Exploring neuronal markers and early social environment influence in divergent quail lines selected for social motivation

Exploring neuronal markers and early social environment influence in divergent quail lines selected for social motivation | 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 Exploring neuronal markers and early social environment influence in divergent quail lines selected for social motivation Lucas Court, Laura Talbottier, Julie Lemarchand, Fabien Cornilleau, and 7 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4521069/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 09 Oct, 2024 Read the published version in Scientific Reports → Version 1 posted 10 You are reading this latest preprint version Abstract Vertebrates, including humans exhibit a wide range of social behaviors that are crucial for the adaptation and survival of most species. The brain organization and function are shaped by genetic and environmental factors, although their precise contributions have been poorly explored in the context of artificial selection. We used divergent lines of quail selected on their high versus low level of motivation to approach a group of conspecifics (S+ and S-, respectively) to investigate the influence of genetic selection and early social environment on sociability. We observed distinct sex- and brain-region-specific expression patterns of three neuronal markers: mesotocin, and vasotocin, the avian homologues of mammalian oxytocin and vasopressin, as well as aromatase, the enzyme that converts androgens into estrogens. These markers displayed pronounced and neuroanatomically specific differences between S+ and S- quail. Additionally, we assessed the influence of early social environment on social skills in adolescent birds. Mixing S+ and S- resulted in more S- males approaching the group without affecting the sociability of S+ or other behaviors, suggesting that the early social environment may influence the results of genetic selection. In conclusion, the divergent quail lines offer a valuable model for unraveling the neuronal and behavioral mechanisms underlying social behaviors. Biological sciences/Neuroscience/Neural circuit Biological sciences/Neuroscience/Social behaviour mesotocin vasotocin aromatase social behavior social environment Japanese quail Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Introduction In vertebrates, social behaviors – the interactions between at least two conspecifics – encompass multiple forms of behavior, including sexual, aggressive and parental behaviors. They play a crucial role for the adaptation and survival of most social species, including humans 1-4 . Conversely, some neurological disorders including autism spectrum disorder (ASD) are characterized by social impairments 5,6 , leading to difficulties in adapting to the social environment. Social experience shapes neural circuits during development and influences social abilities in adults 7,8 . For example, social deprivation alters sociability while enriched social environment improves social skills in models of ASD 9-13 . However, few studies have examined the influence of early social environment and genetic factors and their interplay on sociability in the context of artificial selection. In vertebrates, social behaviors are regulated by an evolutionary conserved set of interconnected brain nuclei forming the social behavior network 14-18 . Oxytocin and vasopressin neuropeptides, as well as mesotocin (MT) and vasotocin (VT) homologs in birds, are primarily synthesized in neurons located in the paraventricular (PVN) and the supraoptic nuclei of the hypothalamus 19-21 . These neurons project to key brain structures within this network, including medial preoptic nucleus (MPN), bed nucleus of the stria terminalis (BNST), lateral septum (LS) and ventromedial nucleus of the hypothalamus (VMN) 22-25 . Moreover, receptors for these neuropeptides, as well as aromatase (ARO) that converts androgens into estrogens, and estrogen receptors are highly conserved and expressed in most brain nuclei within this network, thereby modulating social and socio-sexual behaviors 7,26-37 . Thus, MT, VT and ARO are valuable markers to study sociability. Birds exhibit a wide range of social behaviors that can be modulated through in ovo manipulations 38,39 or genetic selection 40 . They are useful models to study brain and neural mechanisms, as well as environmental and genetic factors affecting social behaviors. Divergent lines of Japanese quail ( Coturnix japonica ) have been selected over 45 generations for either low or high motivation to approach a group of conspecifics and designated as S- and S+, respectively 40 . Subsequent studies have confirmed the social divergence in their approach towards the group while mixed effects were also observed in their expression of sexual behavior, aggressiveness and reaction to humans 41-47 . Notably, S+ quail exhibit increased locomotor activity in response to social isolation 45,48,49 . These lines serve as valuable genetic models to investigate sociability and the influence of distinct social environments. However, the putative neuronal markers of sociability in Japanese quail, particularly in these divergent lines, remain to be tested. In this study, we explore the effects of quail genetic selection on three neurochemical markers that have been associated with sociality: MT, VT and ARO. Results reveal a differential expression of these markers between S+ and S- quail lines, particularly in the PVN and MPN. In addition, we tested the influence of early social environment on social abilities by mixing S+ and S- quail lines. S- male quail exposed to S+ males showed a partial improvement in social motivation when the percentage of birds approaching a social group was examined. These results suggest that divergent quail lines selected for sociability offer a valuable animal model for investigating the interplay between social environment and decades of genetic selection. Results Morphological differences between S+ and S- quail From hatching to adulthood, S+ quail exhibited a smaller body mass than their S- counterparts and their control (CTL) parental line in 7-month-old quail (Fig. 1A and Supplementary Fig. S1A-B and Supplementary Table S1). Similarly, S+ brain mass was decreased compared to S- adult quail (Fig. 1B). In parallel, all brain measurements collected in the PVN, MPN, BNST, and LS were significantly smaller in S+ than in S- brains (Fig. 1C). Therefore, subsequent quantifications were normalized based on body mass or brain measurements. In 7-month-old quail, S+ males exhibited smaller testes than S- but no line-related change was found in the cloacal gland area of males or of ovarian follicle mass and cloacal opening of female quail after correction for body mass (Fig. 1D-E; no difference was similarly present without correction, see Supplementary Table S1). At the age of 3 weeks, the cloacal gland area or opening was significantly larger in S- birds but these differences disappeared after correction for body mass (Supplementary Fig. S1C-D and Supplementary Table S1). Brain region- and sex- specific expression of MT, VT and ARO social markers in divergent quail lines S+ and S- quail