female subjects, respectively. All stimulus animals were obtained via Charles Rivers Laboratories (Les Oncins, France). Mice were kept under controlled laboratory conditions in ventilated shelves with (12:12h light/dark cycle; lights off at 10:30h, 21 ± 1°C, 60 ± 5% humidity, standard mouse nutrition (RM3), and water ad libitum). Stimulus and experimental animals were housed in large mouse cages (42x32x18cm) with sawdust bedding in groups of four. Importantly, after viral injection and prior resident or female intruder (RIT or FIT) tests, subjects were singly housed in individual mouse cages (33x16x13cm) for at least one week to induce territoriality. All procedures described here were approved by the local Animal Ethics Committee of the University of Liège (protocol numbers 22-2469 and 2195). 2.2. Female, Maternal defense, and Resident intruder tests (FIT, MDT, and RIT) All tests took place in the early dark phase under dim red light conditions. Male mice were confronted with an unfamiliar same-sex intruder for 10 minutes. Intruders were 10–20% lighter than residents [ 1 , 32 , 33 ]. Behavior was scored live and later via videos by an observer blind to the experimental condition using JWatcher event recorder Program [ 34 , 35 ]. As described elsewhere[ 35 ] the percentage of time spent on four major sets of behaviors was scored: i) aggressive behavior, consisting of attacks, threats, chases, tail rattles, offensive grooming, offensive up-right; ii) neutral behaviors, consisting of exploring (investigating the home-cage), drinking and eating, autogrooming, immobility; iii) social behaviors (non-aggressive social interactions, sniffing, following); and iv) defensive behavior (submissive posture, kicking a pursuing intruder with hind limb). In addition, we scored the frequency of attacks as well as the latency of the first attack. Females were typically confronted with a juvenile (p16-21) CD-1 female for 10 min, following a combination of the protocols described by Oliveira et. al., 2021[ 35 ] and Hashikawa et. al, 2017[ 16 ]. Typically females did not display attack bites[ 4 ] unless they were single-housed following Oliveira et. al, 2021 (Supplementary Fig. 1, Supplementary video). Therefore we scored all the non-aggressive behaviors mentioned above for males. However aggressive/dominant behavior consisted of the percentage of time spent on threatening, pushing/shoving, mounting, chasing, and, eventually biting the intruder. For lactating females aggressive behavior was scored similarly to males. Maternal aggression was measured using the maternal defense test as described elsewhere[ 36 ]. In brief, females were paired with experienced males for 1 week, and vaginal plugs were assessed to confirm pregnancy. Maternal aggression was tested by introducing a virgin female into the lactating female home cage between lactation day 4 (LD4) and LD7, for 10 min. 2.3. Sexual Behavior Sexual behavior was measured as previously described[ 25 , 37 ]. Briefly, Sexual behavior was videotaped and scored live. Tests also took place in the early hours of the dark phase under dim red light conditions. Typically, for male sexual behavior, male mice were paired with a receptive (estrus) naturally cycling C57BL6/J mouse in their home cage and left free to interact for 10 or 30 minutes, depending on the test day (Please see Figs. 2 and 3 ). Latencies for the first mount and first intromission as well as frequencies of mounts and intromissions were scored. The female estrous cycle was monitored via vaginal smears and histology. Additionally, receptivity was confirmed prior to the experiment by pairing the estrous females with experienced males. Female sexual behavior was assessed via the lordosis quotient (%). For neuronal ablation, experimental females were ovariectomized and implanted with a Silastic capsule containing crystalline 17-β-estradiol (diluted 1:1 with cholesterol) subcutaneously, followed by treatment with 500 µg progesterone 3 hours prior to the sexual behavior test. This protocol was chosen to enable optimal levels of sexual receptivity in order to evaluate neuronal ablation effects. For neuronal activity experiments, naturally cycling and receptive females were tested. Importantly during the sexual behavior test, females were paired with experienced males, and the amount of lordosis and rejections displayed in response to male mount attempts was annotated. The test was terminated after 10 mounts or 10 min whatever came first. 2.4. Mate and social preference Mate and social preferences were assessed using a 3-chamber apparatus[ 12 , 38 – 41 ]. Briefly, the test consisted of 3 phases (each of which lasted 10 min). Habituation consisting of free investigation of the 3-chamber apparatus. Whereas during social preference experimental animals had to choose between an object and an adult unknown same-sex-conspecific, preference was calculated as an index of time investigating the conspecific/(time spent sniffing the conspecific + object). Directly after the social preference test, animals were given the option to choose between a same- and opposite-sex conspecific (mate preference), and preference was calculated as an index of time spent investigating the opposite-sex target/(time spent sniffing the same + opposite sex). Preference was analyzed using a one-sample t-test, and animals were assumed to prefer once their index differed from chance levels (0.5). Mice that did not leave the center chamber or spent more than 5 minutes in the center chamber were excluded from the analysis. 2.5. Experiment overview Experiment A ( Fig. 2 ) Neuronal activity upon social encounter Mice of both sexes were confronted either with an object (control) or an opposite-sex-conspecific for 15 min (mating). One hour later, those animals were again exposed to an object (controls) or a same-sex-conspecific (fighting). Importantly, males were confronted with smaller adult male mice whereas females were confronted with juvenile females[ 16 ] for 10 min (Fig. 2 A). Directly after the last interactions, animals were transcardially perfused with 4% paraformaldehyde (PFA) and had their brains collected and processed for immunohistochemistry targeting neuronal activity markers with different expression/phosphorylation dynamics, e.g. c-FOS for mating and pERK for fighting. Of note, both markers are known to be triggered by social interactions[ 15 , 16 , 37 , 42 , 43 ] including sexual and aggressive behavior, similar approaches have been used by others[ 44 , 45 ]. Lactating[ 36 ] and socially isolated and aggression-trained (IST) females[ 4 , 34 , 35 , 46 ] were used as high female aggression controls. Briefly, IST was followed as described elsewhere[ 35 ]. Female C57BL6/J mice (8–12 weeks) were single-housed for 3 weeks and after social isolation confronted with a same-sex and juvenile intruder for 4 consecutive days in the FIT. Two cohorts were trained in this schedule the first one was used to confirm aggression/dominant behavior levels and detailed behavioral analysis as well as activation of the oxytocin system (Supplementary Fig. 1), as oxytocin has been shown to promote aggression in females[ 34 , 36 , 46 , 47 ]. The second cohort of IST females was confronted with a juvenile and perfused directly after behavioral testing to verify pERK phosphorylation of VMHvl-nNOS neurons upon fighting (Fig. 2 A). As lactating females are known to exhibit high levels of aggression[ 4 , 36 ], they were confronted between LD 04–07 with an adult virgin female and perfused directly after the maternal defense test to check for pERK phosphorylation after fighting (Fig. 2 A). Experiment B ( Fig. 3 ) VMHvl-nNOS ablation in male mice Male nNOS::Cre mice underwent stereotaxic surgery and were injected into the VMHvl bilaterally with either AAV1-flex-taCasp3-TEVp (ablation, 6.4 × 10 13 genomic copies per ml) or AAV1-flex-EF1a-EGFP-WPRE (control, 7.7 × 10 13 genomic copies per ml) virus. After 3 weeks, animals underwent behavioral testing to measure different aspects of social behavior. First, sexual behavior was assessed by exposing experimental males to receptive females (in their home cage) for 10 min on 3 consecutive days. Directly after mating animals were then confronted with a a male in their home cage for 10 minutes, sexual behavior testing prior to aggression was used to induce male territoriality[ 32 , 33 ]. Additionally, 5 days after the last RI, males were tested in a 30-minute mating test,. Finally, 4 days after sexual behavior testing, animals underwent the 3-chamber test to measure social and mate preference. After behavioral testing animals were transcardially perfused with 4% PFA and brains were harvested and used to confirm neuronal ablation via immunohistochemistry (Fig. 3 A). Experiment C ( Fig. 4 ) VMHvl-nNOS ablation in female mice Female nNOS::Cre mice (8–12 weeks) underwent stereotaxic surgery and were injected into the VMHvl bilaterally with either AAV1-flex-taCasp3-TEVp (ablation) or AAV1-flex-EF1a-EGFP-WPRE (control) virus. After 3 weeks, animals underwent behavioral testing to measure different aspects of social behavior. First, all-natural cycling (intact) female mice underwent the 3-chamber test to measure social and mate preference. After the 3-chamber test took place, subjects were split into two cohorts. Cohort A was ovariectomized and implanted with a Silastic capsule containing crystalline 17-β-estradiol (diluted 1:1 with cholesterol) subcutaneously, followed by treatment with 500 µg progesterone 3 hours prior sexual behavior test. Females were tested 3 times with a 4-day interval in between sexual behavior sessions. Cohort B remained ovary intact and underwent 3 FITs in 3 consecutive days. Finally, after the behavior tests were completed, mice from both cohorts were transcardially perfused and brains were harvested and used to confirm neuronal ablation via immunohistochemistry (Fig. 4 A). Experiment D rescue nNOS::Cre mice underwent stereotaxic surgery and were injected into the VMHvl bilaterally with the AAV1-flex-taCasp3-TEVp virus. After 3 weeks, males underwent sexual and aggressive behavior testing whereas females were only tested for sexual behavior. To try to rescue these behavioral impairments we used a combination of NO donor (SNAP) and a guanylate cyclase activator (BAY) 2 hours before the behavioral testing in a within-subjects design[ 22 , 25 ]. Therefore animals were tested for each behavior three times, once to establish baseline levels of behavior and counterbalance drug and vehicle groups (not shown) twice with intraperitoneal (i.p.) injections. 2.6. Stereotaxic surgery Stereotaxic surgery was performed under semi-sterile conditions[ 25 , 35 ]. For detailed information please see supplementary information. 2.7. Pharmacology Mice were injected intraperitoneally with a cocktail of S-NITROSO-N-ACETYL-DL-PENICIL LAMININE (SNAP, N3398, Sigma-Aldrich) and BAY 41-2272 (BAY, B8810, Sigma-Aldrich), in a dose of 8mg/Kg and 10mg/Kg, respectively 2 hours prior behavioral testing[ 25 ]. 2.8. Immunohistochemistry For information regarding immunohistochemistry protocols and antibody concentrations please see supplementary information. 2.9. Statistical Analysis Normality was tested using the Kolmogorov-Smirnov test. Once normality was found data was analyzed using a Student’s t-test, Pearson’s correlations, and Two-way ANOVAs. Once normality was not reached Mann-Whitney and Spearman correlations were performed. For detailed statistics please see Supplementary Tables 1–4. 3. Results VMH-vl nNOS neurons showed a sex-specific co-expression with Erα We confirmed the sex-dimorphic expression of ERα in the VMHvl, i.e. females show a higher number of ERα + -cells (Fig. 1 A)[ 16 , 18 , 48 ]. Additionally, we confirmed previous reports on the absence of sex differences in nNOS neurons within the VMHvl[ 21 ]. Nevertheless, we found some sex differences when looking into the co-expression of those two proteins. In females, around 60% of all nNOS neurons co-expressed ERα compared to 40% in males (Fig. 1 A, Supplementary table 1). Taking together these data revealed that nNOS neurons do not overlap completely with the well-characterized PR/ERα-VMHvl population[ 1 , 2 , 5 , 11 ]. They rather constitute a subpopulation of ERα neurons similar to other genes such as cckar[ 48 , 49 ] or npy2r[ 48 , 50 ] populations. Additionally, these data showed that approximately half of nNOS neurons co-express estrogen receptors which have been shown to be essential for the display of social behaviors in rodents[ 1 , 5 , 51 ]. Finally, we noticed that most nNOS and nNOS-ERα neurons were localized in the posterior-lateral part (pvll) of the VMHvl of females (Fig. 1 C), a subregion present only in female mice that have been linked with female sexual but not aggressive behavior[ 16 , 49 , 50 ]. VMH-vl nNOS neurons exhibit a sex-specific activation pattern after same- and opposite-sex social stimulation Next we investigated whether different social stimuli recruit VMHvl-nNOS neurons in a sex-specific manner (ExperimentA and Fig. 2 A). Regarding females, measuring aggressive behavior in virgin female mice is challenging[ 4 ], as virgin female C57 mice do not readily display aggressive behavior[ 4 , 16 ]. Thus, we decided to use two animal models of exacerbated aggression as positive controls of heightened female aggression: lactating females[ 4 , 36 ] and IST females[ 1 , 4 , 34 ]. The IST protocol entailed high although variable levels of aggression in a separate cohort of animals (Supplementary Fig. 1A and Supplementary Video 1); IST females also showed increased activation of oxytocin neurons in the paraventricular (PVN) and supraoptic nucleus of the hypothalamus (Supplementary Fig. 1B), OXT-c-FOS percentage of colocalization in the PVN also positively correlated with percentage of time spent on aggression (Pearson’s Correlation PVN: r = 0.867, p = 0.0005; SON: r = 0.528, p = 0.09). Those results are in line with previous reports on oxytocin release having pro-aggressive effects in female rodents, independent of the reproductive state[ 4 , 35 , 36 , 46 , 48 ]. Regarding males exposed to mating and fighting, we found that around 11% of the nNOS neurons responded to both social stimuli indistinctly whereas 15% of the neurons colocalized only with pERK (putatively fighting neurons) and 19% colocalized only with c-FOS (putatively mating neurons). Those results showed that around half of VMHvl-nNOS neurons respond to both social stimuli and that those neurons seem to segregate into different neuronal populations (Fig. 2 B-D, Supplementary Table 1). Females exposed to males (who displayed lordosis behavior) showed an increased colocalization of nNOS and cFOS in the VMHvl compared to animals exposed to an object (on average 14% of nNOS neurons were recruited). Interestingly, exposition to a same-sex juvenile did not increase neuronal activity in VMHvl-nNOS neurons of females (Fig. 2 E and