NPRC-Social isolation and aggression training lead to escalated aggression and hypothalamus-pituitary-gonad axis hyperfunction in mice

NPRC-Social isolation and aggression training lead to escalated aggression and hypothalamus-pituitary-gonad axis hyperfunction in mice | 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 NPRC-Social isolation and aggression training lead to escalated aggression and hypothalamus-pituitary-gonad axis hyperfunction in mice Vinícius Oliveira, Florence Evrard, Melanie Faure, Julie Bakker This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-3660090/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 09 Feb, 2024 Read the published version in Neuropsychopharmacology → Version 1 posted You are reading this latest preprint version Abstract Although the participation of sex hormones and sex hormone-responsive neurons in aggressive behavior has been extensively studied, the role of other systems within the hypothalamus-pituitary-gonadal (HPG) axis remains elusive. Here we assessed how the gonadotropin-releasing hormone (GnRH) and kisspeptin systems are impacted by escalated aggression in male mice. We used a combination of social isolation and aggression training (IST) to exacerbate mice’s aggressive behavior. Next, low-aggressive (group-housed, GH) and highly aggressive (IST) mice were compared regarding neuronal activity in the target populations and hormonal levels, using immunohistochemistry and ELISA, respectively. Finally, we used pharmacological and viral approaches to manipulate neuropeptide signaling and expression, subsequently evaluating its effects on behavior. IST mice exhibited enhanced aggressive behavior compared to GH controls, which was accompanied by elevated neuronal activity in GnRH neurons and arcuate nucleus kisspeptin neurons. Remarkably, IST mice presented an increased number of kisspeptin neurons in the anteroventral periventricular nucleus (AVPV). In addition, IST mice exhibited elevated levels of luteinizing hormone (LH) in serum. Accordingly, activation and blockade of GnRH receptors exacerbated and reduced aggression, respectively. Surprisingly, kisspeptin had intricate effects on aggression, i.e., viral ablation of AVPV-kisspeptin neurons impaired the training-induced rise in aggressive behavior whereas kisspeptin itself strongly reduced aggression in IST mice. Our results indicate that IST enhances aggressive behavior in male mice by exacerbating HPG-axis function. Particularly, increased GnRH neuron activity and peptide signaling were found to underlie aggression whereas the relationship with kisspeptin remains puzzling. Biological sciences/Neuroscience/Emotion Biological sciences/Neuroscience/Social neuroscience Biological sciences/Physiology/Neurophysiology Health sciences/Diseases/Psychiatric disorders Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 1. Introduction Aggressive behavior is an innate behavior displayed by animals in order to get access to important resources for their survival [1,2] . Apart from its ecological role, pathological and out-of-context aggression constitutes a core symptom of several psychiatric disorders such as conduct and antisocial personality disorders, schizophrenia, major depression, and post-traumatic stress disorder [3–7] . The fact that violence generates a severe socioeconomic burden on society [3,6] has led scientists to develop preclinical animal models [4,5,7] to understand the neural underpinnings of escalated aggression. Among those models, social isolation and engaging in successive aggressive experiences (aggression training) were found to entail robust effects on inducing exaggerated aggression [7,8] . Additionally, both conditions are known to disrupt social behaviors in humans by triggering psychological distress and violent behaviors [3,9–11] . Regarding neurobiological factors, attention has been given to several systems such as neuropeptides [4,8,12,13] , hypothalamic-pituitary-adrenal (HPA) axis [4,7] , and sex hormones [8,14] . Specifically, the role of sex hormones [8,14–19] and sex hormone-responsive neurons [8,20–23] has been extensively studied in the context of aggression. Yet, the participation of other brain hubs within the hypothalamus-pituitary-gonadal (HPG) axis which regulates sex steroid production and release remains largely unknown. Gonadotropin-releasing hormone (GnRH) neurons are classically found in the rostral preoptic area (rPOA), medial septum (MS), and anterior hypothalamus. Those neurons are the main node of reproduction in animals [24] as they act on the gonadotrophs in the anterior pituitary to trigger luteinizing hormone (LH) and follicle-stimulating hormone release into the circulation. Those glycoproteins will act at the level of the gonads to stimulate sex steroid production and release [24] . Thus, since aggressive behavior itself [25,26] as well as successive aggressive interactions and social isolation are known to increase plasma testosterone [8,14,17,18,25,26] and to some extent LH [25,27] (main hormonal output of GnRH neurons), one could hypothesize that GnRH neurons are recruited during aggressive interactions and might be impacted by the social context. Further evidence of the participation of GnRH in aggressive behavior comes from the finding that either immunocastration using vaccines targeting GnRH or chronic agonism of GnRH receptors (GnRHR), aiming to desensitize the HPG axis, reduce aggression in male pigs [28] and Macaques [29] , respectively. Similar effects have been described in a male patient with autism spectrum disorder and high aggression [30] . Importantly, GnRHRs are found in brain regions classically known to modulate aggression such as the lateral septum (LS), ventromedial hypothalamus (VMH), and the cortical amygdala (CoA) [31]. Of note, GnRH neuronal activity is regulated mainly by two neuropeptidergic populations: kisspeptin neurons in the anteroventral periventricular nucleus of the hypothalamus (AVPV) and K isspeptin- N eurokinin B (NKB)- D ynorphin ( KND y) neurons in the arcuate nucleus of the hypothalamus (ARC) [24,32–35] . Whereas the role of kisspeptin in male social behaviors remains understudied, the blockade of neurokinin receptor 3 (NK3R), which is co-expressed by KNDy neurons [35,36] , was shown to decrease aggression in highly aggressive socially isolated male mice [37] . Here we used a combination of social isolation and aggression training (IST) [12] to induce escalated aggression in male mice in order to evaluate its effects on GnRH, kisspeptin, and KNDy neuron activity as well as HPG-axis function. Additionally, we manipulated peptide signaling and expression via pharmacological and viral tools to assess its impact on aggressive and social behaviors. 2. Methods 2.1 Ethical statement All procedures described here were approved by the local Animal Ethics Committee of the University of Liège (protocol number 22-2469). 2.2 Animals Typically, experiments were carried out in adult (8-12 weeks) C57BL6/J male mice, which were purchased from Charles River Laboratories (Les Oncins, France). In particular, neuronal ablation experiments were conducted in Kiss::cre or WT mice (6-12 months) bred in the animal facilities of the University of Liège. For aggressive behavior experiments, male CD-1 mice (5-6 weeks) were used as intruders whereas for sexual behavior receptive (estrous) females C57BL6/J (6-8 weeks) were used. All stimulus animals were obtained via Charles Rivers Laboratories (Les Oncins, France). Subjects 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, during the experimental phase, mice were singly housed in individual mouse cages (33x16x13cm) in order to enhance their aggressive behavior. 2.3 Resident intruder test (RI) The Rl 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 weighed between 10-20% less than residents [8,38,39]. Behavior was scored live and posteriorly via videos by a blind observer using JWatcher event recorder Program [12,40]. As previously described [12] 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 to the first attack. 2.4 Sexual behavior Sexual behavior was conducted as previously described [41] . For detailed information please see supplementary information. 2.5 Experimental design 2.5.1 The social isolation and aggression training protocol (IST) The social isolation and aggression training (IST) protocol was adapted from the procedure developed by Oliveira and co-authors in female rats, which has shown to induce robust effects on exacerbating aggressive behavior [8,12,42,43] . Therefore, mice from all different strains used to perform this study were assigned to either the IST (high-aggression) or group-housed (GH, control, low-aggression) condition. IST subjects were kept single-housed for 3 weeks in individual cages and were subsequently exposed to 3 consecutive, 10-minute-long RIs, daily. Group-housed animals were kept in groups of 4 animals per cage and were single-housed 2 hours prior to lights off, on experimental days (Figure 1A). Importantly, in pharmacological experiments targeting the IST condition, levels of aggression between vehicle and drug groups were counterbalanced and randomized using the live scoring data of the previous training sessions, to make sure groups did not differ in terms of the total percentage of time spent on aggression, total number of attacks or attack latency before treatments (not shown). Finally, no crossover effects were found when comparing vehicle groups from different treatment days for both housing conditions. 2.5.2 Endogenous measurements Mice were split into two cohorts: cohort A (neuronal activity experiments; n=6) and cohort B (hormonal measurements n=8) (Figure 1A). After either being assigned to the IST or the GH condition animals underwent the protocols as described above (Figure 1A). Animals of Cohort A were then exposed to a 10-minute RI and left undisturbed in their home cage for 1 hour. Next, animals were deeply anesthetized with a solution of Euthasol (pentobarbital sodium, 200mg/Kg) and transcardially perfused using phosphate buffer (PBS, 0.1M) followed by paraformaldehyde (PFA, 4%). Brains were postfixed overnight in PFA 4% followed by dehydration in sucrose (30 %) for 2-3 days and snap-frozen in dry-ice and isopentane (Sigma-Aldrich, PHR166). Mice of Cohort B either were kept GH or underwent the IST protocol as described above. On the test day, those animals were confronted with an intruder (10 min), deeply anesthetized with Euthasol 10 min after the test, decapitated, and had their trunk blood collected. Brains were harvested, postfixed in PFA 4%, dehydrated in sucrose, and snap-frozen. Blood was stored in ice, centrifuged (4°, 1500 G, 15 min), and serum was stored at -20°C until hormonal assays took place. 