lines were compared to determine whether MT, VT, and ARO expression patterns had been impacted by the genetic selection. MT-immunoreactivity (-ir) somas were mainly located in the PVN while the highest density of MT-ir projecting fibers was observed in the MPN, BNST and LS (Fig. 2, Supplementary Fig. S2 and S3). Following the neuroanatomical nomenclature provided by Puelles and colleagues 50 , MT-ir somas and fibers were also identified in regions corresponding to the prethalamus (plates 14-15 of his atlas), the intermediate nucleus of the supraoptic decussation (plate 22), the tectothalamic tract (plate 22), the pregeniculate nucleus (plate 15), the arcuate hypothalamic nucleus (plate 27), the periventricular stratum (plate 27), the intercollicular area (plate 27), the periaqueductal gray (plate 27) and the parvicellular or magnocellular nucleus of the posterior commissure (plate 28) (Supplementary Fig. S3). Quantification revealed a lower absolute number or normalized number of MT-ir neurons in the PVN of S+ compared to S- quail, as well as a lower density of MT-ir fibers in the MPN of S+ males (Fig. 2A, Supplementary Fig S4A and Table S2). No difference in MT-ir was found in the BNST and LS. Although sex differences in oxytocin expression have rarely been described in mammals and birds 51,52 , we observed, for the first time in quail, a higher density of MT-ir fibers in MPN, BNST, and LS in males compared to females, while no sex difference was found in the number of neurons in the PVN (Fig. 2 and Supplementary Table S2). We observed a difference in the number of VT-ir neurons in the PVN but this difference disappeared after normalization (Fig. 3, Supplementary Figure S4B and Table S2). A lower density of VT-ir fibers was also observed in the MPN and BNST of S+ quail compared to S- but not in the LS. In line with previous studies 53-55 , large sex differences were observed in the density of VT-ir fibers, with higher densities of VT fibers being present in the MPN, BNST and LS of male quail compared to females (Fig. 3 and Supplementary Table S2). Finally, a lower absolute number or normalized number of ARO-ir cells was detected in the MPN of S+ quail compared to S-. A difference between lines was also found in the BNST, but this difference became only a statistical tendency following normalization. No difference between lines was observed in the VMN (Fig. 4, Supplementary Figure S4C and Supplementary Table S2). Contrary to previous studies 56,57 , we found no sex differences in the MPN nor in the BNST. This discrepancy might be due to the higher sociability displayed by the female quail in our study, unlike previous studies that used different quail strains, where most females remained immobile when given the choice to engage in social interaction 58 . A higher number of ARO-ir cells was however observed in the VMN of males compared to females (Fig. 4 and Supplementary Table S2). In conclusion, our results provide evidence that the three neuronal markers, MT, VT and ARO, are differentially expressed between the two divergent lines, thus validating the social divergence of these quail lines. Early social environment improved social ability in some S- males After the immunohistochemical studies confirming that these neuronal markers were differentially expressed between S+ and S- lines, we assessed the influence of distinct social environments from hatching to 3-4 weeks of age on social and non-social (not targeted) behaviors in adolescent males and females across 3 social groups (Fig. 5A-B). The first group exclusively consisted of S+ quail [S+], the second group comprised only S- quail [S-] (representing a low social environment for the S- quail), and the third group included quail raised with in a mix of 50% of S+ [S+(-)] and 50% of S- [S-(+)] (representing an enriched social environment for the S- quail). Given the observed differences in locomotion 45,48 , which could potentially influence sociability, the social motivation test was performed without activating the treadmill, unlike the methods used for the selection process 40 . In this adapted behavioral test, S- male quail exhibited a reduced time in the compartment adjacent to the group of conspecifics and a longer latency to reach the group compared to S+ males (Fig. 5C-D and Supplementary Table S3). Furthermore, only 30% of the S- males reached the compartment adjacent to the social group, while 100% of the S+ males rapidly joined the group (Fig. 5E). Although no significant difference was observed between S+ and S- female quail, likely due the high inter-individual variability, the social divergence was confirmed in both sexes when considering the social line in the statistical analysis (Supplementary Table S3). These results, particularly in males, confirmed the divergence in sociability between the two quail lines independently of treadmill use. In the group composed of a mix of S+ and S- quail, S+(-) male quail were not affected by the presence of S- males, while S-(+) inter-individual variability increased (Fig. 5C-D). Indeed, 70% of S-(+) male quail raised with S+ males approached the social group while only 30% S- males did (Fig. 5E). Notably, the proportion of S-(+) males reaching the group was no longer different than S+ and S+(-), suggesting that an early enriched social environment with S+ partially restored S- social abilities. Subsequently, we examined the influence of these distinct early social environments in the open field and in the tonic immobility tests. In the open field, S+ male and female quail traveled a longer distance compared to the S- quail (Fig. 6A), and S-(+) performed similarly to S- quail, despite a high inter-individual variability. Furthermore, irrespective of the social environment they had been exposed, S+ and S- quail lines spent similar time in the center of the open field (Fig. 6B) or immobile (Fig. 6C, Supplementary Table S3), two measures considered as an index of anxious-like behaviors or of an innate behavioral response to predators. In conclusion, our results confirm the social divergence between the S+ and S- lines, particularly in male quail. Furthermore, an early enriched social environment with S+ partially restored S- social abilities in some male quail without affecting non targeted behaviors. Discussion In this study, we found a reduction in body and brain masses in the S+ quail line relative to S-, along with similar differences in the size of the male gonads and brain structures. This difference in body mass was previously reported 45 and might be partly explained by the genetic selection using a treadmill. Indeed, quail lines exhibiting low and high levels of social motivation were selected based on the speed and traveled distance to rejoin the social group 40 . Thus, the observed morphological differences might result from a selection of multiple phenotypic traits including social motivation, but also locomotor performance in the treadmill. It should also be noted that only the S+ line displayed a difference in body mass compared to the parental (S- were not different), in agreement with the idea that the genetic selection possibly concerned locomotor ability. Co-selection of multiple factors occurs during genetic selection and