F). Accordingly, neither IST females nor lactating females showed any differences in the colocalization of nNOS and pERK in response to an aggressive encounter (Fig. 2 G). Taken together, these data suggest a sex difference in how nNOS neurons are recruited by social stimuli. Ablation of VMH-vl nNOS neurons strongly reduces aggression and mildly affects sexual behavior in males To confirm the involvement of nNOS neurons in different aspects of social behaviors, we ablated VMHvl-nNOS neurons and measured different social behaviors in males (ExperimentB and Fig. 3 A). We confirmed that nNOS deleted (nNOS-del) mice had a 3-fold decrease in the number of nNOS neurons in the VMHvl compared to controls (Fig. 3 B and F). Regarding behavioral data, ablated mice exhibited a lower percentage of time on aggression, threat, and attacking the intruder. Additionally, nNOS ablation almost suppressed attack bites as only 40% of the nNOS-del animals displayed attacks in at least one of the 3 RIs, whereas, in the control group, all animals attacked the intruder at least once. Consequently, nNOS-del mice showed a reduced number of attacks and an increased latency to attack the intruder (Fig. 2 B). Finally, the percentage of time spent on aggression (3rd RI) correlated with the number of nNOS neurons in the VMHvl (Fig. 3 B). Interestingly, the effect of nNOS deletion could not be compensated or reversed by re-engaging in aggressive interactions as nNOS-del animals did not show an increase in aggressive behavior after multiple RIs. Furthermore, reduced aggression could not be associated with a lack of social motivation as nNOS-del animals preferred social stimuli over an object (displaying social preference) (Fig. 3 C) and did not show any effects on social investigation in the RI (Supplementary Table 2). Ablation of nNOS neurons mildly affected mounts and intromissions as well as intromission latency when animals were tested for 10 minutes (Fig. 3 D). Additionally, successive testing seemed to recover the effect of deletion as nNOS-del animals showed an increased number of mounts/intromissions on Test 3 compared to Test 1, consequently, there was no difference between nos-del and controls on Test 3, regarding those parameters (Fig. 3 D). As males did not show a strong phenotype after short sexual behavior tests we decided to challenged them by performing a 30 min session. Under those conditions, NOS-del mice displayed a lower number of mounts (p = 0.05) and intromissions as well as a longer latency to mount the receptive female. Accordingly, the number of nNOS neurons in the VMHvl correlated with the number of mounts&intromissions (Fig. 3 E). Accordingly, nNOS-del animals showed impaired mate preference, meaning that they did not prefer an estrous female over a male mouse (Fig. 3 C). Taking together these data suggests that nNOS neurons might not be directly involved with the display of sexual behavior (consummatory behavior) but rather with female recognition (appetitive behavior) in males (Supplementary Table 2). Deletion of VMH-vl nNOS neurons impairs female sexual behavior and social motivation Next, we performed similar experiments in female nNOS::Cre mice and evaluated different aspects of social interactions (Experiment C and Fig. 4 A). Neuronal ablation was confirmed as nNOS-del females showed a reduced number of nNOS neurons in the VMHvl compared to controls (Fig. 4 F). Additionally, we did not find an effect of hormonal treatment or estrous cycle on the number of nNOS neurons (Supplementary Table 3). Surprisingly, we found strong sex differences in the role of nNOS neurons on social behaviors matching our previous findings with neuronal activity. In contrast with males, neuronal ablation did not affect female aggressive/dominant behaviors toward a juvenile (Fig. 4 B). However, social preference was abolished in nNOS-del females (Fig. 4 C). Concerning reproductive behaviors, nNOS ablation in the VMHvl nearly abolished lordosis behavior in females. Of note, successive behavior testing could not rescue the effect of neuronal ablation on lordosis. Accordingly, the number of nNOS neurons in the VMHvl correlated with the lordosis quotient (Fig. 4 E). Finally, the effects of nNOS deletion in females could not be tied to an impairment in mate preference[ 53 ], as mate preference remained unchanged in nNOS-del females. In fact, those females preferred the male over the female even in the non-receptive phases of the estrous cycle (metestrus-diestrus) (Fig. 4 C), indicating that ablation enhanced male preference in the absence of hormonal priming. Increasing NO availability did not rescue the effects of VMHvl-nNOS ablation In the last set of experiments, we attempted to disentangle the role of NO and nNOS neurons in social behavior, to do so we combined viral ablation, pharmacology and behavior (Experiment D). Deleted males displayed a reduced number of mounts&intromissions and attacks as well as spent a lower percentage of time on aggressive behavior, those effects could not be rescued by the administration of SNAP-BAY. Females also exhibited reduced lordosis quotients, this phenotype could not be rescued by SNAP-BAY (Supplementary Table 4). Those results indicated that nNOS neuron deletion rather affects the social behavior circuit instead of NO neurotransmission in the VMHvl. 4. Discussion A myriad of behaviors ranging from social investigation and affiliative approach to mating and fighting emerge from interactions between conspecifics. Generally, those behavioral responses rely on several factors such as conspecific cues, internal states marked for physiological/hormonal alterations, and previous experiences [ 2 , 54 ]. In this manuscript, we investigated how VMHvl-nNOS neurons are affected by same- and opposite-sex conspecific cues as well as how ablation of those neurons affects the display of social behaviors itself. Our data revealed that nNOS neurons in the VMHvl are recruited in a sex-specific manner by male and female cues. In females, those neurons were exclusively activated by mating (male cues) whereas in males both mating with a receptive female and fighting with a smaller male intruder increased the activation of VMHvl-nNOS neurons. Interestingly, those neurons were not activated in lactating females[ 4 , 36 ] or highly aggressive IST females[ 1 , 4 , 35 , 46 ], known to display exacerbated levels of aggression, indicating that the absence of activity of those neurons in females is not reproductive state, social experience, or aggression level-dependent. Furthermore, this is consistent with the majority of the nNOS neurons being located in the VMHvlpvl, tied to sexual but not aggressive behavior in females[ 16 , 50 , 51 ]. Surprisingly, the ablation of VMHvl-nNOS neurons in males nearly abolished aggressive behavior without affecting social preference. Although the loss of function data matches the neuronal activity after aggression, these data are rather unexpected. Previous studies using nNOS-KOs have shown that those animals exhibit exaggerated aggressive behavior[ 23 , 24 ]. Pharmacological inhibition of nNOS activity systemically also strongly increased aggression in male mice, particularly when those animals were single-housed[ 24 , 27 ]. Furthermore, nNOS enzymatic inhibition also decreased social investigation in single-housed mice[ 24 ]. Conversely, a recent study has found that inhibition of NMDAR-dependent PSD95/nNOS activity either i.p. or intracerebroventricularly (i.c.v.) strongly decreases social isolation-induced aggression in male mice, similar to our findings[ 28 ]. Pretreatment with L-arginine a precursor of NO prevented the effect of PSD95/nNOS inhibition on aggression, indicating those effects were NO-dependent[ 28 ]. Here one should differentiate between the role of nNOS-positive neurons and nNOS enzymatic activity and consequently NO neurotransmission on aggression. Early, pharmacological[ 24 , 27 ] and KO[ 23 ] studies particularly focused on the latter whereas our study is centered on the neuronal populations expressing nNOS. In fact, elevating NO levels via the administration of a NO-donor failed to rescue the behavioral deficits seen in aggressive behavior in our study. This suggests that the effect on nNOS neuronal ablation is not mainly underlied by NO-transmission but rather by their role in the neuronal circuitry of aggression. Alternatively, another possible explanation would be that VMHvl-nNOS neurons are particularly engaged in the plasticity-dependent nNOS activity similar to the study of Yang et. al.[ 28 ], in fact, plasticity-dependent enhancement of aggressive behavior has been tied to the VMHvl in male winners[ 9 ]. Future studies should verify that hypothesis. Regarding male sexual behavior, our data revealed a mild effect of nNOS neuron deletion in reducing the number of intromissions and the copulation latency when animals were tested for 30 minutes. On the other hand, multiple tests seemed to rescue the ablation effect on male sexual behavior partially. In line with our observations, a study performed in male rats using L-NAME (an inhibitor of nNOS) i.c.v. has shown that nNOS inhibition reduces mounting and abolishes ejaculation in naïve but not experienced rats[ 29 ], indicating sexual experience might overrule the effects of NO on mating. This mild effect of NO synthesis and VMHvl-nNOS neurons on male sexual behavior might arise from the participation of nNOS in the detection of female cues (appetitive phase) rather than copulation itself. Consistently, we have found that VMHvl-nNOS neurons are activated by female cues (Fig. 2 ). Additionally, nNOS-del males exhibited impaired mate preference and took longer to intromit and mount (Fig. 3 ). Thus, one could hypothesize that males display less consummatory behaviors because they struggle to identify the receptivity cues of a potential mate. Accordingly, nNOS-KO studies have shown that males usually display persistent mounting even towards non-receptive (anestrous) females, compared to WT[ 23 ]. Taking together with our findings, this indicates that NO signaling and nNOS neurons might be involved rather in mate detection/choice than copulatory behaviors per se. Furthermore, other nNOS neuronal populations might be involved in male copulation, for example sexually experienced rats showed increased nNOS mRNA in the PVN compared to naïve rats[ 29 ]. In contrast to males, VMHvl-nNOS neurons were only activated in females after mating with an adult male. Furthermore, females which were exposed to a male but did not display lordosis failed to show increased nNOS activation (not shown). Interestingly, exposition to a juvenile intruder which is known to trigger dominant behavior (mounts) in C57 female mice[ 51 ] as well as aggression displayed by lactating females[ 4 , 36 ] and IST females[ 4 , 35 , 46 ] did not increase pERK expression in VMHvl-nNOS neurons (Fig. 2 ), indicating that neither same-sex cues, dominant behavior, nor fighting recruits nNOS neurons in females. Accordingly, nNOS-del females did not show any differences in their levels of aggression&dominance (Fig. 4 ). Accordingly, nNOS-KO virgin females do not show differences in the display of aggressive behavior[ 23 ]. Lactating nNOS-KO, on the other hand, showed a moderate reduction in maternal aggression[ 26 ]. Pharmacological studies in female prairie voles have shown similar, although milder effects. Inhibition of nNOS activity i.p. decreased maternal aggression after 2 but not 3 days of treatment[ 55 ]. Those results might indicate different mechanisms regulating the involvement of nNOS in aggressive behavior depending on the reproductive state. Another possible explanation is that other nNOS populations might be recruited during maternal aggression, indeed the number of nNOS neurons expressing citrulline (indirect NO synthesis marker) increased in lactating animals after an aggressive encounter, in regions known to regulated maternal aggression[ 56 , 57 ], such as the medial preoptic area (MPOA) and the PVN (where citrulline cells correlated with the display of aggressive behavior)[ 26 , 55 ]. We also found that nNOS-del females did not show a social preference, which is in agreement with previous data in males demonstrating that inhibition of nNOS activity decreases exploration of male urine [ 24 ]. Perhaps, the lack of dominant or aggressive behavior in virgin females arises from the fact those animals show low levels of motivation to interact and explore an intruder, although non-aggressive social investigation did not differ between control and nos-del females (Fig. 4 D). Finally, nNOS-del females showed a striking reduction in lordosis behavior (nearly abolished) which could not be linked to an impairment in mate choice (Fig. 4 ). Furthermore, in contrast to males, sexual experience did not rescue/compensate for the effect of VMHvl-nNOS deletion in females (Fig. 4 ). These data align with i) our neuronal activity data showing that VMHvl-nNOS neurons are active after mating in females (Fig. 2 ), the localization of nNOS neurons in the VMHvlpvl (Fig. 1 ) and previous reports of our group using nNOS-KO[ 25 ] and behavioral pharmacology[ 30 ], as well as from another group showing that inhibition of VMHvl-nNOS neurons via chemogenetics strongly reduces lordosis in mice[ 22 ]. Surprisingly, although the nNOS-KO phenotype could be reversed by elevating NO levels using SNAP-BAY[ 25 ], the same treatment failed to rescue the lordosis deficit of nNOS-del females. This again indicates that this ablation might affect the circuitry of female sexual behavior[ 11 ] rather than NO-neurotransmission. However, further investigation is needed in order to disentangle local NO production and nNOS activity from nNOS neuron activity, as i.p. injections could act in other brain regions other than the VMHvl. Surprisingly, nNOS-del diestrus females preferred a male over a female. This was unexpected, previous data from our lab shows that nNOS enzymatic activity is necessary for mate preference in females[ 25 ] additionally, male odors were able to activate nNOS neurons [ 31 ]. Knowing that i) PR neurons and nNOS neurons are co-expressed[ 22 ], ii) PR is co-expressed in a ratio of 1:1 with ERα in the VMHvl[ 15 , 16 ] iii) PR-VMHvl neurons are involved in mate rejection[ 19 ] and iv) PR signaling in the olfactory system silences male cue detection during diestrus, leading to an absence of male preference [ 58 ], one could hypothesize that by ablating nNOS neurons we partially eliminate some of the PR (ERα) neurons that might either be involved in mate rejection and/or recognition, therefore females start to show a male preference irrespective of the estrous cycle phase. Future studies should investigate the link between mate preference, VMHvl-nNOS, PR- and ERα signaling. Finally, our data confirm previous findings portraiting the VMHvl as a hotspot for social behavior. Additionally, we characterized a novel neuronal population (nNOS neurons) from the behavioral point of view. Our data show that nNOS neurons in the VMHvl show sex differences in co-expression with ERα as well as how they respond to social stimuli. In females, these neurons seem to preferably respond to male cues whereas in males to both sexes. Finally, male VMHvl-nNOS neurons seem to be crucial for aggressive behavior and mate choice whereas in females those neurons seem to be essential for sexual behavior and social motivation (Fig. 5 ). Declarations Acknowledgments We would like to thank Laura de Vries, Chloé Beaudou, and Lozen Thies for their help with the experiments and Dr. Harold Gainer for giving us the anti-oxytocin antibody. Author Contributions V.O: Designed, performed, analyzed experiments, and wrote the manuscript, as well as designed figures and tables. I.B: Assisted with performing and analyzing behavioral experiments. JB: supervised the study, and gave input to experimental design and to the manuscript. All authors contributed to the article and approved the submitted version. Competing Interests and Funding JB is a Research Director of the FNRS, and she discloses a patent on J. Bakker and S. Ouerdi. "Agonists of human kisspeptin receptor for modulating sexual desire." WO2020/151830A1 filed -1 Unspecified -001 and issued 30 July 2020. All authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. This work was supported by the Fonds de la Recherche Scientifique -FNRS Chargé de Recherche (1.B.308.23AGGRESSIONKiNG project to V.O., by the Fondation Léon Fredericq Bourse de fonctionnement Post-Doctorant (Neuromodulation of aggression by kisspeptin, neurokinin B, and GnRH to VO and the FNRS (PDR T.0015.20 to JB). References Oliveira VE de M. Animal Models of Aggression. Handbook of Anger, Aggression, and Violence, Cham: Springer International Publishing; 2023. p. 1–24. Wei D, Talwar V, Lin D. Neural circuits of social behaviors: Innate yet flexible. Neuron. 