2.5.3 Experiment A C57BL6/J mice underwent the IST protocol. After the third training session, on day 25 animals were injected intraperitoneally (i.p.) either with deslorelin (GnRH receptor agonist) or vehicle and tested on the RI 30 min after injection. After a one-day (day 27) washout (WO: no RI or i.p. injection) mice were tested again following a deslorelin or vehicle injection in a cross-over (within-subjects) design. Three days later (WO) subjects were then injected with either kisspeptin-10 or vehicle i.p. 2 hours before the RI. On day 32 (after 1-day WO) animals were again injected either with kisspeptin-10 or vehicle (i.p.) in a crossover (within-subjects) design. Finally on day 35, IST mice were either injected with vehicle or cetrorelix (GnRH receptor antagonist) 40min before the RI in a between-subjects design (Figure 3A). 2.5.4 Experiment B As deslorelin enhanced aggression in IST mice (Figure 4), we decided to test whether GnRHR agonism alone could enhance the mild levels of aggression displayed by GH mice. Thus, GH mice were also injected either with deslorelin or vehicle 30 min before the RI. After the experiment, animals returned to their original groups and left undisturbed for 1 day (WO). On day 3, GH mice were again single-housed (see above) and either injected with deslorelin or vehicle in a within-subjects design (Figure 3A). 2.5.5 Experiment C Similarly to Experiment A, mice underwent the IST protocol. On day 25, IST mice were either injected with senktide (NK3R agonist) or vehicle, 30 min prior to the RI. Following a 1-day washout, animals were again injected with sektide or vehicle in a cross-over within subjects design. On day 30 (after a 3 WO), IST mice were then injected with osanetant (NK3R antagonist) 30 min before the RI in a between-subjects design (Figure 3A). 2.5.6 Experiment D Kiss::cre or WT mice underwent stereotaxic surgery and were injected into the AVPV with AAV1-flex-taCasp3-TEVp (6.94 × 10 11 genomic copies per ml) in order to specifically ablate AVPV-kisspeptin neurons [22,44] . After 3 weeks (day 21), mice started the training phase of the IST protocol, by going through the RI for 3 consecutive days. Three days after the third RI (RI3) animals were allowed to interact with estrous/receptive females for 20-min. After a resting of 4 days, animals were again tested for sexual behavior (Sex 2, Figure 3A) and subsequently perfused to confirm viral ablation. WT mice were also compared with non-surgery animals (not shown, n=3) as there were no behavioral or neuronal number differences between non-surgery and WT those animals were pulled as the control group (Ctrl). 2.6 Stereotaxic surgery Stereotaxic surgery was performed under semi-sterile conditions and according to the procedures described in the animal license [44]. For details please see supplementary information. 2.7 Drugs For information regarding doses and time points please see supplementary information. 2.8 Immunohistochemistry After perfusion, brains were cryo-cutted and slices (40µm) containing the target regions were collected in cryoprotectant solution and stored at -20°C until the experiment took place. A series of 6-8 slices were used for immunostaining. For neuronal activity, slices were stained using the immunoperoxidase substrate method whereas for confirming cell ablation slices were stained using immunofluorescence (for protocol details please see Supplementary information). 2.9 Sex steroid assays Testosterone (Demeditec, DEV9911, detection limit 0.024ng/ml) and Progesterone (Demeditec, DEV9988, detection limit 0.0156 ng/ml) were measured via commercial ELISA kits following the specifics of those kits. 2.10 LH assay LH was measured in serum following the specifics of protocols previously described (for details please see [45,46] , for antibody concentrations please see Supplementary Table 4). Two animals were excluded from the final data analysis (one from the GH and one from the IST group). The earlier due to blood collection and processing issues (it was also a significant outlier) and the latter for being under the detection limit of the assay. 2.11 Statistics Normality was tested using the Kolmogorov-Smirnov test. Once normality was found, data were analyzed using a two-tailed Student’s t-test, Pearson’s correlations, and Two-way ANOVAs. Once normality was not reached, Mann-Whitney U-test, Wilcoxon matched pairs W test, and Spearman correlations were performed. Agonist experiments used within-subjects design whereas antagonist, endogenous measurements, and ablation used between-subjects. For detailed statistics please see Supplementary Tables 1,2 and 3. 3. Results Social isolation and aggression training exacerbate aggression in male mice and hyperactivate the HPG axis IST mice spent more time displaying aggressive behavior compared to GH controls which was reflected in a higher percentage of time displaying aggression, threatening and attacking the intruder as well as a higher number of attacks and decreased latency to attack (Figure 1A). Also, all IST animals displayed attacks whereas only 16% of the GH mice attacked the intruder (Figure 1A, for detailed behavior please see Supplementary Table 1). Regarding neuronal activity, IST animals showed increased colocalization of c-FOS and GnRH in the MS (Figure 1B) and rPOA (1C). Particularly MS-GnRH neuronal activity positively correlated with the percentage of time displaying aggression (Supplementary Table 5). AVPV-kisspeptin neurons, on the other hand, showed very low levels of co-localization with c-FOS. Nevertheless, IST animals exhibited a higher number of kisspeptin neurons in the AVPV compared to GH controls (Figure 1D). Finally, KNDy neurons (NK3R-positive) in the ARC were also found to present higher colocalization with c-FOS, which positively correlated with aggression (Figure 1E, Supplementary Table 5). Finally, as NKB signaling in hypothalamic regions has been associated with enhanced aggression in isolated animals (36), we evaluated NK3R-colocalization in the dorsomedial (DMH) and lateral hypothalamus (LHp), regions known to be involved in the display of aggressive behavior [37,47] . NK3R-positive neurons in both regions showed an increased colocalization with c-FOS in IST mice compared to GH controls (Supplementary Figure 1). In addition, the percentage of time spent on aggressive behavior positively correlated with aggression in both regions (Supplementary Table 5). As the hypothalamic hubs regulating the HPG axis were found to be either highly active (GnRH and KNDy neurons, Figure 1) or showed increased expression of peptide (AVPV-kisspeptin, Figure 1c), we evaluated whether the IST protocol impacts the hormonal outputs of those peptides in a second cohort of animals. Once again IST mice exhibited higher levels of aggressive behavior reflected in a higher percentage of time in total aggression, threatening and attacking the intruder as well as a higher number of attacks and a reduced latency to attack (Figure 2A, Supplementary Table 1). Concerning serum hormonal levels, LH was found to be elevated in IST mice compared to low-aggressive GH controls. Of note LH concentrations positively correlated with the percentage of time displaying aggression (Figure 2B, Supplementary Table 5). Interestingly, neither progesterone nor testosterone levels were found to be changed or correlate with aggressive behavior in IST mice (Figures 2C and D, Supplementary Table 5). However, within the IST group, we found out that animals with a shorter attack latency (SAL, under 100s) showed higher levels of testosterone in serum compared with animals with a higher attack latency (over 100s) (Figure 2C). Altogether, these data indicate that isolation and aggression training lead to a hyperfunction of the HPG axis at the hypothalamic and pituitary levels. NK3R signaling mildly affects aggression in IST mice As activation of NK3R and expression of NKB have been associated with escalated aggression in isolated mice, in the next set of experiments, we assessed whether agonism and antagonism of NK3Rs in IST males would affect aggressive behavior [37] . Pilot data (not shown) of our lab has demonstrated that senktide at the dose of 2 μg/Kg [37] had strong motor effects (crawling, tail rattle followed by immobility) 20 minutes after injection, thus we decided to use a lower dose of 1 μg/Kg and test behavior after 30 minutes. Our data showed that senktide decreased the percentage of time that IST mice spent on aggressive behavior which is mainly reflected by the reduced percentage of time spent on threats without significant changes in the number of attacks and latency to attack (Figure 3B, Supplementary Table 2). Additionally, those animals seem to compensate for decreased time spent on aggression by increasing the percentage of time exploring their homecage (Supplementary Table 2). Contrasting with previous data [37] , the blockade of NK3Rs via osanetant did not affect any of the behaviors (Figure 3C, Supplementary Table 2). High aggression in IST mice is underlied by GnRHR signaling As GnRH neuron activity (Figure 1) and LH levels (Figure 2) were increased in IST mice, we investigated whether manipulating GnRHR signaling via pharmacology affected aggressive behavior in IST mice. Deslorelin (GnRHR agonist) exacerbated aggression in IST mice, those animals spent more time attacking and threatening as well as exhibited a higher number of attacks and decreased latency to attack (Figure 4A, Supplementary Table 2). Additionally, GnRHR agonism reduced the percentage of time IST mice spend in non-aggressive social investigation (Figure 4a, Supplementary Table 2). Consistently, blockade of GnRHRs via cetrorelix strongly reduced the time IST animals spend on aggression which was reflected in decreased time attacking, threatening, and decreased number of attacks and higher attack latency (Figure 3B, Supplementary Table 2). Interestingly, the percentage of time spent on the social investigation was not affected by cetrorelix treatment. As GnRHR activation led to exacerbated aggressive behavior in IST mice we wondered whether deslorelin would also increase aggression in low-aggressive GH mice. Surprisingly, there was no effect of deslorelin on GH mice's aggressive or social behaviors (Figure 4C, Supplementary Table 2). Kisspeptin and AVPV-Kisspeptin neurons differentially affect aggressive behavior induced by IST As we have found i) increased activation of KNDy neurons and ii) a higher number of kisspeptin neurons in the AVPV of IST mice we decided to investigate the role of kisspeptin on aggression using behavioral pharmacology and loss of function experiments. Surprisingly, contrasting with the effects of GnRH, kisspeptin strongly reduced aggression in IST mice. Those animals showed a decreased percentage of time spent on aggressive behavior, threat, and attacking as well as a decreased number of attacks and higher latency to attack (Figure 5A, Supplementary Table 3). Furthermore, kisspeptin administration tended to increase time spent on social investigation (Figure 5A) and increased home cage exploration (Supplementary Table 3). Those pharmacological effects somewhat differed from the effect of ablation of AVPV kisspeptin neurons on aggression. Kiss::cre mice injected with the AAV1-TEV-Casp-3 virus showed a lower number of kisspeptin neurons as well as lower optical density /mm 2 in the AVPV (Figure 5B). According to the finding that IST animals showed a higher number of AVPV-kisspeptin neurons, ablation of those neurons suppressed the rise in time spent on aggression and number of attacks induced by aggression training without affecting baseline (first day of training) levels of aggression (Figure 5C, Supplementary Table 3). Moreover, those effects of AVPV-kisspeptin deletion seem to be specific to aggressive behavior as neuronal deletion did not affect male sexual behavior (Figure 5D) or physiological measures such as testis relative weight (Supplementary Table 3). 