has been described in multiple species 59 . In addition, as these differences were present from hatching and persisted throughout life, we speculate that the genetic selection targeted genes that might be involved in developmental processes. In an attempt to compensate for these size differences, cell numbers were presented as absolute values and after normalization by the length of each brain structure. Note however that differences in immunoreactive cell numbers or fiber densities were not found in all brain regions of the social behavior network (e.g., LS or VMN), suggesting that their differential expression did not systematically mirror the differences in body mass. In the present study, we showed that the length of several brain structures, the number of MT neurons in the PVN, as well as the density of MT or VT fibers and the number of ARO cells in the MPN are reduced in the S+ quail line compared to the S- line. These differences clearly indicate that the genetic selection for high or low sociability had a major impact on the expression of several neurochemical markers in the social behavior network. The direction of the observed changes is however counterintuitive: a priori one would expect that the more social line would display higher densities of markers such as VT or MT (avian homolog of the mammalian OT and VP) that have been shown to be associated with social behavior. Two obvious reasons might explain this apparent discrepancy. First, it must be recalled that the S+ line also displayed a marked decrease in testis size and previous research has demonstrated that in mammals and birds, including quail, the expression of MT/OT, VT/VP and ARO is activated by testosterone (e.g., 27,29,33,53,56,60 ). If as suspected the decreased testis mass was associated with a lower testosterone circulating concentration, this could explain the decreased density of markers in males of the S+ line. This mechanism could also potentially apply to females even if the difference in gonadal development is less obvious in this sex. The second reason that could explain the unexpected changes in neurochemical markers is that the density of an immunocytochemical marker reflects both its production (synthesis) turnover and its rate of release and use by the brain. A lower density of the immunocytochemical signal might thus reflect a decreased synthesis and/or a faster release of the corresponding peptide (MT, VT) or protein (ARO). It is indeed well-known that some neuropeptides are very difficult to reveal by immunocytochemistry if one does not previously block their release by a drug such as colchicine. The lower density of fibers observed here in the S+ quail might thus reflect a higher activity of MT, VT and ARO neurons resulting in a more intensive release. Additional work collecting information on the rate of synthesis of these markers, using quantitative in situ hybridization, quantitative PCR for the corresponding mRNAs or MT/VT peptide dosage, would be needed to test this interpretation. Previous studies using optogenetic or neurochemical manipulations have indeed highlighted the functional role of OT, AVP and ARO on social behaviors across various species. In male mice, inhibiting or activating PVN-OT neurons respectively impaired or facilitated social behavior 61,62 . Similarly, in male rats, activating the magnocellular OT neurons of the PVN, supraoptic and accessory nuclei of the hypothalamus, resulted in a higher social motivation towards an opponent in a social defeat paradigm 63 . Moreover, in zebra finches, knockdown of OT and VT synthesis in the PVN resulted in sex-specific effects on gregariousness and aggressiveness 64 . Similarly in mice, deleting AVP expressing cells in the PVN increased social investigation in females 65 while knockdown of AVP expression in BNST reduced social investigation, sexual and aggressive behaviors in males 66 . Similar approaches should be used to assess MT and VT activity and peptide release in these divergent quail lines. Finally, the role of brain aromatase in the control of social behavior has been poorly investigated. In quail, chronic intracerebroventricular administration of an aromatase inhibitor affected social motivation depending on the sex of the stimuli used in the group 33 . In addition, site-specific inhibition of ARO in the male quail MPN or BNST reduced the expression of sexual behavior 67,68 . Optogenetic or chemogenetic manipulations of aromatase-expressing neurons similarly affect aggressiveness and some components of sexual behavior in mice 69 . However a study examining sexual or aggressive behaviors in the divergent quail lines found no differences between S+ and S- quail at the age of 7 weeks 45 . The present results demonstrating differences in ARO cell number between S+ and S- quail would then suggest that this neuronal marker could be implicated in social motivation besides its potential role in sexual behavior. Overall, further pharmacological or genetic manipulations to target PVN or MPN would be needed to confirm a differential functional role of MT, VT and ARO in the expression of social motivation or in other forms of social behavior. Although the treadmill was not used in the present study and the sociability tests were performed in 6-week-old quail rather than quail chicks, the social divergence between S+ and S- males was confirmed without any associated effect on anxious-like behaviors or on a presumed innate behavioral response to predators. These findings confirm previous studies 40,45,48 and support the idea that the differential expression in MT and VT observed here are specific to social abilities and do not relate to anxious-like behaviors or stress. These neuropeptides are indeed implicated in both processes (review in 70 ). Interestingly, the early enriched social environment mixing S+ and S- male quail led to an intermediary profile of sociability in S-(+) males relative to S+ and S- counterparts, without affecting other behaviors. This suggests that a subset of S-(+) males exhibit behavioral flexibility and can be influenced by their conspecifics. This study thus provides original information on the influence of the early social environment in divergent lines of animals selected for sociability. More generally, few studies have been conducted on the influence of early social environment on genetic models. Consistent with our results, improved social interactions were found in a mouse model of social deficits, the inbred BTBR T1tf/J mouse line or in mice exposed in utero to valproic acid, when reared with control cage mates 11,12 . Furthermore, Shank3 knock-out mice also display differential social skills when exposed to low or enriched early social environment 13 . Overall, the divergent quail lines selected for social motivation are a valuable model to study the genetic, neural, and behavioral aspects underlying social skills and provide insights into potential behavioral strategies to optimize animal welfare and human conditions associated with social deficits. Methods Animals Japanese quail chicks ( Coturnix japonica ) were selected on their high versus low levels of motivation to approach a group of conspecifics (S+ and S-) while ensuring no difference in the