2021;109:1600–1620. Lenschow C, Lima SQ. In the mood for sex: neural circuits for reproduction. Curr Opin Neurobiol. 2020;60:155–168. Oliveira VE de M, Bakker J. Neuroendocrine regulation of female aggression. Front Endocrinol (Lausanne). 2022;13. Hashikawa K, Hashikawa Y, Lischinsky J, Lin D. The Neural Mechanisms of Sexually Dimorphic Aggressive Behaviors. Trends in Genetics. 2018;10:755–776. Zilkha N, Sofer Y, Kashash Y, Kimchi T. The social network: Neural control of sex differences in reproductive behaviors, motivation, and response to social isolation. Curr Opin Neurobiol. 2021;68:137–151. Lenz KM, Nugent BM, McCarthy MM. Sexual differentiation of the rodent brain: Dogma and beyond. Front Neurosci. 2012;6:1–13. Oyegbile TO, Marler CA. Winning fights elevates testosterone levels in California mice and enhances future ability to win fights. Horm Behav. 2005;48:259–267. Stagkourakis S, Spigolon G, Liu G, Anderson DJ. Experience-dependent plasticity in an innate social behavior is mediated by hypothalamic LTP. 2020. 2020. https://doi.org/10.1073/pnas.2011782117/-/DCSupplemental. Koolhaas JM, Schuurman T, Wiepkema PR. The organization of intraspecific agonistic behaviour in the rat. Prog Neurobiol. 1980;15:247–268. Yin L, Lin D. Neural control of female sexual behaviors. Horm Behav. 2023;151:105339. Hellier V, Brock O, Bakker J. The role of kisspeptin in sexual behavior. Semin Reprod Med. 2019;37:84–92. Falkner AL, Lin D. Recent advances in understanding the role of the hypothalamic circuit during aggression. Front Syst Neurosci. 2014;8:1–14. Haller J. The neurobiology of abnormal manifestations of aggression-A review of hypothalamic mechanisms in cats, rodents, and humans. Brain Res Bull. 2013;93:97–109. Lee H, Kim D-W, Remedios R, Anthony TE, Chang A, Madisen L, et al. Scalable control of mounting and attack by Esr1+ neurons in the ventromedial hypothalamus. Nature. 2014;509:627–632. Hashikawa K, Hashikawa Y, Tremblay R, Zhang J, Feng JE, Sabol A, et al. Esr1+cells in the ventromedial hypothalamus control female aggression. Nat Neurosci. 2017;20:1580–1590. Lin D, Boyle MP, Dollar P, Lee H, Lein ES, Perona P, et al. Functional identification of an aggression locus in the mouse hypothalamus. Nature. 2011;470:221–226. Yang CF, Chiang MC, Gray DC, Prabhakaran M, Alvarado M, Juntti SA, et al. Sexually dimorphic neurons in the ventromedial hypothalamus govern mating in both sexes and aggression in males. Cell. 2013;153:896–909. Gutierrez-Castellanos N, Husain BFA, Dias IC, Nomoto K, Duarte MA, Ferreira L, et al. A hypothalamic node for the cyclical control of female sexual rejection. Neuron. 2024. November 2024. https://doi.org/10.1016/j.neuron.2024.10.026. Kammel LG, Correa SM. Selective sexual differentiation of neurone populations may contribute to sex ‐ specific outputs of the ventromedial nucleus of the hypothalamus. J Neuroendocrinol. 2020. 2020. https://doi.org/10.1111/jne.12801. Sica M, Martini M, Viglietti-Panzica C, Panzica GC. Estrous cycle influences the expression of neuronal nitric oxide synthase in the hypothalamus and limbic system of female mice. BMC Neurosci. 2009;10. Silva MSB, Decoster L, Trova S, Mimouni NEH, Delli V, Chachlaki K, et al. Female sexual behavior is disrupted in a preclinical mouse model of PCOS via an attenuated hypothalamic nitric oxide pathway. Proc Natl Acad Sci U S A. 2022;119:1–12. Nelson RJ, Demais GE, Huang PL, Fishman MC, Dawson VL, Dawson TM, et al. Behavioural abnormalities in male mice lacking neuronal nitric oxide synthase. Nature Letters. 1995;378:383–386. Trainor BC, Workman JL, Jessen R, Nelson RJ. Impaired Nitric Oxide Synthase Signaling Dissociates Social Investigation and Aggression. Behavioral Neuroscience. 2007;121:362–369. Hellier V, Brock O, Candlish M, Desroziers E, Aoki M, Mayer C, et al. Female sexual behavior in mice is controlled by kisspeptin neurons. Nat Commun. 2018;9. Gammie SC, Nelson RJ. Maternal aggression is reduced in neuronal nitric oxide synthase- deficient mice. Journal of Neuroscience. 1999;19:8027–8035. Demas GE, Eliasson MJL, Dawson TM, Dawson VL, Kriegsfeld LJ, Nelson RJ, et al. Inhibition of neuronal nitric oxide synthase increases aggressive behavior in mice. Molecular Medicine. 1997;3:610–616. Yang L, Cui J, Zeng L, Lu W. Targeting PSD95/nNOS by ZL006 alleviates social isolation-induced heightened attack behavior in mice. Psychopharmacology (Berl). 2022;239:267–276. Benelli A, Bertolini A, Poggioli R, Cavazzuti E, Calzà L, Giardino L, et al. Nitric oxide is involved in male sexual behavior of rats. Eur J Pharmacol. 1995;294:505–510. Bentefour Y, Bakker J. Kisspeptin signaling and nNOS neurons in the VMHvl modulate lordosis behavior but not mate preference in female mice. Neuropharmacology. 2021;198:108762. Bentefour Y, Bakker J. Stress during pubertal development affects female sociosexual behavior in mice. Nat Commun. 2024;15:3610. Miczek KA, Maxson SC, Fish EW, Faccidomo S. Aggressive behavioral phenotypes in mice. Behavioural Brain Research. 2001;125:167–181. Koolhaas JM, Coppens CM, de Boer SF, Buwalda B, Meerlo P, Timmermans PJ. The resident-intruder paradigm: A standardized test for aggression , violence and social stress. Journal of Visualized Experiments. 2013;77:4367. Blumstein DT, Evans CS, Daniel JC. JWatcher 0.9: An Introductory User’s Guide. Animal Behaviour Laboratory, Macquarie University. 2000. Oliveira VEM, Lukas M, Wolf HN, Durante E, Lorenz A, Mayer A-L, et al. Oxytocin and vasopressin within the ventral and dorsal lateral septum modulate aggression in female rats. Nat Commun. 2021;12:1–15. Bosch OJ. Maternal aggression in rodents: Brain oxytocin and vasopressin mediate pup defence. Philosophical Transactions of the Royal Society B: Biological Sciences. 2013;368. Oliveira VEM, de Jong, Trynke, R, Neumann ID. Modelling sexual violence in male rats : the sexual aggression test ( SxAT ). Transl Psychiatry. 2022;12:1–12. Oliveira VEM, Neumann ID, de Jong TR. Post-weaning social isolation exacerbates aggression in both sexes and affects the vasopressin and oxytocin system in a sex-specific manner. Neuropharmacology. 2019;156:107504. Camats Perna J, Engelmann M. Recognizing others : Rodent’s social memories. In: Wöhr M. KS, editor. Curr Top Behav Neurosci, vol. 30, . Current ToSpringer, Cham; 2017. p. 25–45. Engelmann M, Hädicke J, Noack J. Testing declarative memory in laboratory rats and mice using the nonconditioned social discrimination procedure. Nat Protoc. 2011;6:1152–1162. Khbouz B, de Bournonville C, Court L, Taziaux M, Corona R, Arnal JF, et al. Role for the membrane estrogen receptor alpha in the sexual differentiation of the brain. European Journal of Neuroscience. 2020;52:2627–2645. Trainor BC, Crean KK, Fry WHD, Sweeney C. Activation of extracellular signal-regulated kinases in social behavior circuits during resident-intruder aggression tests. Neuroscience. 2010;165:325–336. De Jong TR, Beiderbeck DI, Neumann ID. Measuring virgin female aggression in the Female Intruder Test (FIT): Effects of oxytocin, estrous cycle, and anxiety. PLoS One. 2014;9:e91701. Grossmann CP, Sommer C, Fahliogullari IB, Neumann ID, Menon R. Mating-induced release of oxytocin in the mouse lateral septum: Implications for social fear extinction. Psychoneuroendocrinology. 2024;166. Kuiper LB, Lucas KA, Mai V, Coolen LM. Enhancement of Drug Seeking Following Drug Taking in a Sexual Context Requires Anterior Cingulate Cortex Activity in Male Rats. Front Behav Neurosci. 2020;14. Oliveira VEM, Jong TR De, Neumann ID. Synthetic Oxytocin and Vasopressin Act Within the Central Amygdala to Exacerbate Aggression in Female Wistar Rats. Front Neurosci. 2022;16:1–12. Oliveira VE de M, Evrard F, Faure MC, Bakker J. Social isolation and aggression training lead to escalated aggression and hypothalamus-pituitary-gonad axis hyperfunction in mice. Neuropsychopharmacology. 2024. 2024. https://doi.org/10.1038/s41386-024-01808-3. Sofer Y, Zilkha N, Gimpel E, Wagner S, Chuartzman SG, Kimchi T. Sexually dimorphic oxytocin circuits drive intragroup social conflict and aggression in wild house mice. Nat Neurosci. 2024. 2024. https://doi.org/10.1038/s41593-024-01685-5. Cortes LR, Sturgeon H, Forger NG. Sexual differentiation of estrogen receptor alpha subpopulations in the ventromedial nucleus of the hypothalamus. Horm Behav. 2023;151. Yin L, Hashikawa K, Hashikawa Y, Osakada T, Lischinsky JE, Diaz V, et al. VMHvllCckar cells dynamically control female sexual behaviors over the reproductive cycle. Neuron. 2022. 21 September 2022. https://doi.org/10.1016/j.neuron.2022.06.026. Liu M, Kim D-W, Zeng H, Anderson DJ. Make war not love: The neural substrate underlying a state-dependent switch in female social behavior. Neuron. 2022:1–16. Trainor BC, Kyomen HH, Marler CA. Estrogenic encounters: How interactions between aromatase and the environment modulate aggression. Front Neuroendocrinol. 2006;27:170–179. McCarthy EA, Naik AS, Coyne AF, Cherry JA, Baum MJ. Effect of ovarian hormones and mating experience on the preference of female mice to investigate male urinary pheromones. Chem Senses. 2018;43:97–104. Insel TR. The Challenge of Translation in Social Neuroscience: A Review of Oxytocin, Vasopressin, and Affiliative Behavior. Neuron. 2010;65:768–779. Gammie SC, Olaghere-Da Silva UB, Nelson RJ. 3-Bromo-7-nitroindazole, a neuronal nitric oxide synthase inhibitor, impairs maternal aggression and citrulline immunoreactivity in prairie voles. Brain Res. 2000;870:80–86. Hasen NS, Gammie SC. Maternal aggression: New insights from Egr-1. Brain Res. 2006;1108:147–156. Hasen NS, Gammie SC. Differential fos activation in virgin and lactating mice in response to an intruder. Physiol Behav. 2005;84:681–695. Dey S, Chamero P, Pru JK, Chien MS, Ibarra-Soria X, Spencer KR, et al. Cyclic regulation of sensory perception by a female hormone alters behavior. Cell. 2015;161:1334–1344. Additional Declarations There is NO Competing Interest. Supplementary Files Supplementaryvideo1.mov Supplementary video 1 Supplementaryfigure1.tiff SupplementaryinfonNOSfinalCommbio.pdf Cite Share Download PDF Status: Published Journal Publication published 01 Dec, 2025 Read the published version in Communications Biology → 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. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-5070073","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":421741302,"identity":"298df894-f9b0-471d-a2a9-064ce698c8e6","order_by":0,"name":"Vinícius Oliveira","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA5klEQVRIiWNgGAWjYDCCA1CaH8rmAXEkiNIi2UCyFoMDSIJ4tfAd7058zLvHLnHzjdyDBxhz7GTM+Q8w3viAR4vkmbObjXmeJSduu5GXcIBxWzKPZcMBZssZeLQY3MjdJs1zgNnY7EaOweG/25h5DA42sEnz4NNy/y1IS72x8YwcA6At9TwGhxnYpP/gtYUXpOWwnIEEWMthHoNjQC34vC95Jnez4ZwDx+UkzrwBaTnOY3CGsdmyB48WvuNnNz54c6Cah789x/gD47Zqe4Pzhw/e+IHPGiyAsYFEDaNgFIyCUTAK0AEAd75PNC/uTH8AAAAASUVORK5CYII=","orcid":"https://orcid.org/0000-0001-9020-6018","institution":"University Medical Center of the Johannes Gutenberg University Mainz","correspondingAuthor":true,"prefix":"","firstName":"Vinícius","middleName":"","lastName":"Oliveira","suffix":""},{"id":421741303,"identity":"c55a9e73-b3cb-4e6b-8d5d-03111804ebe6","order_by":1,"name":"Ioana Bodea","email":"","orcid":"","institution":"University of Liège","correspondingAuthor":false,"prefix":"","firstName":"Ioana","middleName":"","lastName":"Bodea","suffix":""},{"id":421741304,"identity":"76cd4777-b866-447f-86f4-8917fa165a2d","order_by":2,"name":"Julie Bakker","email":"","orcid":"https://orcid.org/0000-0002-3504-9010","institution":"University of Liège","correspondingAuthor":false,"prefix":"","firstName":"Julie","middleName":"","lastName":"Bakker","suffix":""}],"badges":[],"createdAt":"2024-09-11 09:52:45","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-5070073/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-5070073/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1038/s42003-025-09279-y","type":"published","date":"2025-12-01T05:00:00+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":77655137,"identity":"4d255d53-8302-4cd2-b346-e8ad0629a53f","added_by":"auto","created_at":"2025-03-04 03:13:01","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":816989,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eVMH-vl nNOS neurons exhibit a sex-specific co-expression with ERα. \u003c/strong\u003eMaximal z-projection of ERα (magenta, Alexa-fluor-546 anti-rabbit) and nNOS (green, Alexa-fluor-488 anti-goat) in the ventrolateral portion of the ventromedial hypothalamus (VMHvl) in male and female mice (A).\u0026nbsp; ERα was more abundant in the VMHvl of females compared to males (Mann-Whitney U test U= 0.0, p=0.015), whereas nNOS neurons did not differ between sexes (two-tailed Student’s t-test t\u003csub\u003e(7)\u003c/sub\u003e=0.69, p=0.512). Consequently, co-expression of nNOS and ERα was sexually dimorphic with a higher proportion of nNOS neurons expressing ERα in females (t\u003csub\u003e(7)\u003c/sub\u003e=3.44, p=0.01) (B). Most nNOS neurons were located in the lateral part of the VMHvl (pvll) istead of the medial portion (pvlm) (nNOS: t\u003csub\u003e(6)\u003c/sub\u003e=22.02, p\u0026lt;0.0001; nNOS-ERα/nNOS: U=0.0; p=0.028) \u0026nbsp;(C). Data are presented as mean + s.e.m. *p\u0026lt;0.05;***p\u0026lt;0.001; ****p\u0026lt;0.0001. Scale bar 100μm.