4. Discussion We have shown here that the IST has a robust effect in enhancing aggression in male mice, similar to its effects in female rats [12,43] . In addition, IST animals presented a hyperfunction of the HPG-axis, reflected by increased activity of GnRH and KNDy neurons and a greater number of AVPV-Kisspeptin neurons (Figure 1). In line with our findings, increased hyperfunction of the HPG axis peptides has been associated with aggression in vertebrates. In fish, increased GnRH mRNA, particularly in the POA (where GnRH neurons were highly active in IST mice), has been associated with aggressive and dominant behavior [48,49] . Consistently with exacerbated GnRH neuron activity, IST animals exhibited elevated serum LH levels (Figure 2). These data are in line with previous observations (27) in male mice using indirect methods of detection. In this particular study, socially isolated mice that underwent multiple (4 to 16) aggressive interactions, showed elevated plasma LH levels. Furthermore, in our study, LH levels positively correlated with aggression indicating that high levels of LH (and GnRH activity [24] ) might underlie heightened aggressive behavior. In fact, it has been demonstrated [25] that in violent sexual offenders high hostility and violent behavior are positively correlated with LH. Interestingly LH also correlated with recidivism which was not the case for testosterone levels, indicating that LH might be a better predictor of hostility and recidivism (re-engaging in aggression/aggression seeking) than testosterone. Our data on GnRH neurons and LH support a model where hyperactivation of those neurons seen by colocalization with c-FOS and high serum LH leads to escalated aggression. This hypothesis was further confirmed by the finding that deslorelin (GnRHR agonist) enhanced whereas cetrorelix (GnRHR antagonist) decreased aggression in IST animals (Figure 4). Interestingly, seminal studies in pigs and macaques have used GnRH agonists chronically to suppress HPG axis activity, reducing testosterone and consequently aggression [28,29] . Those studies have ignored the fact that GnRH itself might affect behavior by acting in different brain regions independently or in synergism with testosterone. In fact, GnRHRs are widespread in the mouse brain [31] , including in brain regions involved in aggressive behavior such as the VMH [50] , LS (12), and CoA [51] . Further evidence of an independent role of GnRH neurotransmission on aggression comes from the finding that chronic administration of deslorelin exacerbates aggression and decreases social interactions in female Southern Giant Pouched rats (similarly to our acute effects in IST male mice) without affecting vaginal patency [52] . Additionally, blockade of GnRHRs was associated with decreased maternal aggression in lactating rats [53] , another model where testosterone is not particularly involved. Accordingly, compared to GH controls, IST animals showed no difference in testosterone levels in serum. LH and testosterone release are strongly linked [8,14,25,54] , so to some extent those results are surprising. Nevertheless, the lack of rise of testosterone might be due to the timepoint of blood collection (around 15 min after the RI), since previous studies in rats have shown that serum testosterone rises 1 hour after an aggressive encounter [26] . Interestingly, we found that IST animals with SAL (<100s) exhibit higher levels of testosterone in serum, which is consistent with testosterone being needed to “prime” the neuronal circuits of aggression to facilitate winning [8,17,55,56] . However, whether this rise in testosterone levels reflects increased activity of GnRH neurons as seen in our model, remains to be elucidated. Nevertheless, future studies should assess whether the effects of GnRH signaling depend on testosterone levels and vice-versa. In agreement with high GnRH neuron activity, KNDy neurons were found to be highly active in IST mice (Figure 1) [34,57,58] . Curiously, a higher number of AVPV-kisspeptin neurons was found in IST mice (Figure 1). Ablation of those neurons in Kiss::cre mice before the IST protocol abolished the rise in aggressive behavior induced via training without affecting baseline aggression (first RI) or sexual behavior (Figure 5). Those effects i) indicate a sex-specific role of AVP-kisspeptin neurons on innate behaviors, i.e. involved in aggressive but not sexual behavior in males and sexual behavior in females [44] and ii) highlight kisspeptin as a potential factor underlying the aggression training effect. Social isolation seems to strongly affect peptides as isolated rodents were found to present alterations related to peptide mRNA, synthesis, and release in the NKB [37] , oxytocin [12,13,59] , and vasopressin [12,59] systems. As testosterone (and potentially aromatized estrogens) are known to induce plasticity in the aggression circuits of trained animals [8,14] , one could hypothesize that the increased number of kisspeptin neurons might arise from the plastic effects of estrogens or testosterone in kisspeptin synthesis and/or production in the AVPV. In fact, aromatase knockout (ArKO) male mice exhibited an increased number of kisspeptin neurons in the AVPV which were further heightened after castration and treatment with estradiol for 10 days [60,61] . Additionally, testosterone treatment itself increased the promptness for aggression (reduced attack latency) in Peromyscus, similar to our training effect, whereas aromatase inhibition affected baseline aggression [56] . Taking together, one could hypothesize that aggressive experiences and social isolation alter different sex steroid sources to act on AVPV-kisspeptin neurons enhancing synthesis and release in order to generate the training-induced rise in aggression. Future studies should dissect the link between this IST-induced increase in the number of AVPV-kisspeptin neurons, sex steroids, and aggression. Contrasting with the above-described results, administration of kisspeptin-10 i.p. strongly reduced aggression in males (Figure 5). This result was somewhat puzzling and unexpected. However, one could hypothesize that i) kisspeptin acts via other neuromodulatory systems to decrease aggression independently of GnRH or ii) other atypical kisspeptin neuronal populations are responsible for these anti-aggressive effects. Regarding the first hypothesis, kisspeptin was found to act via nitric oxide (NO) and neuronal nitric oxide synthase (nNOS) to stimulate sexual behavior in females [24,44,62] . In males, decreased NO signaling is known to exacerbate aggression [63–66] , thus one could hypothesize that kisspeptin decreases aggression in males by stimulating NO production and neurotransmission. Concerning other kisspeptin neuronal populations, extrahypothalamic populations have been described in the LS and medial amygdala [67] . As both regions are known to send GABAergic projections to the VMH [51,68,69] to suppress aggressive behavior, one could hypothesize that those neurons are also the ones expressing kisspeptin. Future studies should dissect the neural circuits underlying the anti-aggressive effects of kisspeptin using site-specific gain and loss of function approaches. To the best of our knowledge, this is the first study to assess the role of different levels of the hypothalamus-pituitary-gonad axis on aggressive behavior. Here we provided evidence indicating that escalated aggression is underlied by elevated activity of the GnRH system including high neuronal activity and peptide signaling. Additionally, we described a novel role, beyond the reproductive context, for AVPV-kisspeptin neurons as well as kisspeptin signaling. One could assume that social isolation and aggressive experiences create an imbalance in neuronal inhibitory inputs to the HPG axis leading to a hyperfunction of this system. This hyperfunctioning system probably generates exaggerated aggressive behavior seen in our animals, interestingly aggressive interactions themselves might contribute to maintaining an “allostatic” and hyperfunctioning of the HPG axis by elevating levels of sex steroids such as testosterone which feedback directly or indirectly on kisspeptin and GnRH neurons. It remains to be addressed whether those behavioral and neuroendocrine alterations are reversible. Taken together, this study might impact the fields of neuroendocrinology and behavioral neuroscience opening new venues for the role of reproductive-relevant peptides in pathological behavioral conditions involving exacerbated aggression. Declarations Competing interests and Funding This work was supported by the Fonds de la Recherche Scientifique -FNRS Charges des Recherche (CR 40011133 AGGRESSIONKiNG 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). 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. M.F. was supported by a joint project (E4Liberty) with Mithra Pharmaceuticals. 