tonic immobility test 40 over 45 generations. Independent batches of 7-month-old male and female quail were used to characterize potential morphological differences between the divergent quail lines (batch 1 only) and to perform brain measurements and immunohistochemistry for neuronal markers (batches 1 and 2). Male and female quail were raised separately in groups on the ground floor until they reached sexual maturity and then, they were transferred in cages of one male and two females (batches 1 and 2), with colored balls provided as enrichment. A third batch of birds was used to investigate the influence of early social environment on the later sociability of the divergent lines in adolescent quail (batch 3). During the period covering the experiment, quail were housed on the ground floor that was covered with wood chips and enrichments were composed of tunnels made with plastic mesh. In all batches, adolescent or adult quail were maintained under a 12 h light and 12 h dark photoperiod. Food and water were provided ad libitum . We confirm that the experimental protocol was approved by Ethics Committee CNREEA Val de Loire N°19 animal care committee’s regulations and the French and European Directives (Advisory CE 19-2022-2505-1). The experimental procedures were performed following the ARRIVE guidelines. All animal procedures were performed in accordance with the Ethics Committee CNREEA Val de Loire N°19 animal care committee’s regulations, with relevant named guidelines, and French and European Directives (Advisory CE 19-2022-2505-1). Incubation Eggs were selected and gathered by quail lines for incubation at 37.5°C and 55% of relative humidity over 15 days (Egg Incubator Zundel). On incubation day 15, eggs were candled and if considered as developed, they were gathered in stainless steel hatching baskets (93 x 32 x 8 cm) to prevent mixing of chicks from distinct quail lines. Humidity was increased to 75% to facilitate hatching. On incubation day 19, newly hatched quail chicks were labelled with a ring placed on the right wing. Chicks were then placed in a brooder at a temperature of 38-40°C that was gradually decreased over two weeks to reach room temperature (about 22°C). Sex genotyping Sex identification of newly hatched quail was adapted from a previous study 71 . Five down feathers were collected behind the neck of the quail chicks with a pair of forceps washed with 1% liquid detergent RBS followed by distilled water between each bird. Feathers were placed into a 1.5 mL Eppendorf tube and stored at room temperature. On the same day, genomic DNA extraction was performed by incubating feathers in 200 µl of 10% Chelex® 100 Resin (Bio-rad, 142-1253) in distilled water and 2 µl of proteinase K (20mg/ml; Qiagen, RP103B) for 1 hour at 55°C in a heating-shaking dry bath, followed by centrifugation at 1000 rpm and supernatant collection. PCR was performed using 2 µl of 10X PCR DNA-free MgCl 2 buffer, 1 µl of 50mM DNA free MgCl 2 , 0.4 µl of 10 mM dNTP, 0.2 µl of Invitrogen Platinum Taq DNA polymerase (Thermo Fisher Scientific, 10966034), 1 µl of sample and 0.4 µl of the three primers (ZF, WF and P2) mixed in a total reaction volume of 20 µl of nuclease-free water. Primers for sex determination were as followed ZF (5ʹ-CTCTGGGTTTTGACTGTATTG-3ʹ) and WF (5ʹ-CATCTGTTTTCCCCCCCAAA-3ʹ) forward primers for the Z and W allele, respectively, as well as P2 common reverse primer (5ʹ-TCTGCATCGCTAAATCCTTT-3ʹ). The PCR program was set at 95°C for 5 min followed by 30 cycles of 45 s for each step at 95°C, 58°C and 72°C and a final extension at 72°C for 10 min. 5 µL of PCR volume were migrated on a 2% agarose gel stained with GelRed (Biotium). Based on PCR results, the 2-day-old quail chicks were separated by sex. Morphology and brain measurements Total body mass (g) was measured at three distinct time points: hatching, 3 weeks at the end of the social motivation test (batch 3), and at 7 months (batch 1). The cloacal gland area (length x width) and the cloacal opening (width) considered as indirect markers of circulating concentrations of androgens 72-74 and estrogens 75-77 respectively were also assessed at 3 weeks (batch 1) and 7 months (batch 3). Additional measures of the brain mass, the mass of the two testes and the mass of the largest ovarian follicle (g) were performed in the S+, S- and CTL phenotypes (batch 1). Brain measurements were obtained using neuroanatomical markers specific to the brain sections containing the PVN, MPN, BNST, LS and VMN (batch 1 and 2). The rostral part of the PVN is located at the level of the hippocampal pallial commissure and the beginning of the supraoptic decussation, corresponding to plates 15 and 16 of the chick brain atlas 50 . This atlas provides descriptions that are relatively closer, more detailed and updated than the earlier quail brain atlas version 78 . In the brain section containing the PVN, the length between the top of the pallial commissure to the top of the dorsal supraoptic decussation was measured within the medial part of the brain section, at the level of the third ventricle. The left and right caudal MPN, rostral BNST and medial LS are located in brain sections corresponding to plate 14 50 where the anterior commissure reaches its largest extension. In the brain section containing MPN, the length was measured between the bottom of the anterior commissure to the top of the periventricular fibers at the level of the third ventricle (for MT- or VT-immunostained brain sections) or between the bottom of the anterior commissure to the top of the optic chiasm (for the ARO-immunostained brain sections) at the level of the third ventricle. In the same brain section containing the BNST and LS, the length was measured between the internal edge of the left and right lateral ventricles. Additional measures of the area of MPN, BNST and VMN containing ARO-ir cells were obtained by drawing a polygon shape around the population of these immunoreactive cells. Finally, the rostral VMN was located at the beginning of the median eminence when the optic tectum is not fully developed corresponding approximatively to plates 22 to 23 50 . At the VMN level, the length used for standardization was measured between the two central external parts of each hemisphere. Immunohistochemistry Seven-month-old birds were transcardially perfused with 4% formaldehyde (VWR Chemicals BDH, 20910.363) in 0.01 M phosphate-buffered saline (PBS, pH = 7.2) following anesthesia with sodium pentobarbital (Exagon 400mg/ml; 150 µl per quail) and a transcardiac injection of 100 µl heparine (25 000 UI/5 ml; Cheplapharm France). Brains were removed from the skull, post-fixed in formaldehyde 4% overnight and then rinsed three times in PBS (0.01 M, pH 7.4). Brains were then immersed in 30% sucrose with 0.5% sodium azide (Sigma-Aldrich, 8.22335) until they sank (approximately two nights), frozen on dry ice and stored at -70°C until further use. Seven to eight brains of S+ and S- male and female quail (MT, batch 1; VT and ARO, batch 2) were cryosectioned in four series of 30 μm thick coronal slices from the septopalliomesencephalic tract, which marks the rostral end of the medial preoptic nucleus, to the third nerve indicating the caudal end of the hypothalamus