\u003c/p\u003e","description":"","filename":"image1.png","url":"https://assets-eu.researchsquare.com/files/rs-5070073/v1/458355375f9c16e3007c69e0.png"},{"id":77655138,"identity":"f1b7ef2d-f8d0-4c4b-bcad-369bc18720d5","added_by":"auto","created_at":"2025-03-04 03:13:01","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":582050,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSame- and opposite-sex stimuli differentially recruit VMHvl-nNOS neurons in mice.\u003c/strong\u003e Schematic drawings depicting the experimental designs followed in Experiment A. \u0026nbsp;Mice of both sexes were confronted either with an object (control) or an opposite-sex-conspecific for 15 min (mating). One hour later, those animals were again exposed to an object (controls) or a same-sex-conspecific (fighting) for 10 min after the test animals were perfused. \u003cstrong\u003eHigh-aggression female controls\u003c/strong\u003e consisted of isolated and aggression-trained (IST) females who underwent 4 consecutive female intruder tests and lactating females (lactating day, LD, 4-7) who were also confronted with an intruder for 10 min prior to perfusion (A). \u003cstrong\u003e\u0026nbsp;\u003c/strong\u003eMaximal z-projection of pERK (magenta, Alexa-fluor-546 anti-rabbit), cFOS (yellow, Alexa-fluor-488 anti-guinea-pig), (cyan, Alexa-fluor-633 anti-goat) \u0026nbsp;in the ventrolateral portion of the ventromedial hypothalamus (VMHvl) in male and female mice which were confronted with either an object (control) or a conspecific (stimulated) (B).\u0026nbsp; In males, around\u0026nbsp; 15% or 19% of nNOS neurons were exclusively activated by fighting by showing pERK colocalization (magenta pERK\u0026amp;nNOS\u003csup\u003e+\u003c/sup\u003e/nNOS) or mating by showing colocalization with cFos (yellow,cFos\u0026amp;nNOS/nNOS \u003csup\u003e+\u003c/sup\u003e), respectively.\u0026nbsp; Whereas 11% of the neurons showed a colocalization of cFos\u0026amp;nNOS\u0026amp;p-ERK/nNOS (white stars, blue bars C). Thus, both mating or fighting increased the proportion of nNOS neurons active, showing either c-fos (yellow bars in D and arrowheads in B, Mann-Whitney U test U= 0.0, p=0.031) or p-ERK (magenta bars in C, and arrows in B, U=0.0, p=0.015), respectively (D). \u0026nbsp;In females, around\u0026nbsp; 3% or 14% of nNOS neurons were exclusively activated by same-sex-conspecific/dominance (magenta) or mating (yellow), respectively.\u0026nbsp; Whereas 2% of the neurons showed a colocalization of the three markers (E). Thus, only mating (yellow, bars) increased the proportion of nNOS neurons active c-fos (Mann-Whitney U test U= 0.0, p=0.009) (F). Finally, neither aggressive IST nor lactating females showed increased pERK colocalization with nNOS after being exposed to a same-sex intruder (Kruskal-Wallis test, K=6.66, p=0.157, G) Data are presented as mean + s.e.m.*p\u0026lt;0.05;**p\u0026lt;0.01: Scale bar 100μm.\u003c/p\u003e","description":"","filename":"image2.png","url":"https://assets-eu.researchsquare.com/files/rs-5070073/v1/14a4f700ab3dbdb80bb4b3d2.png"},{"id":77655145,"identity":"043bb233-f3b6-47b0-a8a3-2b0042f25897","added_by":"auto","created_at":"2025-03-04 03:13:02","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":810719,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAblation of nNOS neurons in the VMHvl strongly reduced aggression and mildly affected sexual behavior in males.\u003c/strong\u003e Schematic drawings depicting the experimental designs followed in Experiment B. \u0026nbsp;nNOS:cre (8-12 weeks) were injected bilaterally into the VMHvl with either AAV1-flex-taCasp3-TEVp (nNOS-del, blue) or AAV1-flex-EF1a-EGFP-WPRE (control, white) virus. Three weeks later, both groups were tested for sexual behavior for 10 min and directly after for aggressive behavior (10 min) on 3 consecutive days. After 4 days animals were again tested for sexual behavior for 30 min and 4 days later were tested in the three-chamber apparatus for social and mate preferences, before perfusion (A). \u0026nbsp;Deleted animals (nNOS-del, blue) showed a decreased percentage of time spent on aggression (Virus effect: F\u003csub\u003e(1, 13) \u003c/sub\u003e= 28.55, p=0.001; \u0026nbsp;Training effect: F\u003csub\u003e(2, 26)\u003c/sub\u003e = 3.62, p=0.04) which was reflected in a higher latency to attack (Virus effect: F\u003csub\u003e(1,13)\u003c/sub\u003e=19.58, p=0.0007), lower number of attacks (Virus effect: F\u003csub\u003e(1,13)\u003c/sub\u003e= 37.11, p\u0026lt;0.0001) as well as a lower proportion of animals attacking (Fisher’s exact test, p=0.0001). Consequently, aggression in the third test positively correlated with the number of nNOS neurons in the VMHvl (Pearson’s correlation r= 0.636, p=0.001) (B). Social preference was not affected by neuronal ablation (One sample t-test ctrl: t-test t\u003csub\u003e(6)\u003c/sub\u003e=2.49, p=0.047; nNOS-del: t\u003csub\u003e(5)\u003c/sub\u003e=3.35, p=0.01), however, mate preference was absent in nNOS-del males (ctrl: t\u003csub\u003e(5)\u003c/sub\u003e=2.49, p=0.04; nNOS: t-test t\u003csub\u003e(8)\u003c/sub\u003e=0.26, p=0.79) \u0026nbsp;(C). \u0026nbsp;\u0026nbsp;Regarding sexual behavior, the number of mounts and intromissions was decreased on test day 2 compared controls.\u0026nbsp; However, nNOS-del mice showed an increase in mounts and intromission on test day 3 (Virus effect: F\u003csub\u003e(1,13)\u003c/sub\u003e=9.242, p=0.009; Training effect: F\u003csub\u003e(2,26)\u003c/sub\u003e= 5.690, p=0.008) (D). Once animals were tested for 30 min, a reduction in sexual behavior was more evident in nNOS-del mice which displayed fewer mounts ( two-tailed Student’s t-test t\u003csub\u003e(13)\u003c/sub\u003e=2.147, p=0.05) and intromissions (two-tailed Student’s t-test t\u003csub\u003e(13)\u003c/sub\u003e=2.759, p=0.01). Additionally, the latency to mount a female was higher (U=10, p=0.049). Mounts and intromissions also positively correlated with the number of nNOS neurons in those animals (r=0.3634, p=0.038) (E). \u0026nbsp;Finally, deletion was confirmed as nNOS-del mice showed a lower number of nNOS neurons (U= 0.0, p=0.002) in the VMHvl compared to controls (B and F). *p\u0026lt;0.05 vs test or 0.0 (preferences); # p\u0026lt;0.05,## p\u0026lt;0.01;### p\u0026lt;0.001 vs control. \u0026nbsp;Data are presented as mean + s.e.m.\u003c/p\u003e","description":"","filename":"image3.png","url":"https://assets-eu.researchsquare.com/files/rs-5070073/v1/0b7cb36516265c7fa8a7b9eb.png"},{"id":77655164,"identity":"e1d1054a-8201-461d-ba20-097cad376a42","added_by":"auto","created_at":"2025-03-04 03:13:03","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":558036,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAblation of nNOS neurons in the VMHvl strongly reduced sexual behavior and abolished social preference in female mice.\u003c/strong\u003e Schematic drawings depicting the experimental designs followed in Experiment C. \u0026nbsp;nNOS:cre (8-12 weeks) were injected into the VMHvl bilaterally with either AAV1-flex-taCasp3-TEVp (nNOS-del, purple) or AAV1-flex-EF1a-EGFP-WPRE (control, white) virus. Three weeks later, both groups were tested in the three-chamber apparatus for social and mate preferences. Next, animals were split into two Cohorts, cohort A underwent ovariectomy (OVX) and was implanted with Silastic capsules containing crystalline 17-β-estradiol (diluted 1:1 with cholesterol) subcutaneously,\u0026nbsp; those animals were treated s.c. with 500 µg progesterone 3 hours prior being tested for sexual behavior. Cohort B was kept ovary intact and was tested in the female intruder test, by being confronted with a juvenile (p16-21) CD1 mouse (A). \u0026nbsp;Deleted (nNOS-del, purple) and control (white) females showed very mild levels of aggression/dominance which were not affected by neuronal ablation (B). However social preference was abolished in nos-del animals \u0026nbsp;(One sample t-test ctrl: t-test t\u003csub\u003e(11)\u003c/sub\u003e=2.345, p=0.038; nNOS-del: t\u003csub\u003e(13)\u003c/sub\u003e=1.87, p=0.08), this could not be linked to social approach as social investigation deficits in the FIT did not differ between groups (D). Mate preference, on the other hand, remained unchanged in nNOS-del females independently of the estrous cycle status (ctrl P-E: t-test t\u003csub\u003e(5)\u003c/sub\u003e=2.63, p=0.046; ctrl-MD: t-test t\u003csub\u003e(7)\u003c/sub\u003e=0.28, p=0.28; nNOS-del P-E: t-test t\u003csub\u003e(3)\u003c/sub\u003e=0.2093, p=0.12; nNOS-del-MD: t-test t\u003csub\u003e(9)\u003c/sub\u003e=3.971, p=0.003 P-E: proestrus/estrus phase of the estrous cycle; M-D: metaestrus/diestrus phase of the estrous cycle) \u0026nbsp;(C). Female sexual behavior was strongly disrupted by the deletion of VMHvl-nNOS neurons,\u0026nbsp; as females displayed a reduced lordosis quotient (Virus effect: F\u003csub\u003e(1,12)\u003c/sub\u003e= 34.29, p\u0026lt;0.0001; Training effect: F\u003csub\u003e(2,24)\u003c/sub\u003e=1.305, p=0.29; Virus x training effect: F\u003csub\u003e(2,24)\u003c/sub\u003e= 4.969, p=0.01), which negatively correlated with the number of nNOS neurons in the VMHvl (Pearsons correlation r= 0.872, p\u0026lt;0.0001) (E).\u0026nbsp; Finally, deletion was confirmed in both cohorts as nNOS-del females (intact and OVX-E2) showed a lower number of nNOS neurons (CohA (OVX+E2) U= 0.0, p=0.0043; CohB (intact) U= 0.0, p=0.0006) in the VMHvl compared to controls (F). *p\u0026lt;0.05, **p\u0026lt;0.01 vs test or 0.0 (preferences); # p\u0026lt;0.05,## p\u0026lt;0.01;### p\u0026lt;0.001; ;#### p\u0026lt;0.0001 \u0026nbsp;vs control. \u0026nbsp;Data are presented as mean + s.e.m.\u003c/p\u003e","description":"","filename":"image4.png","url":"https://assets-eu.researchsquare.com/files/rs-5070073/v1/bd208404fc3fa471dd669f35.png"},{"id":77655673,"identity":"0942b20c-4674-4781-87f3-e340734d1b6e","added_by":"auto","created_at":"2025-03-04 03:21:02","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":136587,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eVMHvl nNOS neurons modulate social behaviors in a sex-specific manner. \u003c/strong\u003eIn males (right side, blue) nNOS neurons are activated by male and female cues and are scattered throughout the VMHvl additionally those neurons are needed for the display of male sexual and aggressive behavior. In females (left side, purple), nNOS neurons are recruited by male but not female cues. Additionally, those neurons are mostly located in the lateral portion of the VMHvl, an area involved in female sexual behavior. VMHvl-nNOS neurons were found to be crucial for female sexual behavior and social preference.\u003c/p\u003e","description":"","filename":"image5.png","url":"https://assets-eu.researchsquare.com/files/rs-5070073/v1/0b7ee2587bf176c0b1435b05.png"},{"id":97322745,"identity":"2cf8059b-3861-4461-a409-d0d83685141b","added_by":"auto","created_at":"2025-12-03 08:10:56","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3927149,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5070073/v1/29b243bf-199a-4eb3-86bd-fe1113ff826e.pdf"},{"id":77655139,"identity":"d16dad69-cc61-48a0-a532-65a538bc05cc","added_by":"auto","created_at":"2025-03-04 03:13:02","extension":"mov","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":660248,"visible":true,"origin":"","legend":"Supplementary video 1","description":"","filename":"Supplementaryvideo1.mov","url":"https://assets-eu.researchsquare.com/files/rs-5070073/v1/cc4b825547ac37d7aa50c717.mov"},{"id":77655675,"identity":"cc26e48c-96f7-47c1-9013-cc55ac17542a","added_by":"auto","created_at":"2025-03-04 03:21:02","extension":"tiff","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":6646858,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementaryfigure1.tiff","url":"https://assets-eu.researchsquare.com/files/rs-5070073/v1/0043fb401463834a9bd296be.tiff"},{"id":77655142,"identity":"0aff6ea9-7f81-4def-9ae1-8eba6c247b19","added_by":"auto","created_at":"2025-03-04 03:13:02","extension":"pdf","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":858277,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryinfonNOSfinalCommbio.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5070073/v1/93b3e6423c55d71784ffea51.pdf"}],"financialInterests":"There is \u003cb\u003eNO\u003c/b\u003e Competing Interest.","formattedTitle":"Ventromedial hypothalamus (VMHvl) nNOS neurons regulate social behaviors in a sex-specific manner","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eSocial behaviors such as mating, aggression, and prosocial approach are considered innate behaviors as they are displayed readily without the need for previous experience[1\u0026ndash;3]. In general, those behaviors rely on stereotypical motor patterns and are embedded in developmentally hardwired neural circuits[2], which are known to exhibit strong sex differences[2\u0026ndash;6] and are shaped by internal and external factors[1,4,6] such as reproductive states[2\u0026ndash;5,7]\u0026nbsp;and social experience[1,6,8\u0026ndash;10], respectively.\u003c/p\u003e\n\u003cp\u003eParticularly hypothalamic areas have been strongly associated with the display of instinctive behaviors such as mating[3,11,12]\u0026nbsp;and fighting[4,13,14]. \u0026nbsp; Of note estrogen receptor \u0026alpha; (ER\u0026alpha;) and progesterone receptor (PR)-positive neurons (expressed in a ratio of 1:1[15,16]) in the ventrolateral part of the ventromedial nucleus of the hypothalamus (VMHvl) are crucial for the display of aggressive and sexual behavior in both sexes[15\u0026ndash;19]. Although the role of ER\u0026alpha;-PR neurons (accounting for 40-50% of total neurons in the VMHvl) on sociosexual behaviors has been extensively studied, the participation of other neuronal populations within the VMHvl, which is a highly heterogeneous and sexually differentiated region\u003csup\u003e\u0026nbsp;\u003c/sup\u003e[20], remains understudied.\u003c/p\u003e\n\u003cp\u003eOf note, neuronal nitric oxide synthase (nNOS) neurons are found in the VMHvl[21] and are known to co-express PR in female mice\u003cspan lang=\"EN-US\"\u003e[22]\u003c/span\u003e. Furthermore, compelling evidence has tied nNOS expression and possibly nitric oxide (NO) synthesis to a sex-specific regulation of social behaviors. Briefly, nNOS knockout (KO) males exhibit abnormal sexual behavior (excessive mounting) and exaggerated aggression compared to wild types (WT)[23,24] whereas females showed reduced lordosis\u003cspan lang=\"EN-US\"\u003e[25]\u003c/span\u003e and maternal aggression\u003cspan lang=\"EN-US\"\u003e[26]\u003c/span\u003e.