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. Acknowledgments This work was supported by the Fonds de la Recherche Scientifique -FNRS Charges des Recherche (CR 40011133 AGGRESSIONKiNG 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). We would like to thank Philippe Cioffi (anti-NK3R), Erik Hrabovszky (anti-GnRH), and Isabelle Francheschini (anti-kisspeptin) for sharing antibody resources with the authors. We also would like to thank Laura de Vries for her help with perfusion experiments and Mahault Cornelis for her help with immunostaings. Author Contributions V.O: Designed, performed, and analyzed experiments, wrote the manuscript, as well as designed the figures and tables. F.E: Assisted with performing and analyzing behavioral experiments and analyzed the LH assay with the help of M.F.. M.F.: performed the LH assay. J.B: supervised the study, and gave input to experimental design and to the manuscript. All authors contributed to the article and approved the submitted version. References Nelson RJ, Trainor BC. Neural mechanisms of aggression. Nat Rev Neurosci. 2007;8:536–546. Lischinsky JE, Lin D. Neural mechanisms of aggression across species. Nat Neurosci. 2020;23:1317–1328. Reidy DE, Kearns MC, DeGue S, Lilienfeld SO, Massetti G, Kiehl KA. Why psychopathy matters: Implications for public health and violence prevention. Aggress Violent Behav. 2015;24:214–225. Masis-Calvo M, Schmidtner AK, de Moura Oliveira VE, Grossmann CP, de Jong TR, Neumann ID. Animal models of social stress: the dark side of social interactions. Stress. 2018;5:41–432. Haller J. Preclinical models of conduct disorder − principles and pharmacologic perspectives. 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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-3660090","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":253506880,"identity":"99e0e680-3e8e-4f5a-a7ba-ef6d774825f6","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,"submittingAuthor":false,"prefix":"","firstName":"Vinícius","middleName":"","lastName":"Oliveira","suffix":""},{"id":253506881,"identity":"3c043166-eeff-438e-aac0-bffb0fd14fc4","order_by":1,"name":"Florence Evrard","email":"","orcid":"","institution":"University of Liege","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Florence","middleName":"","lastName":"Evrard","suffix":""},{"id":253506882,"identity":"ce79c83e-1276-44f5-9e41-30ea61f7c976","order_by":2,"name":"Melanie Faure","email":"","orcid":"","institution":"University of Liege","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Melanie","middleName":"","lastName":"Faure","suffix":""},{"id":253506883,"identity":"41474fe7-bae0-4f98-bf90-4afb24714797","order_by":3,"name":"Julie Bakker","email":"","orcid":"","institution":"University of Liege","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Julie","middleName":"","lastName":"Bakker","suffix":""}],"badges":[],"createdAt":"2023-11-24 16:20:16","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-3660090/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-3660090/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1038/s41386-024-01808-3","type":"published","date":"2024-02-09T05:00:00+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":47224605,"identity":"65dee9a0-fd2d-4f6f-b718-dbef086c8beb","added_by":"auto","created_at":"2023-11-28 19:21:08","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":7064309,"visible":true,"origin":"","legend":"\u003cp\u003eEight to ten weeks-old male mice were either kept housed in groups (GH) or single-housed and underwent 4 consecutive resident intruder (RI) tests (TR: training). Subjects from both conditions were then split into two cohorts: Cohort A (CohA) was used for neuronal activity experiments whereas Cohort B (CohB) was used for blood collection and hormonal assays (A).\u0026nbsp; Isolated and trained (IST, black bars) mice displayed a higher percentage of time on aggression (two-tailed Student’s t-test t\u003csub\u003e(10)\u003c/sub\u003e= 2.93, p=0.01), a higher number of attacks (Mann-Whitney U test U= 3.0, p=0.01), and shorter attack latency (U= 0.0, p=0.002) \u0026nbsp;when compared to GH controls (white bars). Furthermore, all IST animals attacked the intruder compared to 16% of the GH mice (Fisher exact test, p\u0026lt;0.00001) (A). Regarding neuronal activity, IST mice showed an increased percentage of GnRH neurons co-expressing cFOS in the medial septum (t\u003csub\u003e(9)\u003c/sub\u003e= 2.8, p=0.02) (MS, B) and in the rostral preoptic area (t\u003csub\u003e(9)\u003c/sub\u003e= 2.4, p=0.04) (rPOA, C). IST mice also exhibited a higher number of anteroventral periventricular (AVPV) kisspeptin neurons (t\u003csub\u003e(10)\u003c/sub\u003e= 3.0, p=0.01) (D) and a higher percentage of colocalization between neurokinin-3 receptor (KNDy neuron marker) and cFOS (t\u003csub\u003e(10)\u003c/sub\u003e= 11.7, p\u0026lt;0.0001) \u0026nbsp;(E). Apart from AVPV-Kisspeptin, no other neuronal numbers were changed between GH and IST mice ( MS GnRH: t\u003csub\u003e(9)\u003c/sub\u003e= 0.1, p=0.89; RPOA GnRH: t\u003csub\u003e(9)\u003c/sub\u003e= 1.5, p=0.17; NK3R KNDy: t\u003csub\u003e(10)\u003c/sub\u003e= 0.6, p=0.5; AVPV Kiss-Fos: U= 9, p=0.2. *p\u0026lt;.05; **p\u0026lt;.01;****p\u0026lt;.0001 vs GH. Data are presented as mean + s.e.m. Scale bar 100μm.\u003c/p\u003e","description":"","filename":"Figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-3660090/v1/369a59384c8bbeb660f44a32.png"},{"id":47224602,"identity":"8e799bce-6ea4-44c9-9887-a1f5f4a65089","added_by":"auto","created_at":"2023-11-28 19:21:08","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":1008684,"visible":true,"origin":"","legend":"\u003cp\u003eIsolated and trained (IST, black bars) male mice spent a higher percentage of time on aggression (two-tailed Student’s t-test t\u003csub\u003e(14)\u003c/sub\u003e= 5.2, p=0.0001), exhibited a higher number of attacks (Mann-Whitney U test U= 3.6, p=0.002), and had a shorter attack latency (t\u003csub\u003e(14)\u003c/sub\u003e= 2.8, p=0.01)\u0026nbsp; when compared to GH controls (white bars) (A). Regarding hormonal levels, IST mice exhibited higher levels of LH in serum (t\u003csub\u003e(14)\u003c/sub\u003e= 2.5, p=0.02), which positively correlated with aggression (Pearson’s correlation r=0.53, p=0.04) (B). \u0026nbsp;Testosterone levels remained unchanged in IST animals. However, IST mice with a short attack latency (SAL, under 100s) were found to present higher testosterone compared to long attack latency mice (LAL, over 100s) (Latency to attack: U= 0, p=0.02, Testosterone: U= 0, p=0.02) (C). Finally, progesterone and testosterone/progesterone ratio remained unchanged in IST animals compared to controls. *p\u0026lt;.05, **\u0026lt;.01 vs GH. Data are presented as mean + s.e.m.\u003c/p\u003e","description":"","filename":"Figure2corr.png","url":"https://assets-eu.researchsquare.com/files/rs-3660090/v1/d2b345bba5df785191c8734d.png"},{"id":47224606,"identity":"6984a1d0-e360-4d95-b864-c2140eb2653b","added_by":"auto","created_at":"2023-11-28 19:21:08","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":1374700,"visible":true,"origin":"","legend":"\u003cp\u003eSchematic drawings depicting the detailed protocols followed on experiments (Exp) A to D. Please note that each individual Exp has used different cohorts of animals. \u003cstrong\u003eAVPV:\u003c/strong\u003e anteroventral periventricular nucleus; \u003cstrong\u003eCetr:\u003c/strong\u003e Cetrorelix (GnRHR antagonist, 0.5mg/Kg);\u0026nbsp; \u003cstrong\u003eDeso:\u003c/strong\u003e deslorelin (GnRHR agonist, 300ng/Kg); \u003cstrong\u003eKp-10:\u003c/strong\u003e Kisspeptin-10 (0.52μg/Kg); \u003cstrong\u003eOsan:\u003c/strong\u003e Osanetant (5μg/Kg); \u003cstrong\u003eRI:\u003c/strong\u003e resident-intruder; \u003cstrong\u003eSenkt:\u003c/strong\u003e \u0026nbsp;senktide (NK3R agonist, 1μg/kg); \u0026nbsp;\u003cstrong\u003eSex:\u003c/strong\u003e sexual behavior test; \u003cstrong\u003eWO: \u003c/strong\u003eWashout (neither behavior nor injection) (A). Senktide (light orange bars) reduced the total percentage of time spent on aggression (two-tailed Student’s t-test t\u003csub\u003e(11)\u003c/sub\u003e= 2.96, p=0.01), but it did not affect the number of attacks, attack latency, and social investigation in IST mice (B). Osanetant (dark orange bars) did not affect the total percentage of time spent on aggression, the number of attacks, the latency to attack, or social investigation (C). *p\u0026lt;.05 vs vehicle. Data are presented as mean + s.e.m.\u003c/p\u003e","description":"","filename":"Figure3.png","url":"https://assets-eu.researchsquare.com/files/rs-3660090/v1/2f5374375dcb76d58ad5412b.png"},{"id":47224603,"identity":"96a52b0b-5725-4876-ab3a-b6a5bc441221","added_by":"auto","created_at":"2023-11-28 19:21:08","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":1474574,"visible":true,"origin":"","legend":"\u003cp\u003eDeslorelin (Deso, GnRHR agonist, 300ng/Kg, light purple bars) increased the total percentage of time spent on aggression (two-tailed Student’s t-test t\u003csub\u003e(13)\u003c/sub\u003e= 2.98, p=0.01) as well as the number of attacks (Wilcoxon test W= 82, p=0.007), and reduced both attack latency (t-test t\u003csub\u003e(13)\u003c/sub\u003e= 3.39, p=0.004) and non-aggressive social investigations (t-test t\u003csub\u003e(13)\u003c/sub\u003e= 3.24, p=0.006) in IST mice (A). Cetrorelix (Ctrx, GnRHR antagonist, 0.5mg/Kg, dark purple bars) strongly reduced the total percentage of time spent on aggression (t-test t\u003csub\u003e(6)\u003c/sub\u003e= 6.36, p=0.0007), the number of attacks (Mann-Whitney U test U= 10, p=0.02), and the latency to attack U test (U= 13, p=0.05) in IST mice without affecting social investigation (B). Finally, deslorelin did not affect either aggressive or social behaviors in group-housed (GH) mice (C). *p\u0026lt;.05, **\u0026lt;.01 vs vehicle. Data are presented as mean + s.e.m.\u003c/p\u003e","description":"","filename":"Figure4.png","url":"https://assets-eu.researchsquare.com/files/rs-3660090/v1/966d2aaa3d59d03686c38f8f.png"},{"id":47224609,"identity":"22461008-2f2e-4715-92d5-3a2fc05ae678","added_by":"auto","created_at":"2023-11-28 19:21:09","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":4400736,"visible":true,"origin":"","legend":"\u003cp\u003eKisspeptin (Kp-10, 0.52μg/Kg, pink bars) reduced the total percentage of time spent on aggression (two-tailed Student’s t-test t\u003csub\u003e(10)\u003c/sub\u003e= 2.51, p=0.04) as well as the number of attacks (Wilcoxon test W= -39, p=0.04), increased the attack latency (t\u003csub\u003e(10)\u003c/sub\u003e= 2.53, p=0.02) and tended to elevate time spent on social investigation (t\u003csub\u003e(10)\u003c/sub\u003e= 2.05, p=0.06) in IST mice (A). Maximal z-projection of DAPI (magenta) and kisspeptin (green, Alexa-fluor 488 anti-rabbit) in the anteroventral periventricular nucleus (AVPV) confirming viral deletion of kisspeptin neurons (t\u003csub\u003e(10)\u003c/sub\u003e= 2.29, p=0.02) and reduction on fiber densities (Mann-Whitney U test U= 1, p=0.005) (B). Finally, the deletion of kisspeptin neurons in the AVPV (Kiss-del, pink) abolished the rise in aggression levels seen in controls regarding the percentage of time spent on aggression (Two-way ANOVA Training effect: F\u003csub\u003e(2,22)\u003c/sub\u003e= 20.10, p\u0026lt;0.0001; Deletion effect: F\u003csub\u003e(1,11)\u003c/sub\u003e= 4.16, p=0.06; TrainingxDeletion:\u0026nbsp; F\u003csub\u003e(2,22)\u003c/sub\u003e= 4.55, p=0.02) \u0026nbsp;and number of attacks (Training effect: F\u003csub\u003e(2,22)\u003c/sub\u003e= 3.93, p=0.03; Deletion effect: F\u003csub\u003e(1,11)\u003c/sub\u003e= 0.006, p=0.93; TrainingxDeletion:\u0026nbsp; F\u003csub\u003e(2,22)\u003c/sub\u003e= 0.34, p=0.77) \u0026nbsp;(C) without affecting sexual behavior (D). *p\u0026lt;.05, **\u0026lt;.01 vs vehicle or control (Ctrl); #\u0026lt;0.5 vs RI1; ## \u0026lt;0.1 vs RI1 .Data are presented as mean + s.e.m. Scale bar 100μm.