and stored in antifreeze at -20°C until further use. Antifreeze was prepared using 40% v/v PBS 0.01M, 1% polyvinylpyrrolidone (Sigma-Aldrich, P5288), 30% saccharose (VWR Chemicals BDH, 27478.296), 30% v/v ethylene glycol (VWR Chemicals BDH, 24041.297), which was adjusted with PBS to achieve the final volume. Brain sections were immunostained for each neuronal marker using validated antibodies against MT, VT and ARO according to previous studies 37,51,55 . Sections were first rinsed three times in 0.01 M tris-buffered saline (TBS, pH 7.6) for 5 minutes each (same duration applied for all following rinses). For ARO and MT, peroxidase activity was blocked with 0.6% of hydrogen peroxide (H 2 O 2; Merck Millipore, 1072090250) in TBS at room temperature (RT) for 20 or 30 minutes, respectively. After rinses with TBS (ARO) or TBS containing 0.1% Triton X-100 (TBST; MT), sections were blocked and permeabilized for 1 hur in 5% (ARO) or 20% (MT) normal goat serum (NGS; ARO: Merck Millipore, S26, MT: Cell signaling technology, 5425S) in TBST. For VT, peroxidase activity was blocked with 1% of H 2 O 2 at RT for 30 minutes with 10% NGS (Merck Millipore, S26) in TBST. Sections were then incubated with a rabbit anti-oxytocin primary antibody (1/10000, Immunostar, Ref: 20068), rabbit anti-arginine vasopressin primary antibody (1/5000, Merck Millipore, Ref: AB1565) or a rabbit anti-aromatase primary antibody (1/3000, Harada QR 02/05) with 2% (MT and VT) or 5% (ARO) NGS in TBST for two nights at 4°C. Sections were rinsed in TBS (VT and ARO) or TBST (MT) and incubated for 2 hours at RT with a goat anti-rabbit biotinylated secondary antibody (1/250 for MT and 1/400 for VT and ARO respectively, Jackson ImmunoResearch, Ref: 111-065-003) in TBST. Sections were washed in TBS (VT and ARO) or TBST (MT) and incubated for one hour and a half in the vectastain Elite ABC-HRP Kit, Peroxidase in TBST (1/200, 1/1000, 1/800 of each reagent for MT, VT and ARO, respectively; Vector Laboratories, PK-6100) at RT, then rinsed in TBS (VT and ARO) or TBST (MT). The peroxidase was visualized with 0.04% of 3,3’-diaminobenzidine tetrahydrochloride hydrate (Sigma-Aldrich, D537) used as chromogen along with 0.012% of H 2 O 2 in TBS at RT. Sections were finally rinsed, mounted on slides, dried overnight, left 10 min in xylene and cover-slipped using Eukitt (Sigma-Aldrich, 03989). Image acquisition and analysis Photographs of brain sections were acquired using a microscope slide scanner (Zeiss, Axio Scan.Z1) with an objective 20x/0.8 and a HV-F202SCL Hitachi camera. High-resolution images were generated by the Zen Blue 3.1 software (Carl Zeiss, Oberkochen, Germany) and visualized with QuPath software, version 0.5.0 79 . Specific brain regions were delimitated by rectangular quantification fields of identical dimensions across social lines and sexes in the left and right hemispheres for each brain region of interest using QuPath. These rectangular fields were then exported for further analyses in ImageJ 1.54f 80 . For the MPN, the rectangular quantification field was placed in the corner formed by the ventral edge of the anterior commissure and the lateral edge of the third ventricle. For MT and VT staining, small anatomical inter-individual differences prevented the positioning of a standardized quantification field covering the entire immunoreactive (ir) fibers of the MPN, without including magnocellular MT- or VT-ir cells and fibers from the periventricular region. Fiber density was thus quantified in a rectangular field covering the dorsal portion of the MPN, which remained identical across all brain sections regardless of social line and sex. For the ARO staining, the quantification field included the entire MPN, as no interfering signal was present in adjacent regions. In the BNST, LS and VMN, the quantification fields were placed to cover the entire immunoreactive part of the brain nucleus located dorsally to the most lateral edge of the anterior commissure, approximately under the lateral ventricle (BNST), adjacent to the lateral ventricle (LS), or in the corner formed by the lateral edge of the third ventricle and the ventral limit of the brain (VMN). The number of MT- and VT-ir cells within the rostral PVN was determined manually using the cell counter plugin in ImageJ. The number of ARO-ir cells in the left and right part of the caudal MPN, rostral BNST and rostral VMN was determined by an automatic method using the cyto2 model from the Cellpose deep learning-based segmentation method 81 in ImageJ. The percentage of area covered by MT- or VT-ir fibers in the left and right part of the caudal MPN, rostral BNST and medial LS was determined by a semi-automatic method using the threshold method in ImageJ where threshold was manually adjusted for each subject. The values from the left and right quantification field were averaged to obtain one single value for each brain region. The number of cells was normalized based on the length between neuroanatomical markers identified in the brain sections containing each brain nucleus of interest (detailed in the Morphology and brain measurements section). For all measurements and analyses, the investigator was blind to the social line or the sex of the birds. Early social environment Two-day-old quail chicks were segregated by sex from hatching to the age of 6 week in 6 social groups of 32 quail, generating 8 statistical groups: two groups (males and females) comprised solely S+ quail [S+], two groups only S- quail [S-] and two groups contained a ratio of 50% of S+ [S+(-)] and 50% of S- [S-(+)] quail. Nineteen days after hatching, quail were transferred in larger spaces comprising two rooms of 5.6 x 3.6 meters (one for each sex) divided in three equal compartments to receive the three distinct social groups (S+, S- and S+(-) with S-(+) respectively). Compartments were separated by wooden board at the bottom and wire mesh at the top to prevent visual contacts with the other groups. A third room was dedicated to the parental quail line (N= 25 quail of each sex) whose individuals were used as stimuli in subsequent behavioral tests. Behaviors Three- to four-week-old adolescent quail were used to conduct the behavioral tests in order to avoid the surge of testosterone production and the activation of sexual behaviors 82-84 . Ten to seventeen birds from each social group and sex were tested for social motivation, open field activity and tonic immobility with three and four resting days between each test. Birds were carried individually to the testing room in a transport box (15 [length] x 15 [width] x 20 [height] cm) made of opaque polycarbonate, alternating between sex and social group. The testing apparatus was cleaned using water and paper towels between subjects. Behavioral scoring and analyses were carried out by the investigator who was unaware of the social group to which the birds belonged. Social motivation test Three-week-old experimental quail placed at one end of a long corridor (245 [length] x 30 [width] x 30 [height] cm) was offered the choice to approach and stay close to a group of 5 age- and sex-matched quail from the parental line located at the other end. The corridor made with wire mesh was divided into five virtual