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003ePharmacological evidence further demonstrated the role of nNOS activity and possibly NO signaling in social behaviors. In male mice, pharmacological inhibition of nNOS activity increased and decreased aggression depending on factors such as social experience\u003cspan lang=\"EN-US\"\u003e[24,27]\u003c/span\u003e and synaptic plasticity\u003cspan lang=\"EN-US\"\u003e[28]\u003c/span\u003e. In contrast to KO studies, inhibition of nNOS activity decreased sexual behavior in naive male rats without affecting sexually experienced rats\u003cspan lang=\"EN-US\"\u003e[29]\u003c/span\u003e. On the other hand, in females, KO and pharmacological studies seem to be aligned as inhibition of VMHvl-nNOS neurons\u003cspan lang=\"EN-US\"\u003e[22]\u003c/span\u003e or nNOS activity\u003cspan lang=\"EN-US\"\u003e[30,31]\u003c/span\u003e in the VMHvl both reduced lordosis. Yet the role of nNOS neurons in virgin female aggression\u003cspan lang=\"EN-US\"\u003e[4]\u003c/span\u003e as well as the participation of specific nNOS populations in various social behaviors such as social motivation, mate choice, mating, and fighting in a sex-specific context remains elusive.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eHere we first assessed whether nNOS expression in the VMHvl overlaps with the well-characterized ER\u0026alpha; population in a sex-specific manner. Next, we exposed mice of both sexes to different social stimuli in order to trigger different behavioral responses and measured co-expression of nNOS and neuronal activity markers with different temporal dynamics, i.e. c-Fos (mating) and pERK (fighting), taking into account the effect of reproductive states and social experience. Finally, in the last set of experiments, we performed loss of function experiments using nNOS::cre mice and viral injections to specifically ablate nNOS neurons within the VMHvl and measure its consequences on behavior as well as used NO donors to rescue the behavioral deficits induced by deletion.\u003c/p\u003e"},{"header":"2. Material and Methods","content":"\u003cdiv id=\"Sec2\" class=\"Section2\"\u003e \u003ch2\u003e2.1. Animals\u003c/h2\u003e \u003cp\u003eExperiments were carried out in adult (8\u0026ndash;12 weeks) male and female mice, which were either purchased from Charles River Laboratories (Les Oncins, France) or bred in the animal facilities of the University of Li\u0026egrave;ge. Viral ablation experiments were conducted in nNOS::Cre mice (Figs.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e,\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e and Supplementary Table\u0026nbsp;3) whereas immunohistochemistry experiments were conducted in C57BL6/J mice (Figs.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e, \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e, and Supplementary Fig.\u0026nbsp;1). For aggressive behavior testing, adult male (5\u0026ndash;6 weeks) and juvenile female (p16-21) CD-1 mice were used as intruders for male and female residents, respectively. For sexual behavior receptive (estrous) females (6\u0026ndash;8 weeks) and experienced breeders (8\u0026ndash;12 weeks old) C57BL6/J were used as stimuli to test male and female subjects, respectively.\u003c/p\u003e \u003cp\u003eAll stimulus animals were obtained via Charles Rivers Laboratories (Les Oncins, France). Mice were kept under controlled laboratory conditions in ventilated shelves with (12:12h light/dark cycle; lights off at 10:30h, 21\u0026thinsp;\u0026plusmn;\u0026thinsp;1\u0026deg;C, 60\u0026thinsp;\u0026plusmn;\u0026thinsp;5% humidity, standard mouse nutrition (RM3), and water ad libitum). Stimulus and experimental animals were housed in large mouse cages (42x32x18cm) with sawdust bedding in groups of four. Importantly, after viral injection and prior resident or female intruder (RIT or FIT) tests, subjects were singly housed in individual mouse cages (33x16x13cm) for at least one week to induce territoriality. All procedures described here were approved by the local Animal Ethics Committee of the University of Li\u0026egrave;ge (protocol numbers 22-2469 and 2195).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.2. Female, Maternal defense, and Resident intruder tests (FIT, MDT, and RIT)\u003c/h2\u003e \u003cp\u003eAll tests took place in the early dark phase under dim red light conditions. Male mice were confronted with an unfamiliar same-sex intruder for 10 minutes. Intruders were 10\u0026ndash;20% lighter than residents [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e, \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. Behavior was scored live and later via videos by an observer blind to the experimental condition using JWatcher event recorder Program [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e, \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. As described elsewhere[\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e] the percentage of time spent on four major sets of behaviors was scored: i) aggressive behavior, consisting of attacks, threats, chases, tail rattles, offensive grooming, offensive up-right; ii) neutral behaviors, consisting of exploring (investigating the home-cage), drinking and eating, autogrooming, immobility; iii) social behaviors (non-aggressive social interactions, sniffing, following); and iv) defensive behavior (submissive posture, kicking a pursuing intruder with hind limb). In addition, we scored the frequency of attacks as well as the latency of the first attack.\u003c/p\u003e \u003cp\u003eFemales were typically confronted with a juvenile (p16-21) CD-1 female for 10 min, following a combination of the protocols described by Oliveira et. al., 2021[\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e] and Hashikawa et. al, 2017[\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. Typically females did not display attack bites[\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e] unless they were single-housed following Oliveira et. al, 2021 (Supplementary Fig.\u0026nbsp;1, Supplementary video). Therefore we scored all the non-aggressive behaviors mentioned above for males. However aggressive/dominant behavior consisted of the percentage of time spent on threatening, pushing/shoving, mounting, chasing, and, eventually biting the intruder. For lactating females aggressive behavior was scored similarly to males.\u003c/p\u003e \u003cp\u003eMaternal aggression was measured using the maternal defense test as described elsewhere[\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. In brief, females were paired with experienced males for 1 week, and vaginal plugs were assessed to confirm pregnancy. Maternal aggression was tested by introducing a virgin female into the lactating female home cage between lactation day 4 (LD4) and LD7, for 10 min.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.3. Sexual Behavior\u003c/h2\u003e \u003cp\u003eSexual behavior was measured as previously described[\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e, \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. Briefly, Sexual behavior was videotaped and scored live. Tests also took place in the early hours of the dark phase under dim red light conditions. Typically, for male sexual behavior, male mice were paired with a receptive (estrus) naturally cycling C57BL6/J mouse in their home cage and left free to interact for 10 or 30 minutes, depending on the test day (Please see Figs.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e and \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). Latencies for the first mount and first intromission as well as frequencies of mounts and intromissions were scored. The female estrous cycle was monitored via vaginal smears and histology. Additionally, receptivity was confirmed prior to the experiment by pairing the estrous females with experienced males.\u003c/p\u003e \u003cp\u003eFemale sexual behavior was assessed via the lordosis quotient (%). For neuronal ablation, experimental females were ovariectomized and implanted with a Silastic capsule containing crystalline 17-β-estradiol (diluted 1:1 with cholesterol) subcutaneously, followed by treatment with 500 \u0026micro;g progesterone 3 hours prior to the sexual behavior test. This protocol was chosen to enable optimal levels of sexual receptivity in order to evaluate neuronal ablation effects. For neuronal activity experiments, naturally cycling and receptive females were tested. Importantly during the sexual behavior test, females were paired with experienced males, and the amount of lordosis and rejections displayed in response to male mount attempts was annotated. The test was terminated after 10 mounts or 10 min whatever came first.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.4. Mate and social preference\u003c/h2\u003e \u003cp\u003eMate and social preferences were assessed using a 3-chamber apparatus[\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan additionalcitationids=\"CR39 CR40\" citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]. Briefly, the test consisted of 3 phases (each of which lasted 10 min). Habituation consisting of free investigation of the 3-chamber apparatus. Whereas during social preference experimental animals had to choose between an object and an adult unknown same-sex-conspecific, preference was calculated as an index of time investigating the conspecific/(time spent sniffing the conspecific\u0026thinsp;+\u0026thinsp;object). Directly after the social preference test, animals were given the option to choose between a same- and opposite-sex conspecific (mate preference), and preference was calculated as an index of time spent investigating the opposite-sex target/(time spent sniffing the same\u0026thinsp;+\u0026thinsp;opposite sex). Preference was analyzed using a one-sample t-test, and animals were assumed to prefer once their index differed from chance levels (0.5). Mice that did not leave the center chamber or spent more than 5 minutes in the center chamber were excluded from the analysis.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.5. Experiment overview\u003c/h2\u003e \u003cp\u003e \u003cb\u003eExperiment A (\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e\u003cb\u003e) Neuronal activity upon social encounter\u003c/b\u003e\u003c/p\u003e \u003cp\u003eMice of both sexes were confronted either with an object (control) or an opposite-sex-conspecific for 15 min (mating). One hour later, those animals were again exposed to an object (controls) or a same-sex-conspecific (fighting). Importantly, males were confronted with smaller adult male mice whereas females were confronted with juvenile females[\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e] for 10 min (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). Directly after the last interactions, animals were transcardially perfused with 4% paraformaldehyde (PFA) and had their brains collected and processed for immunohistochemistry targeting neuronal activity markers with different expression/phosphorylation dynamics, e.g. c-FOS for mating and pERK for fighting. Of note, both markers are known to be triggered by social interactions[\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e, \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e, \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e] including sexual and aggressive behavior, similar approaches have been used by others[\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e, \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eLactating[\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e] and socially isolated and aggression-trained (IST) females[\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e, \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e, \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e] were used as high female aggression controls. Briefly, IST was followed as described elsewhere[\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. Female C57BL6/J mice (8\u0026ndash;12 weeks) were single-housed for 3 weeks and after social isolation confronted with a same-sex and juvenile intruder for 4 consecutive days in the FIT. Two cohorts were trained in this schedule the first one was used to confirm aggression/dominant behavior levels and detailed behavioral analysis as well as activation of the oxytocin system (Supplementary Fig.\u0026nbsp;1), as oxytocin has been shown to promote aggression in females[\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e, \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e, \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e, \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e]. The second cohort of IST females was confronted with a juvenile and perfused directly after behavioral testing to verify pERK phosphorylation of VMHvl-nNOS neurons upon fighting (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA).\u003c/p\u003e \u003cp\u003eAs lactating females are known to exhibit high levels of aggression[\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e], they were confronted between LD 04\u0026ndash;07 with an adult virgin female and perfused directly after the maternal defense test to check for pERK phosphorylation after fighting (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA).\u003c/p\u003e \u003cp\u003e \u003cb\u003eExperiment B (\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e\u003cb\u003e) VMHvl-nNOS ablation in male mice\u003c/b\u003e\u003c/p\u003e \u003cp\u003eMale nNOS::Cre mice underwent stereotaxic surgery and were injected into the VMHvl bilaterally with either AAV1-flex-taCasp3-TEVp (ablation, 6.4 \u0026times; 10\u003csup\u003e13\u003c/sup\u003e genomic copies per ml) or AAV1-flex-EF1a-EGFP-WPRE (control, 7.7 \u0026times; 10\u003csup\u003e13\u003c/sup\u003e genomic copies per ml) virus. After 3 weeks, animals underwent behavioral testing to measure different aspects of social behavior. First, sexual behavior was assessed by exposing experimental males to receptive females (in their home cage) for 10 min on 3 consecutive days. Directly after mating animals were then confronted with a a male in their home cage for 10 minutes, sexual behavior testing prior to aggression was used to induce male territoriality[\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e, \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. Additionally, 5 days after the last RI, males were tested in a 30-minute mating test,. Finally, 4 days after sexual behavior testing, animals underwent the 3-chamber test to measure social and mate preference. After behavioral testing animals were transcardially perfused with 4% PFA and brains were harvested and used to confirm neuronal ablation via immunohistochemistry (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA).\u003c/p\u003e \u003cp\u003e \u003cb\u003eExperiment C (\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e\u003cb\u003e) VMHvl-nNOS ablation in female mice\u003c/b\u003e\u003c/p\u003e \u003cp\u003eFemale nNOS::Cre mice (8\u0026ndash;12 weeks) underwent stereotaxic surgery and were injected into the VMHvl bilaterally with either AAV1-flex-taCasp3-TEVp (ablation) or AAV1-flex-EF1a-EGFP-WPRE (control) virus. After 3 weeks, animals underwent behavioral testing to measure different aspects of social behavior. First, all-natural cycling (intact) female mice underwent the 3-chamber test to measure social and mate preference. After the 3-chamber test took place, subjects were split into two cohorts. Cohort A was ovariectomized and implanted with a Silastic capsule containing crystalline 17-β-estradiol (diluted 1:1 with cholesterol) subcutaneously, followed by treatment with 500 \u0026micro;g progesterone 3 hours prior sexual behavior test. Females were tested 3 times with a 4-day interval in between sexual behavior sessions. Cohort B remained ovary intact and underwent 3 FITs in 3 consecutive days. Finally, after the behavior tests were completed, mice from both cohorts were transcardially perfused and brains were harvested and used to confirm neuronal ablation via immunohistochemistry (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA).\u003c/p\u003e \u003cp\u003e \u003cb\u003eExperiment D rescue\u003c/b\u003e \u003c/p\u003e \u003cp\u003enNOS::Cre mice underwent stereotaxic surgery and were injected into the VMHvl bilaterally with the AAV1-flex-taCasp3-TEVp virus. After 3 weeks, males underwent sexual and aggressive behavior testing whereas females were only tested for sexual behavior. To try to rescue these behavioral impairments we used a combination of NO donor (SNAP) and a guanylate cyclase activator (BAY) 2 hours before the behavioral testing in a within-subjects design[\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. Therefore animals were tested for each behavior three times, once to establish baseline levels of behavior and counterbalance drug and vehicle groups (not shown) twice with intraperitoneal (i.p.) injections.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e2.6. Stereotaxic surgery\u003c/h2\u003e \u003cp\u003eStereotaxic surgery was performed under semi-sterile conditions[\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e, \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. For detailed information please see supplementary information.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e2.7. Pharmacology\u003c/h2\u003e \u003cp\u003eMice were injected intraperitoneally with a cocktail of S-NITROSO-N-ACETYL-DL-PENICIL LAMININE (SNAP, N3398, Sigma-Aldrich) and BAY 41-2272 (BAY, B8810, Sigma-Aldrich), in a dose of 8mg/Kg and 10mg/Kg, respectively 2 hours prior behavioral testing[\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003e2.8. Immunohistochemistry\u003c/h2\u003e \u003cp\u003eFor information regarding immunohistochemistry protocols and antibody concentrations please see supplementary information.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003e2.9. Statistical Analysis\u003c/h2\u003e \u003cp\u003eNormality was tested using the Kolmogorov-Smirnov test. Once normality was found data was analyzed using a Student\u0026rsquo;s t-test, Pearson\u0026rsquo;s correlations, and Two-way ANOVAs. Once normality was not reached Mann-Whitney and Spearman correlations were performed. For detailed statistics please see Supplementary Tables\u0026nbsp;1\u0026ndash;4.\u003c/p\u003e \u003c/div\u003e"},{"header":"3. Results","content":"\u003cp\u003e \u003cb\u003eVMH-vl nNOS neurons showed a sex-specific co-expression with Erα\u003c/b\u003e \u003c/p\u003e \u003cp\u003eWe confirmed the sex-dimorphic expression of ERα in the VMHvl, i.e. females show a higher number of ERα\u003csup\u003e+\u003c/sup\u003e-cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA)[\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e]. Additionally, we confirmed previous reports on the absence of sex differences in nNOS neurons within the VMHvl[\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. Nevertheless, we found some sex differences when looking into the co-expression of those two proteins. In females, around 60% of all nNOS neurons co-expressed ERα compared to 40% in males (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA, Supplementary table 1). Taking together these data revealed that nNOS neurons do not overlap completely with the well-characterized PR/ERα-VMHvl population[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. They rather constitute a subpopulation of ERα neurons similar to other genes such as cckar[\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e, \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e] or npy2r[\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e, \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e] populations. Additionally, these data showed that approximately half of nNOS neurons co-express estrogen receptors which have been shown to be essential for the display of social behaviors in rodents[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eFinally, we noticed that most nNOS and nNOS-ERα neurons were localized in the posterior-lateral part (pvll) of the VMHvl of females (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC), a subregion present only in female mice that have been linked with female sexual but not aggressive behavior[\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e, \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e].\u003c/p\u003e \u003cp\u003e \u003cb\u003eVMH-vl nNOS neurons exhibit a sex-specific activation pattern after same- and opposite-sex social stimulation\u003c/b\u003e \u003c/p\u003e \u003cp\u003eNext we investigated whether different social stimuli recruit VMHvl-nNOS neurons in a sex-specific manner (ExperimentA and Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA).\u003c/p\u003e \u003cp\u003eRegarding females, measuring aggressive behavior in virgin female mice is challenging[\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e], as virgin female C57 mice do not readily display aggressive behavior[\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. Thus, we decided to use two animal models of exacerbated aggression as positive controls of heightened female aggression: lactating females[\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e] and IST females[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. The IST protocol entailed high although variable levels of aggression in a separate cohort of animals (Supplementary Fig.\u0026nbsp;1A and Supplementary Video 1); IST females also showed increased activation of oxytocin neurons in the paraventricular (PVN) and supraoptic nucleus of the hypothalamus (Supplementary Fig.\u0026nbsp;1B), OXT-c-FOS percentage of colocalization in the PVN also positively correlated with percentage of time spent on aggression (Pearson\u0026rsquo;s Correlation PVN: r\u0026thinsp;=\u0026thinsp;0.867, p\u0026thinsp;=\u0026thinsp;0.0005; SON: r\u0026thinsp;=\u0026thinsp;0.528, p\u0026thinsp;=\u0026thinsp;0.09). Those results are in line with previous reports on oxytocin release having pro-aggressive effects in female rodents, independent of the reproductive state[\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e, \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e, \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e, \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eRegarding males exposed to mating and fighting, we found that around 11% of the nNOS neurons responded to both social stimuli indistinctly whereas 15% of the neurons colocalized only with pERK (putatively fighting neurons) and 19% colocalized only with c-FOS (putatively mating neurons). Those results showed that around half of VMHvl-nNOS neurons respond to both social stimuli and that those neurons seem to segregate into different neuronal populations (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB-D, Supplementary Table\u0026nbsp;1).\u003c/p\u003e \u003cp\u003eFemales exposed to males (who displayed lordosis behavior) showed an increased colocalization of nNOS and cFOS in the VMHvl compared to animals exposed to an object (on average 14% of nNOS neurons were recruited). Interestingly, exposition to a same-sex juvenile did not increase neuronal activity in VMHvl-nNOS neurons of females (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eE and F). Accordingly, neither IST females nor lactating females showed any differences in the colocalization of nNOS and pERK in response to an aggressive encounter (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eG). Taken together, these data suggest a sex difference in how nNOS neurons are recruited by social stimuli.\u003c/p\u003e \u003cp\u003e \u003cb\u003eAblation of VMH-vl nNOS neurons strongly reduces aggression and mildly affects sexual behavior in males\u003c/b\u003e \u003c/p\u003e \u003cp\u003eTo confirm the involvement of nNOS neurons in different aspects of social behaviors, we ablated VMHvl-nNOS neurons and measured different social behaviors in males (ExperimentB and Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA).\u003c/p\u003e \u003cp\u003eWe confirmed that nNOS deleted (nNOS-del) mice had a 3-fold decrease in the number of nNOS neurons in the VMHvl compared to controls (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB and F). Regarding behavioral data, ablated mice exhibited a lower percentage of time on aggression, threat, and attacking the intruder. Additionally, nNOS ablation almost suppressed attack bites as only 40% of the nNOS-del animals displayed attacks in at least one of the 3 RIs, whereas, in the control group, all animals attacked the intruder at least once. Consequently, nNOS-del mice showed a reduced number of attacks and an increased latency to attack the intruder (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB). Finally, the percentage of time spent on aggression (3rd RI) correlated with the number of nNOS neurons in the VMHvl (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB). Interestingly, the effect of nNOS deletion could not be compensated or reversed by re-engaging in aggressive interactions as nNOS-del animals did not show an increase in aggressive behavior after multiple RIs. Furthermore, reduced aggression could not be associated with a lack of social motivation as nNOS-del animals preferred social stimuli over an object (displaying social preference) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC) and did not show any effects on social investigation in the RI (Supplementary Table\u0026nbsp;2).\u003c/p\u003e \u003cp\u003eAblation of nNOS neurons mildly affected mounts and intromissions as well as intromission latency when animals were tested for 10 minutes (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD). Additionally, successive testing seemed to recover the effect of deletion as nNOS-del animals showed an increased number of mounts/intromissions on Test 3 compared to Test 1, consequently, there was no difference between nos-del and controls on Test 3, regarding those parameters (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD). As males did not show a strong phenotype after short sexual behavior tests we decided to challenged them by performing a 30 min session. Under those conditions, NOS-del mice displayed a lower number of mounts (p\u0026thinsp;=\u0026thinsp;0.05) and intromissions as well as a longer latency to mount the receptive female. Accordingly, the number of nNOS neurons in the VMHvl correlated with the number of mounts\u0026amp;intromissions (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eE). Accordingly, nNOS-del animals showed impaired mate preference, meaning that they did not prefer an estrous female over a male mouse (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC). Taking together these data suggests that nNOS neurons might not be directly involved with the display of sexual behavior (consummatory behavior) but rather with female recognition (appetitive behavior) in males (Supplementary Table\u0026nbsp;2).\u003c/p\u003e \u003cp\u003e \u003cb\u003eDeletion of VMH-vl nNOS neurons impairs female sexual behavior and social motivation\u003c/b\u003e \u003c/p\u003e \u003cp\u003eNext, we performed similar experiments in female nNOS::Cre mice and evaluated different aspects of social interactions (Experiment C and Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA).\u003c/p\u003e \u003cp\u003eNeuronal ablation was confirmed as nNOS-del females showed a reduced number of nNOS neurons in the VMHvl compared to controls (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eF). Additionally, we did not find an effect of hormonal treatment or estrous cycle on the number of nNOS neurons (Supplementary Table\u0026nbsp;3). Surprisingly, we found strong sex differences in the role of nNOS neurons on social behaviors matching our previous findings with neuronal activity. In contrast with males, neuronal ablation did not affect female aggressive/dominant behaviors toward a juvenile (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB). However, social preference was abolished in nNOS-del females (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC).\u003c/p\u003e \u003cp\u003eConcerning reproductive behaviors, nNOS ablation in the VMHvl nearly abolished lordosis behavior in females. Of note, successive behavior testing could not rescue the effect of neuronal ablation on lordosis. Accordingly, the number of nNOS neurons in the VMHvl correlated with the lordosis quotient (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eE). Finally, the effects of nNOS deletion in females could not be tied to an impairment in mate preference[\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e], as mate preference remained unchanged in nNOS-del females. In fact, those females preferred the male over the female even in the non-receptive phases of the estrous cycle (metestrus-diestrus) (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC), indicating that ablation enhanced male preference in the absence of hormonal priming.\u003c/p\u003e \u003cp\u003e \u003cb\u003eIncreasing NO availability did not rescue the effects of VMHvl-nNOS ablation\u003c/b\u003e \u003c/p\u003e \u003cp\u003eIn the last set of experiments, we attempted to disentangle the role of NO and nNOS neurons in social behavior, to do so we combined viral ablation, pharmacology and behavior (Experiment D).\u003c/p\u003e \u003cp\u003eDeleted males displayed a reduced number of mounts\u0026amp;intromissions and attacks as well as spent a lower percentage of time on aggressive behavior, those effects could not be rescued by the administration of SNAP-BAY. Females also exhibited reduced lordosis quotients, this phenotype could not be rescued by SNAP-BAY (Supplementary Table\u0026nbsp;4). Those results indicated that nNOS neuron deletion rather affects the social behavior circuit instead of NO neurotransmission in the VMHvl.