\u003c/p\u003e","description":"","filename":"Figure5.png","url":"https://assets-eu.researchsquare.com/files/rs-3660090/v1/349b188f5fb80a18d8811e56.png"},{"id":51070159,"identity":"b1dd301f-bcbe-450b-a5fb-e36aa8e3537a","added_by":"auto","created_at":"2024-02-13 16:05:54","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2521683,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3660090/v1/8fcf0821-4b38-4560-aa8c-ae20ea867346.pdf"},{"id":47224604,"identity":"a3b76d8a-0c70-48fd-be4c-d8011de611d3","added_by":"auto","created_at":"2023-11-28 19:21:08","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":1250223,"visible":true,"origin":"","legend":"Supplementary information","description":"","filename":"Supplementaryinfocorrectedrevised.docx","url":"https://assets-eu.researchsquare.com/files/rs-3660090/v1/bb62bcdc47a11aebf2a02d96.docx"}],"financialInterests":"There is \u003cb\u003eNO\u003c/b\u003e conflict of interest to disclose.","formattedTitle":"NPRC-Social isolation and aggression training lead to escalated aggression and hypothalamus-pituitary-gonad axis hyperfunction in mice","fulltext":[{"header":"1.\tIntroduction","content":"\u003cp\u003eAggressive behavior is an innate behavior displayed by animals in order to get access to important resources for their survival \u003cspan lang=\"EN-US\"\u003e[1,2]\u003c/span\u003e. Apart from its ecological role, pathological and out-of-context aggression constitutes a core symptom of several psychiatric disorders such as conduct and antisocial personality disorders, schizophrenia, major depression, and post-traumatic stress disorder \u003cspan lang=\"EN-US\"\u003e[3\u0026ndash;7]\u003c/span\u003e.\u003c/p\u003e\n\u003cp\u003eThe fact that violence generates a severe socioeconomic burden on society \u003cspan lang=\"EN-US\"\u003e[3,6]\u003c/span\u003e has led scientists to develop preclinical animal models \u003cspan lang=\"EN-US\"\u003e[4,5,7]\u003c/span\u003e to understand the neural underpinnings of escalated aggression. Among those models, social isolation and engaging in successive aggressive experiences (aggression training) were found to entail robust effects on inducing exaggerated aggression \u003cspan lang=\"EN-US\"\u003e[7,8]\u003c/span\u003e. Additionally, both conditions are known to disrupt social behaviors in humans by triggering psychological distress and violent behaviors \u003cspan lang=\"EN-US\"\u003e[3,9\u0026ndash;11]\u003c/span\u003e.\u003c/p\u003e\n\u003cp\u003eRegarding neurobiological factors, attention has been given to several systems such as neuropeptides \u003cspan lang=\"EN-US\"\u003e[4,8,12,13]\u003c/span\u003e, hypothalamic-pituitary-adrenal (HPA) axis \u003cspan lang=\"EN-US\"\u003e[4,7]\u003c/span\u003e, and sex hormones \u003cspan lang=\"EN-US\"\u003e[8,14]\u003c/span\u003e. Specifically, the role of sex hormones \u003cspan lang=\"EN-US\"\u003e[8,14\u0026ndash;19]\u003c/span\u003e and sex hormone-responsive neurons \u003cspan lang=\"EN-US\"\u003e[8,20\u0026ndash;23]\u003c/span\u003e has been extensively studied in the context of aggression. Yet, the participation of other brain hubs within the hypothalamus-pituitary-gonadal (HPG) axis which regulates sex steroid production and release remains largely unknown. \u003c/p\u003e\n\u003cp\u003eGonadotropin-releasing hormone (GnRH) neurons are classically found in the rostral preoptic area (rPOA), medial septum (MS), and anterior hypothalamus. Those neurons are the main node of reproduction in animals \u003cspan lang=\"EN-US\"\u003e[24]\u003c/span\u003e as they act on the gonadotrophs in the anterior pituitary to trigger luteinizing hormone (LH) and follicle-stimulating hormone release into the circulation. Those glycoproteins will act at the level of the gonads to stimulate sex steroid production and release \u003cspan lang=\"EN-US\"\u003e[24]\u003c/span\u003e. Thus, since aggressive behavior itself \u003cspan lang=\"EN-US\"\u003e[25,26]\u003c/span\u003e as well as successive aggressive interactions and social isolation are known to increase plasma testosterone \u003cspan lang=\"EN-US\"\u003e[8,14,17,18,25,26]\u003c/span\u003e and to some extent LH \u003cspan lang=\"EN-US\"\u003e[25,27]\u003c/span\u003e (main hormonal output of GnRH neurons), one could hypothesize that GnRH neurons are recruited during aggressive interactions and might be impacted by the social context.\u003c/p\u003e\n\u003cp\u003eFurther evidence of the participation of GnRH in aggressive behavior comes from the finding that either immunocastration using vaccines targeting GnRH or chronic agonism of GnRH receptors (GnRHR), aiming to desensitize the HPG axis, reduce aggression in male pigs \u003cspan lang=\"EN-US\"\u003e[28]\u003c/span\u003e and Macaques \u003cspan lang=\"EN-US\"\u003e[29]\u003c/span\u003e, respectively. Similar effects have been described in a male patient with autism spectrum disorder and high aggression \u003cspan lang=\"EN-US\"\u003e[30]\u003c/span\u003e. Importantly, GnRHRs are found in brain regions classically known to modulate aggression such as the lateral septum (LS), ventromedial hypothalamus (VMH), and the cortical amygdala (CoA) [31]. \u003c/p\u003e\n\u003cp\u003eOf note, GnRH neuronal activity is regulated mainly by two neuropeptidergic populations: kisspeptin neurons in the anteroventral periventricular nucleus of the hypothalamus (AVPV) and \u003cstrong\u003eK\u003c/strong\u003eisspeptin-\u003cstrong\u003eN\u003c/strong\u003eeurokinin B (NKB)-\u003cstrong\u003eD\u003c/strong\u003eynorphin (\u003cstrong\u003eKND\u003c/strong\u003ey) neurons in the arcuate nucleus of the hypothalamus (ARC) \u003cspan lang=\"EN-US\"\u003e[24,32\u0026ndash;35]\u003c/span\u003e. Whereas the role of kisspeptin in male social behaviors remains understudied, the blockade of neurokinin receptor 3 (NK3R), which is co-expressed by KNDy neurons \u003cspan lang=\"EN-US\"\u003e[35,36]\u003c/span\u003e, was shown to decrease aggression in highly aggressive socially isolated male mice \u003cspan lang=\"EN-US\"\u003e[37]\u003c/span\u003e.\u003c/p\u003e\n\u003cp\u003eHere we used a combination of social isolation and aggression training (IST) \u003cspan lang=\"EN-US\"\u003e[12]\u003c/span\u003e to induce escalated aggression in male mice in order to evaluate its effects on GnRH, kisspeptin, and KNDy neuron activity as well as HPG-axis function. Additionally, we manipulated peptide signaling and expression via pharmacological and viral tools to assess its impact on aggressive and social behaviors. \u003c/p\u003e"},{"header":"2. Methods","content":"\u003cp\u003e\u003cstrong\u003e2.1 Ethical statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll procedures described here were approved by the local Animal Ethics Committee of the University of Li\u0026egrave;ge (protocol number 22-2469).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.2 Animals\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTypically, experiments were carried out in adult (8-12 weeks) C57BL6/J male mice, which were purchased from Charles River Laboratories (Les Oncins, France). In particular, neuronal ablation experiments were conducted in Kiss::cre or WT mice (6-12 months) bred in the animal facilities of the University of Li\u0026egrave;ge. For aggressive behavior experiments, male CD-1 mice (5-6 weeks) were used as intruders whereas for sexual behavior receptive (estrous) females C57BL6/J (6-8 weeks) were used. All stimulus animals were obtained via Charles Rivers Laboratories (Les Oncins, France). Subjects were kept under controlled laboratory conditions in ventilated shelves with (12:12h light/dark cycle; lights off at 10:30h, 21\u0026plusmn;1\u0026deg;C, 60\u0026plusmn;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, during the experimental phase, mice were singly housed in individual mouse cages (33x16x13cm) in order to enhance their aggressive behavior. \u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.3 Resident intruder test (RI)\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe Rl 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 weighed between 10-20% less than residents [8,38,39]. Behavior was scored live and posteriorly via videos by a blind observer using JWatcher event recorder Program [12,40]. As previously described \u003cspan lang=\"EN-US\"\u003e[12]\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 to the first attack. \u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.4 Sexual behavior\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSexual behavior was conducted as previously described \u003cspan lang=\"EN-US\"\u003e[41]\u003c/span\u003e. For detailed information please see supplementary information.