compartments (C1-5) of equal length. The experimental quail was placed for 10 seconds for habituation in C1 defined by a transparent Plexiglas. Then, the partition was removed, and the experimental quail could freely move for 5 minutes within compartments C1 to C4 and get closer visual access to the group confined in C5 at the opposite end of the corridor. The full test was recorded. The time spent in each compartment was measured, as well as the latency to reach the compartment adjacent to the social group (C4). Open field test Four-week-old experimental quail were individually placed for 5 min in the center of a square arena (80 [length] x 80 [width] x 50 [height] cm) made of wood with the floor covered by a plastic surface and surrounded by an opaque green curtain. A light and a camera positioned above the center of the arena was used to record the behavior. Locomotor activity, also used in previous studies as a proxy for social response induced by short-term isolation 48,85 and anxious-like behaviors were assessed by quantifying the mean distance traveled and the time spent in the squared central zone, respectively. These measures were automatically measured using the EthoVision ® XT tracking software. Tonic immobility test Tonic immobility is an innate behavioral response to predators in birds 86-88 , which has been counter-selected during the divergence process in these two lines. A quail was placed and maintained on its back by the experimenter’s hand for 10 seconds (induction) in a plastic U-shaped cradle (20 [length] x 10 [width] x 10 [height] cm) covered with a cloth. Then, the experimenter’s hand was gently removed from the bird and the time spent by the bird to stand up was quantified (tonic immobility duration per se ). When the induction phase failed, the attempt was repeated. After 5 attempts, the tonic immobility duration was scored as 0 second. Conversely, a score of 300 seconds was attributed when a quail failed to stand up after the maximal duration of the test set at 5 minutes. The tonic immobility duration and the number of induction attempts were quantified in this test. Statistics and writing Considering social line and sex as independent factors, morphological and behavioral data were analyzed by two-way ANOVAs followed by Sidak’s or Tukey’s post-hoc tests when significant. The data from the cloacal gland area, the cloacal opening and the testes or ovarian follicle masses were analyzed by ordinary one-way ANOVAs followed by Holm-Sidak’s post-hoc tests when significant. The percentage of male quail reaching the compartment adjacent to the social group in the social motivation test were compared using the Fisher’s exact probability test followed by a Bonferroni correction for multiple comparisons and thus, all p values were multiplied by the number of comparisons (adjusted p or p adj ). All analyses were performed with GraphPad Prism version 8.0.2. Results were considered significant for p value <0.05 and were presented by boxplots with individual data points, median, first and third quartiles and whiskers indicating the smallest and largest value. English language editing was partly provided by ChatGPT 3.5 developed by OpenAI. Declarations Acknowledgements This work was supported by INRAE “SOCIALOME” project (PAF_29). Quail breeding and care was conducted in Poultry Experimental Facility (PEAT) from the INRAE experimental unit 1295 (F-37380 Nouzilly, France, DOI: 10.15454/1.5572326250887292E12). Microscopy was performed through the facilities and expertise of the "Plateforme d'Imagerie Cellulaire" (PIC) of the UMR PRC, INRAE. The authors thank Dr. Nobuhiro Harada for providing the aromatase antibodies. Author Contributions statement Lucas Court (LC), JL, FC, JB, CAC, MK, Ludovic Calandreau (LCC) and LP conceived the experiments; LC, LT, JL, EP, FC conducted the experiments; LC, JL, MK, LCC and LP analyzed the results; LC and LP wrote the paper; MCB contributed analytic tools. All authors reviewed the manuscript. Additional Information Competing interests The authors declare no competing interests. Data availability All data generated and analyzed during this study are included in the Supplementary Tables 1 to 3. References Lee, V. E., Arnott, G. & Turner, S. P. Social behavior in farm animals: Applying fundamental theory to improve animal welfare. Frontiers in Veterinary Science 9 , doi:10.3389/fvets.2022.932217 (2022). Regan, A., Radošić, N. & Lyubomirsky, S. Experimental effects of social behavior on well-being. Trends in Cognitive Sciences 26 , 987-998, doi:10.1016/j.tics.2022.08.006 (2022). Ferrara, N. C. et al. Neural Circuit Transitions Supporting Developmentally Specific Social Behavior. The Journal of neuroscience 43 , 7456-7462, doi:10.1523/jneurosci.1377-23.2023 (2023). Chen, P. & Hong, W. Neural Circuit Mechanisms of Social Behavior. Neuron 98 , 16-30, doi:10.1016/j.neuron.2018.02.026 (2018). Kennedy, D. P. & Adolphs, R. The social brain in psychiatric and neurological disorders. Trends in Cognitive Sciences 16 , 559-572, doi:10.1016/j.tics.2012.09.006 (2012). Barak, B. & Feng, G. Neurobiology of social behavior abnormalities in autism and Williams syndrome. Nature Neuroscience 19 , 647-655, doi:10.1038/nn.4276 (2016). Cushing, B. S. & Kramer, K. M. Mechanisms underlying epigenetic effects of early social experience: the role of neuropeptides and steroids. Neuroscience and biobehavioral reviews 29 , 1089-1105, doi:10.1016/j.neubiorev.2005.04.001 (2005). Veenema, A. H. Toward understanding how early-life social experiences alter oxytocin- and vasopressin-regulated social behaviors. Hormones and Behavior 61 , 304-312, doi:10.1016/j.yhbeh.2011.12.002 (2012). Burke, A. R., McCormick, C. M., Pellis, S. M. & Lukkes, J. L. Impact of adolescent social experiences on behavior and neural circuits implicated in mental illnesses. Neuroscience and biobehavioral reviews 76 , 280-300, doi:10.1016/j.neubiorev.2017.01.018 (2017). Strathearn, L. The elusive etiology of autism: nature and nurture? Frontiers in Behavioral Neuroscience 3 , 11, doi:10.3389/neuro.08.011.2009 (2009). Yang, M., Perry, K., Weber, M. D., Katz, A. M. & Crawley, J. N. Social peers rescue autism-relevant sociability deficits in adolescent mice. Autism research : official journal of the International Society for Autism Research 4 , 17-27, doi:10.1002/aur.163 (2011). Campolongo, M. et al. Sociability deficits after prenatal exposure to valproic acid are rescued by early social enrichment. Molecular Autism 9 , 36, doi:10.1186/s13229-018-0221-9 (2018). Gora, C. et al. Effect of the social environment on olfaction and social skills in WT and a mouse model of autism. Translational Psychiatry. , doi:10.21203/rs.3.rs-3759429/v1 (2024). O'Connell, L. A. & Hofmann, H. A. The vertebrate mesolimbic reward system and social behavior network: a comparative synthesis. The Journal of comparative neurology 519 , 3599-3639, doi:10.1002/cne.22735 (2011). O'Connell, L. A. & Hofmann, H. A. Evolution of a vertebrate social decision-making network. Science 336 , 1154-1157, doi:10.1126/science.1218889 (2012). Goodson, J. L. The vertebrate social behavior network: Evolutionary themes and variations. Hormones and behavior 48 , 11-22, doi:10.1016/j.yhbeh.2005.02.003 (2005). Goodson, J. L. & Kabelik, D. Dynamic limbic networks and social diversity in vertebrates: From neural context to neuromodulatory patterning. Frontiers in Neuroendocrinology 30 , 429-441, doi:10.1016/j.yfrne.2009.05.007 (2009). Robinson, G. E., Fernald, R. D. & Clayton, D. F. Genes and Social Behavior. Science 322 , 896-900, doi:10.1126/science.1159277 (2008). Donaldson, Z. R. & Young, L. J. Oxytocin, vasopressin, and the neurogenetics of sociality. Science 322 , 900-904, doi:10.1126/science.1158668 (2008). Liutkeviciute, Z., Koehbach, J., Eder, T., Gil-Mansilla, E. & Gruber, C. W. Global map of oxytocin/vasopressin-like neuropeptide signalling in insects. Scientific Reports 6 , 39177, doi:10.1038/srep39177 (2016). Goodson, J. L., Kelly, A. M. & Kingsbury, M. A. Evolving nonapeptide mechanisms of gregariousness and social diversity in birds. Horm Behav 61 , 239-250, doi:10.1016/j.yhbeh.2012.01.005 (2012). Caldwell, H. K. Oxytocin and Vasopressin: Powerful Regulators of Social Behavior. The Neuroscientist : a review journal bringing neurobiology, neurology and psychiatry 23 , 517-528, doi:10.1177/1073858417708284 (2017). Marlin, B. J. & Froemke, R. C. Oxytocin modulation of neural circuits for social behavior. Developmental Neurobiology 77 , 169-189, doi:10.1002/dneu.22452 (2017). Rigney, N., de Vries, G. J., Petrulis, A. & Young, L. J. Oxytocin, Vasopressin, and Social Behavior: From Neural Circuits to Clinical Opportunities. Endocrinology 163 , doi:10.1210/endocr/bqac111 (2022). Rigney, N., de Vries, G. J. & Petrulis, A. Modulation of social behavior by distinct vasopressin sources. Frontiers in Endocrinology 14 , 1127792, doi:10.3389/fendo.2023.1127792 (2023). Bons, N. The topography of mesotocin and vasotocin systems in the brain of the domestic mallard and japanese quail: Immunocytochemical identification. Cell and tissue research 213 , 37-51, doi:10.1007/BF00236919 (1980). Panzica, G., Aste, N., Castagna, C., Viglietti-Panzica, C. & Balthazart, J. Steroid-induced plasticity in the sexually dimorphic vasotocinergic innervation of the avian brain: behavioral implications. Brain research reviews 37 , 178-200, doi:10.1016/s0165-0173(01)00118-7 (2001). Balthazart, J., Absil, P., Viglietti-Panzica, C. & Panzica, G. C. Vasotocinergic innervation of areas containing aromatase-immunoreactive cells in the quail forebrain. Journal of Neurobiology 33 , 45-60 (1997). Absil, P., Baillien, M., Ball, G. F., Panzica, G. C. & Balthazart, J. The control of preoptic aromatase activity by afferent inputs in Japanese quail. Brain research. Brain research reviews 37 , 38-58, doi:10.1016/s0165-0173(01)00122-9 (2001). Stanić, D. et al. Characterization of aromatase expression in the adult male and female mouse brain. I. Coexistence with oestrogen receptors α and β, and androgen receptors. PloS one 9 , e90451-e90451, doi:10.1371/journal.pone.0090451 (2014). Soumier, A., Habart, M., Lio, G., Demily, C. & Sirigu, A. Differential fate between oxytocin and vasopressin cells in the developing mouse brain. iScience 25 , 103655, doi:10.1016/j.isci.2021.103655 (2022). Goodson, J. L., Schrock, S. E., Klatt, J. D., Kabelik, D. & Kingsbury, M. A. Mesotocin and Nonapeptide Receptors Promote Estrildid Flocking Behavior. Science 325 , 862-866 (2009). Court, L., Balthazart, J., Ball, G. F. & Cornil, C. A. Effect of chronic intracerebroventricular administration of an aromatase inhibitor on the expression of socio-sexual behaviors in male Japanese quail. Behavioural Brain Research , 113315, doi:10.1016/j.bbr.2021.113315 (2021). Albers, H. E. Species, sex and individual differences in the vasotocin/vasopressin system: Relationship to neurochemical signaling in the social behavior neural network. Frontiers in Neuroendocrinology 36 , 49-71, doi:10.1016/j.yfrne.2014.07.001 (2015). Dhakar, M. B., Stevenson, E. L. & Caldwell, H. K. in Oxytocin, Vasopressin and Related Peptides in the Regulation of Behavior (eds Donald Pfaff, Elena Choleris, & Martin Kavaliers) Ch. 1, 3 - 26 (Cambridge University Press, 2013). Wu, M. V. et al. Estrogen Masculinizes Neural Pathways and Sex-Specific Behaviors. Cell 139 , 61-72 (2009). Foidart, A. et al. Critical re-examination of the distribution of aromatase-immunoreactive cells in the quail forebrain using antibodies raised against human placental aromatase and against the recombinant quail, mouse or human enzyme. Journal of chemical neuroanatomy 8 , 267-282, doi:10.1016/0891-0618(95)00054-b (1995). Csillag, A., Ádám, Á. & Zachar, G. Avian models for brain mechanisms underlying altered social behavior in autism. Frontiers in physiology 13 , 1032046, doi:10.3389/fphys.2022.1032046 (2022). Balthazart, J. & Ball, G. F. The Japanese quail as a model system for the investigation of steroid-catecholamine interactions mediating appetitive and consummatory aspects of male sexual behavior. Annual Review of Sex Research 9 , 96-176 (1998). Mills, A. D. & Faure, J. M. Divergent selection for duration of tonic immobility and social reinstatement behavior in Japanese quail (Coturnix coturnix japonica) chicks. Journal of Comparative Psychology 105 , 25-38, doi:10.1037/0735-7036.105.1.25 (1991). Launay, F., Mills, A. D. & Faure, J. M. Social motivation in Japanese quail coturnix coturnix japonica chicks selected for high or low levels of treadmill behaviour. Behavioural Processes 24 , 95-110, doi:10.1016/0376-6357(91)90002-H (1991). Launay, F., Mills, A. D. & Faure, J. M. Effects of test age, line and sex on tonic immobility responses and social reinstatement behaviour in Japanese quail Coturnix japonica. Behavioural Processes 29 , 1-16, doi:10.1016/0376-6357(93)90023-k (1993). Mills, A. D., Jones, R. B. & Faure, J. M. Species specificity of social reinstatement in Japanese quail Coturnix japonica genetically selected for high or low levels of social reinstatement behaviour. Behavioural Processes 34 , 13-22, doi:10.1016/0376-6357(94)00044-h (1995). Jones, R. B., Mills, A. D. & Faure, J. M. Social discrimination in Japanese quail Coturnix japonica chicks genetically selected for low or high social reinstatement motivation. Behavioural Processes 36 , 117-124, doi:10.1016/0376-6357(95)00024-0 (1996). Recoquillay, J. et al. Evidence of phenotypic and genetic relationships between sociality, emotional reactivity and production traits in Japanese quail. PloS one 8 , e82157, doi:10.1371/journal.pone.0082157 (2013). Burns, M., Domjan, M. & Mills, A. D. Effects of genetic selection for fearfulness or social reinstatement behavior on adult social and sexual behavior in domestic quail (Coturnix japonica). Psychobiology 26 , 249-257, doi:10.3758/BF03330613 (1998). François, N., Decros, S., Picard, M., Faure, J. M. & Mills, A. D. Effect of group disruption on social behaviour in lines of Japanese quail (Coturnix japonica) selected for high or low levels of social reinstatement behaviour. Behavioural Processes 48 , 171-181, doi:10.1016/S0376-6357(99)00081-9 (2000). Mills, A. D., Jones, R. B., Faure, J. M. & Williams, J. B. Responses to isolation in Japanese quail genetically selected for high or low sociality. Physiology & Behavior 53 , 183-189, doi:10.1016/0031-9384(93)90029-f (1993). Schweitzer, C., Houdelier, C., Lumineau, S., Lévy, F. & Arnould, C. Social motivation does not go hand in hand with social bonding between two familiar Japanese quail chicks, Coturnix japonica. Animal Behaviour 79 , 571-578, doi:10.1016/j.anbehav.2009.11.023 (2010). Puelles, L., Martinez-de-la-Torre, M., Martinez, S., Watson, C. & Paxinos, G. The Chick Brain in Stereotaxic Coordinates and Alternate Stains: Featuring Neuromeric Divisions and Mammalian Homologies . 