\u003c/p\u003e"},{"header":"4. Discussion","content":"\u003cp\u003eA myriad of behaviors ranging from social investigation and affiliative approach to mating and fighting emerge from interactions between conspecifics. Generally, those behavioral responses rely on several factors such as conspecific cues, internal states marked for physiological/hormonal alterations, and previous experiences [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e]. In this manuscript, we investigated how VMHvl-nNOS neurons are affected by same- and opposite-sex conspecific cues as well as how ablation of those neurons affects the display of social behaviors itself.\u003c/p\u003e \u003cp\u003eOur data revealed that nNOS neurons in the VMHvl are recruited in a sex-specific manner by male and female cues. In females, those neurons were exclusively activated by mating (male cues) whereas in males both mating with a receptive female and fighting with a smaller male intruder increased the activation of VMHvl-nNOS neurons. Interestingly, those neurons were not activated in lactating females[\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e] or highly aggressive IST females[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e, \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e], known to display exacerbated levels of aggression, indicating that the absence of activity of those neurons in females is not reproductive state, social experience, or aggression level-dependent. Furthermore, this is consistent with the majority of the nNOS neurons being located in the VMHvlpvl, tied to sexual but not aggressive behavior in females[\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e, \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eSurprisingly, the ablation of VMHvl-nNOS neurons in males nearly abolished aggressive behavior without affecting social preference. Although the loss of function data matches the neuronal activity after aggression, these data are rather unexpected. Previous studies using nNOS-KOs have shown that those animals exhibit exaggerated aggressive behavior[\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. Pharmacological inhibition of nNOS activity systemically also strongly increased aggression in male mice, particularly when those animals were single-housed[\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. Furthermore, nNOS enzymatic inhibition also decreased social investigation in single-housed mice[\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. Conversely, a recent study has found that inhibition of NMDAR-dependent PSD95/nNOS activity either i.p. or intracerebroventricularly (i.c.v.) strongly decreases social isolation-induced aggression in male mice, similar to our findings[\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. Pretreatment with L-arginine a precursor of NO prevented the effect of PSD95/nNOS inhibition on aggression, indicating those effects were NO-dependent[\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eHere one should differentiate between the role of nNOS-positive neurons and nNOS enzymatic activity and consequently NO neurotransmission on aggression. Early, pharmacological[\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e] and KO[\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e] studies particularly focused on the latter whereas our study is centered on the neuronal populations expressing nNOS. In fact, elevating NO levels via the administration of a NO-donor failed to rescue the behavioral deficits seen in aggressive behavior in our study. This suggests that the effect on nNOS neuronal ablation is not mainly underlied by NO-transmission but rather by their role in the neuronal circuitry of aggression. Alternatively, another possible explanation would be that VMHvl-nNOS neurons are particularly engaged in the plasticity-dependent nNOS activity similar to the study of Yang et. al.[\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e], in fact, plasticity-dependent enhancement of aggressive behavior has been tied to the VMHvl in male winners[\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. Future studies should verify that hypothesis.\u003c/p\u003e \u003cp\u003eRegarding male sexual behavior, our data revealed a mild effect of nNOS neuron deletion in reducing the number of intromissions and the copulation latency when animals were tested for 30 minutes. On the other hand, multiple tests seemed to rescue the ablation effect on male sexual behavior partially. In line with our observations, a study performed in male rats using L-NAME (an inhibitor of nNOS) i.c.v. has shown that nNOS inhibition reduces mounting and abolishes ejaculation in na\u0026iuml;ve but not experienced rats[\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e], indicating sexual experience might overrule the effects of NO on mating. This mild effect of NO synthesis and VMHvl-nNOS neurons on male sexual behavior might arise from the participation of nNOS in the detection of female cues (appetitive phase) rather than copulation itself. Consistently, we have found that VMHvl-nNOS neurons are activated by female cues (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). Additionally, nNOS-del males exhibited impaired mate preference and took longer to intromit and mount (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). Thus, one could hypothesize that males display less consummatory behaviors because they struggle to identify the receptivity cues of a potential mate. Accordingly, nNOS-KO studies have shown that males usually display persistent mounting even towards non-receptive (anestrous) females, compared to WT[\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. Taking together with our findings, this indicates that NO signaling and nNOS neurons might be involved rather in mate detection/choice than copulatory behaviors per se. Furthermore, other nNOS neuronal populations might be involved in male copulation, for example sexually experienced rats showed increased nNOS mRNA in the PVN compared to na\u0026iuml;ve rats[\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIn contrast to males, VMHvl-nNOS neurons were only activated in females after mating with an adult male. Furthermore, females which were exposed to a male but did not display lordosis failed to show increased nNOS activation (not shown). Interestingly, exposition to a juvenile intruder which is known to trigger dominant behavior (mounts) in C57 female mice[\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e] as well as aggression displayed by lactating females[\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e] and IST females[\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e, \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e] did not increase pERK expression in VMHvl-nNOS neurons (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e), indicating that neither same-sex cues, dominant behavior, nor fighting recruits nNOS neurons in females. Accordingly, nNOS-del females did not show any differences in their levels of aggression\u0026amp;dominance (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eAccordingly, nNOS-KO virgin females do not show differences in the display of aggressive behavior[\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. Lactating nNOS-KO, on the other hand, showed a moderate reduction in maternal aggression[\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. Pharmacological studies in female prairie voles have shown similar, although milder effects. Inhibition of nNOS activity i.p. decreased maternal aggression after 2 but not 3 days of treatment[\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e]. Those results might indicate different mechanisms regulating the involvement of nNOS in aggressive behavior depending on the reproductive state. Another possible explanation is that other nNOS populations might be recruited during maternal aggression, indeed the number of nNOS neurons expressing citrulline (indirect NO synthesis marker) increased in lactating animals after an aggressive encounter, in regions known to regulated maternal aggression[\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e, \u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e], such as the medial preoptic area (MPOA) and the PVN (where citrulline cells correlated with the display of aggressive behavior)[\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e, \u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eWe also found that nNOS-del females did not show a social preference, which is in agreement with previous data in males demonstrating that inhibition of nNOS activity decreases exploration of male urine [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. Perhaps, the lack of dominant or aggressive behavior in virgin females arises from the fact those animals show low levels of motivation to interact and explore an intruder, although non-aggressive social investigation did not differ between control and nos-del females (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD).\u003c/p\u003e \u003cp\u003eFinally, nNOS-del females showed a striking reduction in lordosis behavior (nearly abolished) which could not be linked to an impairment in mate choice (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). Furthermore, in contrast to males, sexual experience did not rescue/compensate for the effect of VMHvl-nNOS deletion in females (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). These data align with i) our neuronal activity data showing that VMHvl-nNOS neurons are active after mating in females (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e), the localization of nNOS neurons in the VMHvlpvl (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e) and previous reports of our group using nNOS-KO[\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e] and behavioral pharmacology[\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e], as well as from another group showing that inhibition of VMHvl-nNOS neurons via chemogenetics strongly reduces lordosis in mice[\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. Surprisingly, although the nNOS-KO phenotype could be reversed by elevating NO levels using SNAP-BAY[\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e], the same treatment failed to rescue the lordosis deficit of nNOS-del females. This again indicates that this ablation might affect the circuitry of female sexual behavior[\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e] rather than NO-neurotransmission. However, further investigation is needed in order to disentangle local NO production and nNOS activity from nNOS neuron activity, as i.p. injections could act in other brain regions other than the VMHvl.\u003c/p\u003e \u003cp\u003eSurprisingly, nNOS-del diestrus females preferred a male over a female. This was unexpected, previous data from our lab shows that nNOS enzymatic activity is necessary for mate preference in females[\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e] additionally, male odors were able to activate nNOS neurons [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. Knowing that i) PR neurons and nNOS neurons are co-expressed[\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e], ii) PR is co-expressed in a ratio of 1:1 with ERα in the VMHvl[\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e] iii) PR-VMHvl neurons are involved in mate rejection[\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e] and iv) PR signaling in the olfactory system silences male cue detection during diestrus, leading to an absence of male preference [\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e], one could hypothesize that by ablating nNOS neurons we partially eliminate some of the PR (ERα) neurons that might either be involved in mate rejection and/or recognition, therefore females start to show a male preference irrespective of the estrous cycle phase. Future studies should investigate the link between mate preference, VMHvl-nNOS, PR- and ERα signaling.\u003c/p\u003e \u003cp\u003eFinally, our data confirm previous findings portraiting the VMHvl as a hotspot for social behavior. Additionally, we characterized a novel neuronal population (nNOS neurons) from the behavioral point of view. Our data show that nNOS neurons in the VMHvl show sex differences in co-expression with ERα as well as how they respond to social stimuli. In females, these neurons seem to preferably respond to male cues whereas in males to both sexes. Finally, male VMHvl-nNOS neurons seem to be crucial for aggressive behavior and mate choice whereas in females those neurons seem to be essential for sexual behavior and social motivation (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe would like to thank Laura de Vries, Chlo\u0026eacute; Beaudou, and Lozen Thies for their help with the experiments and Dr. Harold Gainer for giving us the anti-oxytocin antibody.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor Contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eV.O: Designed, performed, analyzed experiments, and wrote the manuscript, as well as designed figures and tables. I.B: Assisted with performing and analyzing behavioral experiments. JB: supervised the study, and gave input to experimental design and to the manuscript. \u0026nbsp;All authors contributed to the article and approved the submitted version.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting Interests and Funding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eJB is a Research Director of the FNRS, and she discloses a patent on J. Bakker and S. Ouerdi. \u0026quot;Agonists of human kisspeptin receptor for modulating sexual desire.\u0026quot; WO2020/151830A1 filed -1 Unspecified -001 and issued 30 July 2020. All authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. This work was supported by the Fonds de la Recherche Scientifique -FNRS Charg\u0026eacute; de Recherche (1.B.308.23AGGRESSIONKiNG project to V.O., by the Fondation L\u0026eacute;on Fredericq Bourse de fonctionnement Post-Doctorant (Neuromodulation of aggression by kisspeptin, neurokinin B, and GnRH to VO and the FNRS (PDR T.0015.20 to JB).\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eOliveira VE de M. Animal Models of Aggression. Handbook of Anger, Aggression, and Violence, Cham: Springer International Publishing; 2023. p. 1\u0026ndash;24.\u003c/li\u003e\n\u003cli\u003eWei D, Talwar V, Lin D. Neural circuits of social behaviors: Innate yet flexible. Neuron. 2021;109:1600\u0026ndash;1620.\u003c/li\u003e\n\u003cli\u003eLenschow C, Lima SQ. In the mood for sex: neural circuits for reproduction. Curr Opin Neurobiol. 2020;60:155\u0026ndash;168.\u003c/li\u003e\n\u003cli\u003eOliveira VE de M, Bakker J. Neuroendocrine regulation of female aggression. Front Endocrinol (Lausanne). 2022;13.