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.5 Experimental design\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.5.1 The social isolation and aggression training protocol (IST)\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe social isolation and aggression training (IST) protocol was adapted from the procedure developed by Oliveira and co-authors in female rats, which has shown to induce robust effects on exacerbating aggressive behavior \u003cspan lang=\"EN-US\"\u003e[8,12,42,43]\u003c/span\u003e. Therefore, mice from all different strains used to perform this study were assigned to either the IST (high-aggression) or group-housed (GH, control, low-aggression) condition. IST subjects were kept single-housed for 3 weeks in individual cages and were subsequently exposed to 3 consecutive, 10-minute-long RIs, daily. Group-housed animals were kept in groups of 4 animals per cage and were single-housed 2 hours prior to lights off, on experimental days (Figure 1A). Importantly, in pharmacological experiments targeting the IST condition, levels of aggression between vehicle and drug groups were counterbalanced and \u003cstrong\u003erandomized\u003c/strong\u003e using the live scoring data of the previous training sessions, to make sure groups did not differ in terms of the total percentage of time spent on aggression, total number of attacks or attack latency before treatments (not shown). Finally, no crossover effects were found when comparing vehicle groups from different treatment days for both housing conditions. \u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.5.2 Endogenous measurements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eMice were split into two cohorts: cohort A (neuronal activity experiments; n=6) and cohort B (hormonal measurements n=8) (Figure 1A). After either being assigned to the IST or the GH condition animals underwent the protocols as described above (Figure 1A). Animals of \u003cem\u003eCohort A\u003c/em\u003e were then exposed to a 10-minute RI and left undisturbed in their home cage for 1 hour. Next, animals were deeply anesthetized with a solution of Euthasol (pentobarbital sodium, 200mg/Kg) and transcardially perfused using phosphate buffer (PBS, 0.1M) followed by paraformaldehyde (PFA, 4%). Brains were postfixed overnight in PFA 4% followed by dehydration in sucrose (30 %) for 2-3 days and snap-frozen in dry-ice and isopentane (Sigma-Aldrich, PHR166). Mice of \u003cem\u003eCohort B\u003c/em\u003e either were kept GH or underwent the IST protocol as described above. On the test day, those animals were confronted with an intruder (10 min), deeply anesthetized with Euthasol 10 min after the test, decapitated, and had their trunk blood collected. Brains were harvested, postfixed in PFA 4%, dehydrated in sucrose, and snap-frozen. Blood was stored in ice, centrifuged (4\u0026deg;, 1500 G, 15 min), and serum was stored at -20\u0026deg;C until hormonal assays took place. \u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.5.3 Experiment A\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eC57BL6/J mice underwent the IST protocol. After the third training session, on day 25 animals were injected intraperitoneally (i.p.) either with deslorelin (GnRH receptor agonist) or vehicle and tested on the RI 30 min after injection. After a one-day (day 27) washout (WO: no RI or i.p. injection) mice were tested again following a deslorelin or vehicle injection in a cross-over (within-subjects) design. Three days later (WO) subjects were then injected with either kisspeptin-10 or vehicle i.p. 2 hours before the RI. On day 32 (after 1-day WO) animals were again injected either with kisspeptin-10 or vehicle (i.p.) in a crossover (within-subjects) design. Finally on day 35, IST mice were either injected with vehicle or cetrorelix (GnRH receptor antagonist) 40min before the RI in a between-subjects design (Figure 3A). \u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.5.4 Experiment B\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAs deslorelin enhanced aggression in IST mice (Figure 4), we decided to test whether GnRHR agonism alone could enhance the mild levels of aggression displayed by GH mice. Thus, GH mice were also injected either with deslorelin or vehicle 30 min before the RI. After the experiment, animals returned to their original groups and left undisturbed for 1 day (WO). On day 3, GH mice were again single-housed (see above) and either injected with deslorelin or vehicle in a within-subjects design (Figure 3A). \u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.5.5 Experiment C\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSimilarly to Experiment A, mice underwent the IST protocol. On day 25, IST mice were either injected with senktide (NK3R agonist) or vehicle, 30 min prior to the RI. Following a 1-day washout, animals were again injected with sektide or vehicle in a cross-over within subjects design. On day 30 (after a 3 WO), IST mice were then injected with osanetant (NK3R antagonist) 30 min before the RI in a between-subjects design (Figure 3A). \u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.5.6 Experiment D\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eKiss::cre or WT mice underwent stereotaxic surgery and were injected into the AVPV with AAV1-flex-taCasp3-TEVp (6.94 \u0026times; 10\u003csup\u003e11\u003c/sup\u003e genomic copies per ml) in order to specifically ablate AVPV-kisspeptin neurons \u003cspan lang=\"EN-US\"\u003e[22,44]\u003c/span\u003e. After 3 weeks (day 21), mice started the training phase of the IST protocol, by going through the RI for 3 consecutive days. Three days after the third RI (RI3) animals were allowed to interact with estrous/receptive females for 20-min. After a resting of 4 days, animals were again tested for sexual behavior (Sex 2, Figure 3A) and subsequently perfused to confirm viral ablation. WT mice were also compared with non-surgery animals (not shown, n=3) as there were no behavioral or neuronal number differences between non-surgery and WT those animals were pulled as the control group (Ctrl). \u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.6 Stereotaxic surgery\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eStereotaxic surgery was performed under semi-sterile conditions and according to the procedures described in the animal license [44]. For details please see supplementary information. \u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.7 Drugs \u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFor information regarding doses and time points please see supplementary information. \u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.8 Immunohistochemistry\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAfter perfusion, brains were cryo-cutted and slices (40\u0026micro;m) containing the target regions were collected in cryoprotectant solution and stored at -20\u0026deg;C until the experiment took place. A series of 6-8 slices were used for immunostaining. For neuronal activity, slices were stained using the immunoperoxidase substrate method whereas for confirming cell ablation slices were stained using immunofluorescence (for protocol details please see Supplementary information). \u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.9 Sex steroid assays\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTestosterone (Demeditec, DEV9911, detection limit 0.024ng/ml) and Progesterone (Demeditec, DEV9988, detection limit 0.0156 ng/ml) were measured via commercial ELISA kits following the specifics of those kits. \u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.10 LH assay\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eLH was measured in serum following the specifics of protocols previously described (for details please see \u003cspan lang=\"EN-US\"\u003e[45,46]\u003c/span\u003e, for antibody concentrations please see Supplementary Table 4). Two animals were excluded from the final data analysis (one from the GH and one from the IST group). The earlier due to blood collection and processing issues (it was also a significant outlier) and the latter for being under the detection limit of the assay. \u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.11 Statistics \u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNormality was tested using the Kolmogorov-Smirnov test. Once normality was found, data were analyzed using a two-tailed Student\u0026rsquo;s t-test, Pearson\u0026rsquo;s correlations, and Two-way ANOVAs. Once normality was not reached, Mann-Whitney U-test, Wilcoxon matched pairs W test, and Spearman correlations were performed. Agonist experiments used within-subjects design whereas antagonist, endogenous measurements, and ablation used between-subjects. For detailed statistics please see Supplementary Tables 1,2 and 3.\u003c/p\u003e"},{"header":"3.\tResults","content":"\u003cp\u003e\u003cstrong\u003eSocial isolation and aggression training exacerbate aggression in male mice and hyperactivate the HPG axis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIST mice spent more time displaying aggressive behavior compared to GH controls which was reflected in a higher percentage of time displaying aggression, threatening and attacking the intruder as well as a higher number of attacks and decreased latency to attack (Figure 1A). Also, all IST animals displayed attacks whereas only 16% of the GH mice attacked the intruder (Figure 1A, for detailed behavior please see Supplementary Table 1).\u003c/p\u003e\n\u003cp\u003eRegarding neuronal activity, IST animals showed increased colocalization of c-FOS and GnRH in the MS (Figure 1B) and rPOA (1C). Particularly MS-GnRH neuronal activity positively correlated with the percentage of time displaying aggression (Supplementary Table 5). AVPV-kisspeptin neurons, on the other hand, showed very low levels of co-localization with c-FOS. Nevertheless, IST animals exhibited a higher number of kisspeptin neurons in the AVPV compared to GH controls (Figure 1D). Finally, KNDy neurons (NK3R-positive) in the ARC were also found to present higher colocalization with c-FOS, which positively correlated with aggression (Figure 1E, Supplementary Table 5). \u003c/p\u003e\n\u003cp\u003eFinally, as NKB signaling in hypothalamic regions has been associated with enhanced aggression in isolated animals \u003cspan lang=\"EN-US\"\u003e(36),\u003c/span\u003e we evaluated NK3R-colocalization in the dorsomedial (DMH) and lateral hypothalamus (LHp), regions known to be involved in the display of aggressive behavior \u003cspan lang=\"EN-US\"\u003e[37,47]\u003c/span\u003e. NK3R-positive neurons in both regions showed an increased colocalization with c-FOS in IST mice compared to GH controls (Supplementary Figure 1). In addition, the percentage of time spent on aggressive behavior positively correlated with aggression in both regions (Supplementary Table 5). \u003c/p\u003e\n\u003cp\u003eAs the hypothalamic hubs regulating the HPG axis were found to be either highly active (GnRH and KNDy neurons, Figure 1) or showed increased expression of peptide (AVPV-kisspeptin, Figure 1c), we evaluated whether the IST protocol impacts the hormonal outputs of those peptides in a second cohort of animals. Once again IST mice exhibited higher levels of aggressive behavior reflected in a higher percentage of time in total aggression, threatening and attacking the intruder as well as a higher number of attacks and a reduced latency to attack (Figure 2A, Supplementary Table 1).\u003c/p\u003e\n\u003cp\u003eConcerning serum hormonal levels, LH was found to be elevated in IST mice compared to low-aggressive GH controls. Of note LH concentrations positively correlated with the percentage of time displaying aggression (Figure 2B, Supplementary Table 5). Interestingly, neither progesterone nor testosterone levels were found to be changed or correlate with aggressive behavior in IST mice (Figures 2C and D, Supplementary Table 5). However, within the IST group, we found out that animals with a shorter attack latency (SAL, under 100s) showed higher levels of testosterone in serum compared with animals with a higher attack latency (over 100s) (Figure 2C). \u003c/p\u003e\n\u003cp\u003eAltogether, these data indicate that isolation and aggression training lead to a hyperfunction of the HPG axis at the hypothalamic and pituitary levels.