\u003c/li\u003e\n\u003cli\u003eHashikawa K, Hashikawa Y, Lischinsky J, Lin D. The Neural Mechanisms of Sexually Dimorphic Aggressive Behaviors. Trends in Genetics. 2018;10:755\u0026ndash;776.\u003c/li\u003e\n\u003cli\u003eZilkha N, Sofer Y, Kashash Y, Kimchi T. The social network: Neural control of sex differences in reproductive behaviors, motivation, and response to social isolation. Curr Opin Neurobiol. 2021;68:137\u0026ndash;151.\u003c/li\u003e\n\u003cli\u003eLenz KM, Nugent BM, McCarthy MM. Sexual differentiation of the rodent brain: Dogma and beyond. Front Neurosci. 2012;6:1\u0026ndash;13.\u003c/li\u003e\n\u003cli\u003eOyegbile TO, Marler CA. Winning fights elevates testosterone levels in California mice and enhances future ability to win fights. Horm Behav. 2005;48:259\u0026ndash;267.\u003c/li\u003e\n\u003cli\u003eStagkourakis S, Spigolon G, Liu G, Anderson DJ. Experience-dependent plasticity in an innate social behavior is mediated by hypothalamic LTP. 2020. 2020. https://doi.org/10.1073/pnas.2011782117/-/DCSupplemental.\u003c/li\u003e\n\u003cli\u003eKoolhaas JM, Schuurman T, Wiepkema PR. The organization of intraspecific agonistic behaviour in the rat. Prog Neurobiol. 1980;15:247\u0026ndash;268.\u003c/li\u003e\n\u003cli\u003eYin L, Lin D. Neural control of female sexual behaviors. Horm Behav. 2023;151:105339.\u003c/li\u003e\n\u003cli\u003eHellier V, Brock O, Bakker J. The role of kisspeptin in sexual behavior. Semin Reprod Med. 2019;37:84\u0026ndash;92.\u003c/li\u003e\n\u003cli\u003eFalkner AL, Lin D. Recent advances in understanding the role of the hypothalamic circuit during aggression. Front Syst Neurosci. 2014;8:1\u0026ndash;14.\u003c/li\u003e\n\u003cli\u003eHaller J. The neurobiology of abnormal manifestations of aggression-A review of hypothalamic mechanisms in cats, rodents, and humans. Brain Res Bull. 2013;93:97\u0026ndash;109.\u003c/li\u003e\n\u003cli\u003eLee H, Kim D-W, Remedios R, Anthony TE, Chang A, Madisen L, et al. Scalable control of mounting and attack by Esr1+ neurons in the ventromedial hypothalamus. Nature. 2014;509:627\u0026ndash;632.\u003c/li\u003e\n\u003cli\u003eHashikawa K, Hashikawa Y, Tremblay R, Zhang J, Feng JE, Sabol A, et al. Esr1+cells in the ventromedial hypothalamus control female aggression. Nat Neurosci. 2017;20:1580\u0026ndash;1590.\u003c/li\u003e\n\u003cli\u003eLin D, Boyle MP, Dollar P, Lee H, Lein ES, Perona P, et al. Functional identification of an aggression locus in the mouse hypothalamus. Nature. 2011;470:221\u0026ndash;226.\u003c/li\u003e\n\u003cli\u003eYang CF, Chiang MC, Gray DC, Prabhakaran M, Alvarado M, Juntti SA, et al. Sexually dimorphic neurons in the ventromedial hypothalamus govern mating in both sexes and aggression in males. Cell. 2013;153:896\u0026ndash;909.\u003c/li\u003e\n\u003cli\u003eGutierrez-Castellanos N, Husain BFA, Dias IC, Nomoto K, Duarte MA, Ferreira L, et al. A hypothalamic node for the cyclical control of female sexual rejection. Neuron. 2024. November 2024. https://doi.org/10.1016/j.neuron.2024.10.026.\u003c/li\u003e\n\u003cli\u003eKammel LG, Correa SM. Selective sexual differentiation of neurone populations may contribute to sex ‐ specific outputs of the ventromedial nucleus of the hypothalamus. J Neuroendocrinol. 2020. 2020. https://doi.org/10.1111/jne.12801.\u003c/li\u003e\n\u003cli\u003eSica M, Martini M, Viglietti-Panzica C, Panzica GC. Estrous cycle influences the expression of neuronal nitric oxide synthase in the hypothalamus and limbic system of female mice. BMC Neurosci. 2009;10.\u003c/li\u003e\n\u003cli\u003eSilva MSB, Decoster L, Trova S, Mimouni NEH, Delli V, Chachlaki K, et al. Female sexual behavior is disrupted in a preclinical mouse model of PCOS via an attenuated hypothalamic nitric oxide pathway. Proc Natl Acad Sci U S A. 2022;119:1\u0026ndash;12.\u003c/li\u003e\n\u003cli\u003eNelson RJ, Demais GE, Huang PL, Fishman MC, Dawson VL, Dawson TM, et al. Behavioural abnormalities in male mice lacking neuronal nitric oxide synthase. Nature Letters. 1995;378:383\u0026ndash;386.\u003c/li\u003e\n\u003cli\u003eTrainor BC, Workman JL, Jessen R, Nelson RJ. Impaired Nitric Oxide Synthase Signaling Dissociates Social Investigation and Aggression. Behavioral Neuroscience. 2007;121:362\u0026ndash;369.\u003c/li\u003e\n\u003cli\u003eHellier V, Brock O, Candlish M, Desroziers E, Aoki M, Mayer C, et al. Female sexual behavior in mice is controlled by kisspeptin neurons. Nat Commun. 2018;9.\u003c/li\u003e\n\u003cli\u003eGammie SC, Nelson RJ. Maternal aggression is reduced in neuronal nitric oxide synthase- deficient mice. Journal of Neuroscience. 1999;19:8027\u0026ndash;8035.\u003c/li\u003e\n\u003cli\u003eDemas GE, Eliasson MJL, Dawson TM, Dawson VL, Kriegsfeld LJ, Nelson RJ, et al. Inhibition of neuronal nitric oxide synthase increases aggressive behavior in mice. Molecular Medicine. 1997;3:610\u0026ndash;616.\u003c/li\u003e\n\u003cli\u003eYang L, Cui J, Zeng L, Lu W. Targeting PSD95/nNOS by ZL006 alleviates social isolation-induced heightened attack behavior in mice. Psychopharmacology (Berl). 2022;239:267\u0026ndash;276.\u003c/li\u003e\n\u003cli\u003eBenelli A, Bertolini A, Poggioli R, Cavazzuti E, Calz\u0026agrave; L, Giardino L, et al. Nitric oxide is involved in male sexual behavior of rats. Eur J Pharmacol. 1995;294:505\u0026ndash;510.\u003c/li\u003e\n\u003cli\u003eBentefour Y, Bakker J. Kisspeptin signaling and nNOS neurons in the VMHvl modulate lordosis behavior but not mate preference in female mice. Neuropharmacology. 2021;198:108762.\u003c/li\u003e\n\u003cli\u003eBentefour Y, Bakker J. Stress during pubertal development affects female sociosexual behavior in mice. Nat Commun. 2024;15:3610.\u003c/li\u003e\n\u003cli\u003eMiczek KA, Maxson SC, Fish EW, Faccidomo S. Aggressive behavioral phenotypes in mice. Behavioural Brain Research. 2001;125:167\u0026ndash;181.\u003c/li\u003e\n\u003cli\u003eKoolhaas JM, Coppens CM, de Boer SF, Buwalda B, Meerlo P, Timmermans PJ. The resident-intruder paradigm: A standardized test for aggression , violence and social stress. Journal of Visualized Experiments. 2013;77:4367.\u003c/li\u003e\n\u003cli\u003eBlumstein DT, Evans CS, Daniel JC. JWatcher 0.9: An Introductory User\u0026rsquo;s Guide. Animal Behaviour Laboratory, Macquarie University. 2000.\u003c/li\u003e\n\u003cli\u003eOliveira VEM, Lukas M, Wolf HN, Durante E, Lorenz A, Mayer A-L, et al. Oxytocin and vasopressin within the ventral and dorsal lateral septum modulate aggression in female rats. Nat Commun. 2021;12:1\u0026ndash;15.\u003c/li\u003e\n\u003cli\u003eBosch OJ. Maternal aggression in rodents: Brain oxytocin and vasopressin mediate pup defence. Philosophical Transactions of the Royal Society B: Biological Sciences. 2013;368.\u003c/li\u003e\n\u003cli\u003eOliveira VEM, de Jong, Trynke, R, Neumann ID. Modelling sexual violence in male rats : the sexual aggression test ( SxAT ). Transl Psychiatry. 2022;12:1\u0026ndash;12.\u003c/li\u003e\n\u003cli\u003eOliveira VEM, Neumann ID, de Jong TR. Post-weaning social isolation exacerbates aggression in both sexes and affects the vasopressin and oxytocin system in a sex-specific manner. Neuropharmacology. 2019;156:107504.\u003c/li\u003e\n\u003cli\u003eCamats Perna J, Engelmann M. Recognizing others : Rodent\u0026rsquo;s social memories. In: W\u0026ouml;hr M. KS, editor. Curr Top Behav Neurosci, vol. 30, . Current ToSpringer, Cham; 2017. p. 25\u0026ndash;45.\u003c/li\u003e\n\u003cli\u003eEngelmann M, H\u0026auml;dicke J, Noack J. Testing declarative memory in laboratory rats and mice using the nonconditioned social discrimination procedure. Nat Protoc. 2011;6:1152\u0026ndash;1162.\u003c/li\u003e\n\u003cli\u003eKhbouz B, de Bournonville C, Court L, Taziaux M, Corona R, Arnal JF, et al. Role for the membrane estrogen receptor alpha in the sexual differentiation of the brain. European Journal of Neuroscience. 2020;52:2627\u0026ndash;2645.\u003c/li\u003e\n\u003cli\u003eTrainor BC, Crean KK, Fry WHD, Sweeney C. Activation of extracellular signal-regulated kinases in social behavior circuits during resident-intruder aggression tests. Neuroscience. 2010;165:325\u0026ndash;336.\u003c/li\u003e\n\u003cli\u003eDe Jong TR, Beiderbeck DI, Neumann ID. Measuring virgin female aggression in the Female Intruder Test (FIT): Effects of oxytocin, estrous cycle, and anxiety. PLoS One. 2014;9:e91701.\u003c/li\u003e\n\u003cli\u003eGrossmann CP, Sommer C, Fahliogullari IB, Neumann ID, Menon R. Mating-induced release of oxytocin in the mouse lateral septum: Implications for social fear extinction. Psychoneuroendocrinology. 2024;166.\u003c/li\u003e\n\u003cli\u003eKuiper LB, Lucas KA, Mai V, Coolen LM. Enhancement of Drug Seeking Following Drug Taking in a Sexual Context Requires Anterior Cingulate Cortex Activity in Male Rats. Front Behav Neurosci. 2020;14.\u003c/li\u003e\n\u003cli\u003eOliveira VEM, Jong TR De, Neumann ID. Synthetic Oxytocin and Vasopressin Act Within the Central Amygdala to Exacerbate Aggression in Female Wistar Rats. Front Neurosci. 2022;16:1\u0026ndash;12.\u003c/li\u003e\n\u003cli\u003eOliveira VE de M, Evrard F, Faure MC, Bakker J. Social isolation and aggression training lead to escalated aggression and hypothalamus-pituitary-gonad axis hyperfunction in mice. Neuropsychopharmacology. 2024. 2024. https://doi.org/10.1038/s41386-024-01808-3.\u003c/li\u003e\n\u003cli\u003eSofer Y, Zilkha N, Gimpel E, Wagner S, Chuartzman SG, Kimchi T. Sexually dimorphic oxytocin circuits drive intragroup social conflict and aggression in wild house mice. Nat Neurosci. 2024. 2024. https://doi.org/10.1038/s41593-024-01685-5.\u003c/li\u003e\n\u003cli\u003eCortes LR, Sturgeon H, Forger NG. Sexual differentiation of estrogen receptor alpha subpopulations in the ventromedial nucleus of the hypothalamus. Horm Behav. 2023;151.\u003c/li\u003e\n\u003cli\u003eYin L, Hashikawa K, Hashikawa Y, Osakada T, Lischinsky JE, Diaz V, et al. VMHvllCckar cells dynamically control female sexual behaviors over the reproductive cycle. Neuron. 2022. 21 September 2022. https://doi.org/10.1016/j.neuron.2022.06.026.\u003c/li\u003e\n\u003cli\u003eLiu M, Kim D-W, Zeng H, Anderson DJ. Make war not love: The neural substrate underlying a state-dependent switch in female social behavior. Neuron. 2022:1\u0026ndash;16.\u003c/li\u003e\n\u003cli\u003eTrainor BC, Kyomen HH, Marler CA. Estrogenic encounters: How interactions between aromatase and the environment modulate aggression. Front Neuroendocrinol. 2006;27:170\u0026ndash;179.\u003c/li\u003e\n\u003cli\u003eMcCarthy EA, Naik AS, Coyne AF, Cherry JA, Baum MJ. Effect of ovarian hormones and mating experience on the preference of female mice to investigate male urinary pheromones. Chem Senses. 2018;43:97\u0026ndash;104.\u003c/li\u003e\n\u003cli\u003eInsel TR. The Challenge of Translation in Social Neuroscience: A Review of Oxytocin, Vasopressin, and Affiliative Behavior. Neuron. 2010;65:768\u0026ndash;779.\u003c/li\u003e\n\u003cli\u003eGammie SC, Olaghere-Da Silva UB, Nelson RJ. 3-Bromo-7-nitroindazole, a neuronal nitric oxide synthase inhibitor, impairs maternal aggression and citrulline immunoreactivity in prairie voles. Brain Res. 2000;870:80\u0026ndash;86.\u003c/li\u003e\n\u003cli\u003eHasen NS, Gammie SC. Maternal aggression: New insights from Egr-1. Brain Res. 2006;1108:147\u0026ndash;156.\u003c/li\u003e\n\u003cli\u003eHasen NS, Gammie SC. Differential fos activation in virgin and lactating mice in response to an intruder. Physiol Behav. 2005;84:681\u0026ndash;695.\u003c/li\u003e\n\u003cli\u003eDey S, Chamero P, Pru JK, Chien MS, Ibarra-Soria X, Spencer KR, et al. Cyclic regulation of sensory perception by a female hormone alters behavior. Cell. 2015;161:1334\u0026ndash;1344.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"nature-portfolio","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"","title":"Nature Portfolio","twitterHandle":"","acdcEnabled":false,"dfaEnabled":false,"editorialSystem":"ejp","reportingPortfolio":"","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-5070073/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-5070073/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"Neuronal nitric oxide synthase (nNOS) neurons are ubiquitously spread in the rodent brain. Data using knockouts and pharmacology have revealed that nNOS is essential for the display of sexual and aggressive behavior. Yet, the specific neuronal populations regulating those behaviors remain elusive. Here, we aimed to study the role of the ventromedial hypothalamus (VMHvl)-nNOS neurons in social behaviors in both sexes. First, we evaluated whether the expression of nNOS overlaps with the well-characterized estrogen receptor alpha (ERα+)-VMHvl population. Next, we assessed how different social stimuli affected VMHvl-nNOS neurons' activity. Lastly, we used transgenic mice and viral approaches to ablate VMHvl-nNOS neurons and evaluate their impact on behavior. Our findings suggest that nNOS neurons constitute a small cluster within the VMHvl-ERα+ population which regulates social behaviors in a sex-specific manner. In males, those neurons seem to be essential for aggression whereas in females for sexual behavior and social motivation.","manuscriptTitle":"Ventromedial hypothalamus (VMHvl) nNOS neurons regulate social behaviors in a sex-specific manner","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-03-04 03:12:56","doi":"10.21203/rs.3.rs-5070073/v1","editorialEvents":[],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"communications-biology","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"commsbio","sideBox":"Learn more about [Communications Biology](http://www.nature.com/commsbio/)","snPcode":"","submissionUrl":"","title":"Communications Biology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Communications Series","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"05796d26-6b14-4e39-8072-be175d3c8d7b","owner":[],"postedDate":"March 4th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":44953047,"name":"Biological sciences/Neuroscience/Social behaviour"},{"id":44953048,"name":"Biological sciences/Neuroscience/Sexual behaviour/Sexual dimorphism"},{"id":44953049,"name":"Biological sciences/Neuroscience/Emotion/Aggression"},{"id":44953050,"name":"Biological sciences/Neuroscience/Neural circuits"},{"id":44953051,"name":"Biological sciences/Neuroscience/Sexual behaviour/Oxytocin"}],"tags":[],"updatedAt":"2025-12-03T08:10:50+00:00","versionOfRecord":{"articleIdentity":"rs-5070073","link":"https://doi.org/10.1038/s42003-025-09279-y","journal":{"identity":"communications-biology","isVorOnly":false,"title":"Communications Biology"},"publishedOn":"2025-12-01 05:00:00","publishedOnDateReadable":"December 1st, 2025"},"versionCreatedAt":"2025-03-04 03:12:56","video":"","vorDoi":"10.1038/s42003-025-09279-y","vorDoiUrl":"https://doi.org/10.1038/s42003-025-09279-y","workflowStages":[]},"version":"v1","identity":"rs-5070073","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-5070073","identity":"rs-5070073","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}