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eNK3R signaling mildly affects aggression in IST mice\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAs activation of NK3R and expression of NKB have been associated with escalated aggression in isolated mice, in the next set of experiments, we assessed whether agonism and antagonism of NK3Rs in IST males would affect aggressive behavior \u003cspan lang=\"EN-US\"\u003e[37]\u003c/span\u003e. Pilot data (not shown) of our lab has demonstrated that senktide at the dose of 2 \u0026mu;g/Kg \u003cspan lang=\"EN-US\"\u003e[37]\u003c/span\u003e had strong motor effects (crawling, tail rattle followed by immobility) 20 minutes after injection, thus we decided to use a lower dose of 1 \u0026mu;g/Kg and test behavior after 30 minutes.\u003c/p\u003e\n\u003cp\u003eOur data showed that senktide decreased the percentage of time that IST mice spent on aggressive behavior which is mainly reflected by the reduced percentage of time spent on threats without significant changes in the number of attacks and latency to attack (Figure 3B, Supplementary Table 2). Additionally, those animals seem to compensate for decreased time spent on aggression by increasing the percentage of time exploring their homecage (Supplementary Table 2).\u003c/p\u003e\n\u003cp\u003eContrasting with previous data \u003cspan lang=\"EN-US\"\u003e[37]\u003c/span\u003e, the blockade of NK3Rs via osanetant did not affect any of the behaviors (Figure 3C, Supplementary Table 2). \u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eHigh aggression in IST mice is underlied by GnRHR signaling\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAs GnRH neuron activity (Figure 1) and LH levels (Figure 2) were increased in IST mice, we investigated whether manipulating GnRHR signaling via pharmacology affected aggressive behavior in IST mice. \u003c/p\u003e\n\u003cp\u003eDeslorelin (GnRHR agonist) exacerbated aggression in IST mice, those animals spent more time attacking and threatening as well as exhibited a higher number of attacks and decreased latency to attack (Figure 4A, Supplementary Table 2). Additionally, GnRHR agonism reduced the percentage of time IST mice spend in non-aggressive social investigation (Figure 4a, Supplementary Table 2). Consistently, blockade of GnRHRs via cetrorelix strongly reduced the time IST animals spend on aggression which was reflected in decreased time attacking, threatening, and decreased number of attacks and higher attack latency (Figure 3B, Supplementary Table 2). Interestingly, the percentage of time spent on the social investigation was not affected by cetrorelix treatment.\u003c/p\u003e\n\u003cp\u003eAs GnRHR activation led to exacerbated aggressive behavior in IST mice we wondered whether deslorelin would also increase aggression in low-aggressive GH mice. Surprisingly, there was no effect of deslorelin on GH mice\u0026apos;s aggressive or social behaviors (Figure 4C, Supplementary Table 2).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eKisspeptin and AVPV-Kisspeptin neurons differentially affect aggressive behavior induced by IST\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAs we have found i) increased activation of KNDy neurons and ii) a higher number of kisspeptin neurons in the AVPV of IST mice we decided to investigate the role of kisspeptin on aggression using behavioral pharmacology and loss of function experiments. \u003c/p\u003e\n\u003cp\u003eSurprisingly, contrasting with the effects of GnRH, kisspeptin strongly reduced aggression in IST mice. Those animals showed a decreased percentage of time spent on aggressive behavior, threat, and attacking as well as a decreased number of attacks and higher latency to attack (Figure 5A, Supplementary Table 3). Furthermore, kisspeptin administration tended to increase time spent on social investigation (Figure 5A) and increased home cage exploration (Supplementary Table 3).\u003c/p\u003e\n\u003cp\u003eThose pharmacological effects somewhat differed from the effect of ablation of AVPV kisspeptin neurons on aggression. Kiss::cre mice injected with the AAV1-TEV-Casp-3 virus showed a lower number of kisspeptin neurons as well as lower optical density /mm\u003csup\u003e2\u003c/sup\u003e in the AVPV (Figure 5B). According to the finding that IST animals showed a higher number of AVPV-kisspeptin neurons, ablation of those neurons suppressed the rise in time spent on aggression and number of attacks induced by aggression training without affecting baseline (first day of training) levels of aggression (Figure 5C, Supplementary Table 3). Moreover, those effects of AVPV-kisspeptin deletion seem to be specific to aggressive behavior as neuronal deletion did not affect male sexual behavior (Figure 5D) or physiological measures such as testis relative weight (Supplementary Table 3). \u003c/p\u003e"},{"header":"4.\tDiscussion","content":"\u003cp\u003eWe have shown here that the IST has a robust effect in enhancing aggression in male mice, similar to its effects in female rats \u003cspan lang=\"EN-US\"\u003e[12,43]\u003c/span\u003e. In addition, IST animals presented a hyperfunction of the HPG-axis, reflected by increased activity of GnRH and KNDy neurons and a greater number of AVPV-Kisspeptin neurons (Figure 1). In line with our findings, increased hyperfunction of the HPG axis peptides has been associated with aggression in vertebrates. In fish, increased GnRH mRNA, particularly in the POA (where GnRH neurons were highly active in IST mice), has been associated with aggressive and dominant behavior \u003cspan lang=\"EN-US\"\u003e[48,49]\u003c/span\u003e. \u003c/p\u003e\n\u003cp\u003eConsistently with exacerbated GnRH neuron activity, IST animals exhibited elevated serum LH levels (Figure 2). These data are in line with previous observations (27) in male mice using indirect methods of detection. In this particular study, socially isolated mice that underwent multiple (4 to 16) aggressive interactions, showed elevated plasma LH levels. Furthermore, in our study, LH levels positively correlated with aggression indicating that high levels of LH (and GnRH activity\u003cspan lang=\"EN-US\"\u003e[24]\u003c/span\u003e) might underlie heightened aggressive behavior. In fact, it has been demonstrated \u003cspan lang=\"EN-US\"\u003e[25]\u003c/span\u003e that in violent sexual offenders high hostility and violent behavior are positively correlated with LH. Interestingly LH also correlated with recidivism which was not the case for testosterone levels, indicating that LH might be a better predictor of hostility and recidivism (re-engaging in aggression/aggression seeking) than testosterone.\u003c/p\u003e\n\u003cp\u003eOur data on GnRH neurons and LH support a model where hyperactivation of those neurons seen by colocalization with c-FOS and high serum LH leads to escalated aggression. This hypothesis was further confirmed by the finding that deslorelin (GnRHR agonist) enhanced whereas cetrorelix (GnRHR antagonist) decreased aggression in IST animals (Figure 4). Interestingly, seminal studies in pigs and macaques have used GnRH agonists chronically to suppress HPG axis activity, reducing testosterone and consequently aggression \u003cspan lang=\"EN-US\"\u003e[28,29]\u003c/span\u003e. Those studies have ignored the fact that GnRH itself might affect behavior by acting in different brain regions independently or in synergism with testosterone. In fact, GnRHRs are widespread in the mouse brain \u003cspan lang=\"EN-US\"\u003e[31]\u003c/span\u003e, including in brain regions involved in aggressive behavior such as the VMH \u003cspan lang=\"EN-US\"\u003e[50]\u003c/span\u003e, LS \u003cspan lang=\"EN-US\"\u003e(12),\u003c/span\u003e and CoA \u003cspan lang=\"EN-US\"\u003e[51]\u003c/span\u003e. Further evidence of an independent role of GnRH neurotransmission on aggression comes from the finding that chronic administration of deslorelin exacerbates aggression and decreases social interactions in female Southern Giant Pouched rats (similarly to our acute effects in IST male mice) without affecting vaginal patency \u003cspan lang=\"EN-US\"\u003e[52]\u003c/span\u003e. Additionally, blockade of GnRHRs was associated with decreased maternal aggression in lactating rats \u003cspan lang=\"EN-US\"\u003e[53]\u003c/span\u003e, another model where testosterone is not particularly involved. \u003c/p\u003e\n\u003cp\u003eAccordingly, compared to GH controls, IST animals showed no difference in testosterone levels in serum. LH and testosterone release are strongly linked \u003cspan lang=\"EN-US\"\u003e[8,14,25,54]\u003c/span\u003e, so to some extent those results are surprising. Nevertheless, the lack of rise of testosterone might be due to the timepoint of blood collection (around 15 min after the RI), since previous studies in rats have shown that serum testosterone rises 1 hour after an aggressive encounter \u003cspan lang=\"EN-US\"\u003e[26]\u003c/span\u003e. Interestingly, we found that IST animals with SAL (\u0026lt;100s) exhibit higher levels of testosterone in serum, which is consistent with testosterone being needed to \u0026ldquo;prime\u0026rdquo; the neuronal circuits of aggression to facilitate winning \u003cspan lang=\"EN-US\"\u003e[8,17,55,56]\u003c/span\u003e. However, whether this rise in testosterone levels reflects increased activity of GnRH neurons as seen in our model, remains to be elucidated. Nevertheless, future studies should assess whether the effects of GnRH signaling depend on testosterone levels and vice-versa. \u003c/p\u003e\n\u003cp\u003eIn agreement with high GnRH neuron activity, KNDy neurons were found to be highly active in IST mice (Figure 1) \u003cspan lang=\"EN-US\"\u003e[34,57,58]\u003c/span\u003e. Curiously, a higher number of AVPV-kisspeptin neurons was found in IST mice (Figure 1). Ablation of those neurons in Kiss::cre mice before the IST protocol abolished the rise in aggressive behavior induced via training without affecting baseline aggression (first RI) or sexual behavior (Figure 5). Those effects i) indicate a sex-specific role of AVP-kisspeptin neurons on innate behaviors, i.e. involved in aggressive but not sexual behavior in males and sexual behavior in females \u003cspan lang=\"EN-US\"\u003e[44]\u003c/span\u003e and ii) highlight kisspeptin as a potential factor underlying the aggression training effect. Social isolation seems to strongly affect peptides as isolated rodents were found to present alterations related to peptide mRNA, synthesis, and release in the NKB\u003cspan lang=\"EN-US\"\u003e[37]\u003c/span\u003e, oxytocin\u003cspan lang=\"EN-US\"\u003e[12,13,59]\u003c/span\u003e, and vasopressin\u003cspan lang=\"EN-US\"\u003e[12,59]\u003c/span\u003e systems. As testosterone (and potentially aromatized estrogens) are known to induce plasticity in the aggression circuits of trained animals \u003cspan lang=\"EN-US\"\u003e[8,14]\u003c/span\u003e, one could hypothesize that the increased number of kisspeptin neurons might arise from the plastic effects of estrogens or testosterone in kisspeptin synthesis and/or production in the AVPV. In fact, aromatase knockout (ArKO) male mice exhibited an increased number of kisspeptin neurons in the AVPV which were further heightened after castration and treatment with estradiol for 10 days \u003cspan lang=\"EN-US\"\u003e[60,61]\u003c/span\u003e. Additionally, testosterone treatment itself increased the promptness for aggression (reduced attack latency) in Peromyscus, similar to our training effect, whereas aromatase inhibition affected baseline aggression\u003cspan lang=\"EN-US\"\u003e[56]\u003c/span\u003e. Taking together, one could hypothesize that aggressive experiences and social isolation alter different sex steroid sources to act on AVPV-kisspeptin neurons enhancing synthesis and release in order to generate the training-induced rise in aggression. Future studies should dissect the link between this IST-induced increase in the number of AVPV-kisspeptin neurons, sex steroids, and aggression.\u003c/p\u003e\n\u003cp\u003eContrasting with the above-described results, administration of kisspeptin-10 i.p. strongly reduced aggression in males (Figure 5). This result was somewhat puzzling and unexpected. However, one could hypothesize that i) kisspeptin acts via other neuromodulatory systems to decrease aggression independently of GnRH or ii) other atypical kisspeptin neuronal populations are responsible for these anti-aggressive effects. Regarding the first hypothesis, kisspeptin was found to act via nitric oxide (NO) and neuronal nitric oxide synthase (nNOS) to stimulate sexual behavior in females \u003cspan lang=\"EN-US\"\u003e[24,44,62]\u003c/span\u003e. In males, decreased NO signaling is known to exacerbate aggression \u003cspan lang=\"EN-US\"\u003e[63\u0026ndash;66]\u003c/span\u003e, thus one could hypothesize that kisspeptin decreases aggression in males by stimulating NO production and neurotransmission.\u003c/p\u003e\n\u003cp\u003eConcerning other kisspeptin neuronal populations, extrahypothalamic populations have been described in the LS and medial amygdala \u003cspan lang=\"EN-US\"\u003e[67]\u003c/span\u003e. As both regions are known to send GABAergic projections to the VMH \u003cspan lang=\"EN-US\"\u003e[51,68,69]\u003c/span\u003e to suppress aggressive behavior, one could hypothesize that those neurons are also the ones expressing kisspeptin. Future studies should dissect the neural circuits underlying the anti-aggressive effects of kisspeptin using site-specific gain and loss of function approaches.\u003c/p\u003e\n\u003cp\u003eTo the best of our knowledge, this is the first study to assess the role of different levels of the hypothalamus-pituitary-gonad axis on aggressive behavior. Here we provided evidence indicating that escalated aggression is underlied by elevated activity of the GnRH system including high neuronal activity and peptide signaling. Additionally, we described a novel role, beyond the reproductive context, for AVPV-kisspeptin neurons as well as kisspeptin signaling.\u003c/p\u003e\n\u003cp\u003eOne could assume that social isolation and aggressive experiences create an imbalance in neuronal inhibitory inputs to the HPG axis leading to a hyperfunction of this system. This hyperfunctioning system probably generates exaggerated aggressive behavior seen in our animals, interestingly aggressive interactions themselves might contribute to maintaining an \u0026ldquo;allostatic\u0026rdquo; and hyperfunctioning of the HPG axis by elevating levels of sex steroids such as testosterone which feedback directly or indirectly on kisspeptin and GnRH neurons. It remains to be addressed whether those behavioral and neuroendocrine alterations are reversible. Taken together, this study might impact the fields of neuroendocrinology and behavioral neuroscience opening new venues for the role of reproductive-relevant peptides in pathological behavioral conditions involving exacerbated aggression.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eCompeting interests and Funding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by the Fonds de la Recherche Scientifique -FNRS Charges des Recherche (CR 40011133 AGGRESSIONKiNG 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\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003eVO and the FNRS (PDR T.0015.20 to JB). JB 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. M.F. was supported by a joint project (E4Liberty) with Mithra Pharmaceuticals. 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.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by the Fonds de la Recherche Scientifique -FNRS Charges des Recherche (CR 40011133 AGGRESSIONKiNG 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\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003eVO and the FNRS (PDR T.0015.20 to JB). We would like to thank Philippe Cioffi (anti-NK3R), Erik Hrabovszky (anti-GnRH), and Isabelle Francheschini (anti-kisspeptin) for sharing antibody resources with the authors. We also would like to thank Laura de Vries for her help with perfusion experiments and Mahault Cornelis for her help with immunostaings.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor Contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eV.O: Designed, performed, and analyzed experiments, wrote the manuscript, as well as designed the figures and tables. F.E: Assisted with performing and analyzing behavioral experiments and analyzed the LH assay with the help of M.F.. M.F.: performed the LH assay. J.B: supervised the study, and gave input to experimental design and to the manuscript. All authors contributed to the article and approved the submitted version.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eNelson RJ, Trainor BC. Neural mechanisms of aggression. 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Estradiol-dependent and -independent stimulation of KIss1 expression in the amygdala, BNST, and lateral septum of mice. Endocrinology. 2018;159:3389\u0026ndash;3402.\u003c/li\u003e\n\u003cli\u003eMenon R, S\u0026uuml;\u0026szlig; T, Oliveira VE de M, Neumann ID, Bludau A. Neurobiology of the lateral septum: regulation of social behavior. Trends Neurosci. 2021:1\u0026ndash;14.\u003c/li\u003e\n\u003cli\u003eWong LC, Wang L, D\u0026rsquo;Amour JA, Yumita T, Chen G, Yamaguchi T, et al. Effective modulation of male aggression through lateral septum to medial hypothalamus projection. Current Biology. 2016;26:593\u0026ndash;604.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":true,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-3660090/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-3660090/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eAlthough the participation of sex hormones and sex hormone-responsive neurons in aggressive behavior has been extensively studied, the role of other systems within the hypothalamus-pituitary-gonadal (HPG) axis remains elusive. Here we assessed how the gonadotropin-releasing hormone (GnRH) and kisspeptin systems are impacted by escalated aggression in male mice. We used a combination of social isolation and aggression training (IST) to exacerbate mice’s aggressive behavior. Next, low-aggressive (group-housed, GH) and highly aggressive (IST) mice were compared regarding neuronal activity in the target populations and hormonal levels, using immunohistochemistry and ELISA, respectively. Finally, we used pharmacological and viral approaches to manipulate neuropeptide signaling and expression, subsequently evaluating its effects on behavior. IST mice exhibited enhanced aggressive behavior compared to GH controls, which was accompanied by elevated neuronal activity in GnRH neurons and arcuate nucleus kisspeptin neurons. Remarkably, IST mice presented an increased number of kisspeptin neurons in the anteroventral periventricular nucleus (AVPV). In addition, IST mice exhibited elevated levels of luteinizing hormone (LH) in serum. Accordingly, activation and blockade of GnRH receptors exacerbated and reduced aggression, respectively. Surprisingly, kisspeptin had intricate effects on aggression, i.e., viral ablation of AVPV-kisspeptin neurons impaired the training-induced rise in aggressive behavior whereas kisspeptin itself strongly reduced aggression in IST mice. Our results indicate that IST enhances aggressive behavior in male mice by exacerbating HPG-axis function. Particularly, increased GnRH neuron activity and peptide signaling were found to underlie aggression whereas the relationship with kisspeptin remains puzzling.\u003c/p\u003e","manuscriptTitle":"NPRC-Social isolation and aggression training lead to escalated aggression and hypothalamus-pituitary-gonad axis hyperfunction in mice","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2023-11-28 19:21:03","doi":"10.21203/rs.3.rs-3660090/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"05796d26-6b14-4e39-8072-be175d3c8d7b","owner":[],"postedDate":"November 28th, 2023","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":26754322,"name":"Biological sciences/Neuroscience/Emotion"},{"id":26754323,"name":"Biological sciences/Neuroscience/Social neuroscience"},{"id":26754324,"name":"Biological sciences/Physiology/Neurophysiology"},{"id":26754325,"name":"Health sciences/Diseases/Psychiatric disorders"}],"tags":[],"updatedAt":"2024-02-13T15:59:57+00:00","versionOfRecord":{"articleIdentity":"rs-3660090","link":"https://doi.org/10.1038/s41386-024-01808-3","journal":{"identity":"neuropsychopharmacology","isVorOnly":false,"title":"Neuropsychopharmacology"},"publishedOn":"2024-02-09 05:00:00","publishedOnDateReadable":"February 9th, 2024"},"versionCreatedAt":"2023-11-28 19:21:03","video":"","vorDoi":"10.1038/s41386-024-01808-3","vorDoiUrl":"https://doi.org/10.1038/s41386-024-01808-3","workflowStages":[]},"version":"v1","identity":"rs-3660090","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-3660090","identity":"rs-3660090","version":["v1"]},"buildId":"WrCJVZZCHTDjtuVLN7oU0","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}