2nd Edition Featuring Neuromeric Divisions and Mammalian Homologies edn, (Academic Press, 2018). Haakenson, C. M., Balthazart, J., Madison, F. N. & Ball, G. F. The neural distribution of the avian homologue of oxytocin, mesotocin, in two songbird species, the zebra finch and the canary: A potential role in song perception and production. Journal of Comparative Neurology 530 , 2402-2414, doi:10.1002/cne.25338 (2022). Dumais, K. M. & Veenema, A. H. Vasopressin and oxytocin receptor systems in the brain: Sex differences and sex-specific regulation of social behavior. Frontiers in Neuroendocrinology 40 , 1-23, doi:10.1016/j.yfrne.2015.04.003 (2016). Viglietti-Panzica, C., Anselmetti, G. C., Balthazart, J., Aste, N. & Panzica, G. C. Vasotocinergic innervation of the septal region in the Japanese quail: sexual differences and the influence of testosterone. Cell and tissue research 267 , 261-265, doi:10.1007/bf00302963 (1992). Panzica, G. C. et al. Organizational effects of estrogens on brain vasotocin and sexual behavior in quail. Developmental Neurobiology 37 , 684-699, doi:10.1002/(sici)1097-4695(199812)37:43.0.co;2-u (1998). Court, L., Vandries, L., Balthazart, J. & Cornil, C. A. Key role of estrogen receptor β in the organization of brain and behavior of the Japanese quail. Hormones and Behavior , 104827, doi:10.1016/j.yhbeh.2020.104827 (2020). Foidart, A., de Clerck, A., Harada, N. & Balthazart, J. Aromatase-immunoreactive cells in the quail brain: effects of testosterone and sex dimorphism. Physiology & Behavior 55 , 453-464, doi:10.1016/0031-9384(94)90100-7 (1994). Balthazart, J., Tlemçani, O. & Harada, N. Localization of testosterone-sensitive and sexually dimorphic aromatase-immunoreactive cells in the quail preoptic area. Journal of Chemical Neuroanatomy 11 , 147-171, doi:10.1016/0891-0618(96)00149-4 (1996). Court, L. Role of neuroestrogens in the regulation of social behaviors Doctor thesis, ULiège, (2022). Rauw, W. M., Kanis, E., Noordhuizen-Stassen, E. N. & Grommers, F. J. Undesirable side effects of selection for high production efficiency in farm animals: a review. Livestock Production Science 56 , 15-33, doi:10.1016/S0301-6226(98)00147-X (1998). Zhou, L., Blaustein, J. D. & De Vries, G. J. Distribution of androgen receptor immunoreactivity in vasopressin- and oxytocin-immunoreactive neurons in the male rat brain. Endocrinology 134 , 2622-2627, doi:10.1210/en.134.6.2622 (1994). Anpilov, S. et al. Wireless Optogenetic Stimulation of Oxytocin Neurons in a Semi-natural Setup Dynamically Elevates Both Pro-social and Agonistic Behaviors. Neuron 107 , 644-655.e647, doi:10.1016/j.neuron.2020.05.028 (2020). Resendez, S. L. et al. Social Stimuli Induce Activation of Oxytocin Neurons Within the Paraventricular Nucleus of the Hypothalamus to Promote Social Behavior in Male Mice. Journal of Neuroscience 40 , 2282-2295, doi:10.1523/jneurosci.1515-18.2020 (2020). Grund, T. et al. Chemogenetic activation of oxytocin neurons: Temporal dynamics, hormonal release, and behavioral consequences. Psychoneuroendocrinology 106 , 77-84, doi:10.1016/j.psyneuen.2019.03.019 (2019). Kelly, A. M. & Goodson, J. L. Hypothalamic oxytocin and vasopressin neurons exert sex-specific effects on pair bonding, gregariousness, and aggression in finches. Proceedings of the National Academy of Sciences 111 , 6069-6074, doi:10.1073/pnas.1322554111 (2014). Rigney, N., Whylings, J., de Vries, Geert J. & Petrulis, A. Sex Differences in the Control of Social Investigation and Anxiety by Vasopressin Cells of the Paraventricular Nucleus of the Hypothalamus. Neuroendocrinology 111 , 521-535, doi:10.1159/000509421 (2020). Rigney, N., Zbib, A., de Vries, G. J. & Petrulis, A. Knockdown of sexually differentiated vasopressin expression in the bed nucleus of the stria terminalis reduces social and sexual behaviour in male, but not female, mice. Journal of Neuroendocrinology 34 , e13083, doi:10.1111/jne.13083 (2022). Court, L., Balthazart, J., Ball, G. F. & Cornil, C. A. Role of aromatase in distinct brain nuclei of the social behaviour network in the expression of sexual behaviour in male Japanese quail. Journal of Neuroendocrinology 34 , e13127, doi:10.1111/jne.13127 (2022). de Bournonville, M. P., Vandries, L. M., Ball, G. F., Balthazart, J. & Cornil, C. A. Site-specific effects of aromatase inhibition on the activation of male sexual behavior in male Japanese quail (Coturnix japonica). Hormones and Behavior 108 , 42-49, doi:10.1016/j.yhbeh.2018.12.015 (2019). Bayless, D. W. et al. Limbic Neurons Shape Sex Recognition and Social Behavior in Sexually Naive Males. Cell , doi:10.1016/j.cell.2018.12.041 (2019). Neumann, I. D. & Landgraf, R. Balance of brain oxytocin and vasopressin: implications for anxiety, depression, and social behaviors. Trends in Neurosciences 35 , 649-659, doi:10.1016/j.tins.2012.08.004 (2012). Coustham, V., Godet, E. & Beauclair, L. A simple PCR method for sexing Japanese quail Coturnix japonica at hatching. British poultry science 58 , 59-62, doi:10.1080/00071668.2016.1246708 (2017). Sachs, B. D. Photoperiodic Control of the Cloacal Gland of the Japanese Quail. Science 157 , 201-203, doi:10.1126/science.157.3785.201 (1967). Balthazart, J., Schumacher, M. & Ottinger, M. A. Sexual differences in the Japanese quail: behavior, morphology, and intracellular metabolism of testosterone. General and Comparative Endocrinology 51 , 191-207, doi:10.1016/0016-6480(83)90072-2 (1983). Biswas, A., Ranganatha, O. S., Mohan, J. & Sastry, K. V. H. Relationship of cloacal gland with testes, testosterone and fertility in different lines of male Japanese quail. Animal reproduction science 97 , 94-102, doi:10.1016/j.anireprosci.2005.12.012 (2007). Noble, R. The effects of estrogen and progesterone on copulation in female quail (Coturnix coturnix japonica) housed in continuous dark. Hormones and Behavior 3 , 199-204, doi:10.1016/0018-506X(72)90032-3 (1972). Noble, R. Hormonal Control of Receptivity in Female Quail (Coturnix coturnix japonica). Hormones and Behavior 4 , 61-72, doi:10.1016/0018-506X(73)90017-2 (1973). Delville, Y. & Balthazart, J. Hormonal Control of Female Sexual Behavior in the Japanese Quail. Hormones and behavior 21 , 288-309, doi:10.1016/0018-506x(87)90016-x (1987). Baylé, J.-D., Ramade, F. & Oliver, J. Stereotaxic topography of the brain of the quail (Coturnix coturnix japonica) . Vol. 68 219-241 (The Journal of Physiology, 1974). Bankhead, P. et al. QuPath: Open source software for digital pathology image analysis. Scientific Reports 7 , 16878, doi:10.1038/s41598-017-17204-5 (2017). Schindelin, J. et al. Fiji: an open-source platform for biological-image analysis. Nature methods 9 , 676-682, doi:10.1038/nmeth.2019 (2012). Stringer, C., Wang, T., Michaelos, M. & Pachitariu, M. Cellpose: a generalist algorithm for cellular segmentation. Nature methods 18 , 100-106, doi:10.1038/s41592-020-01018-x (2021). Ottinger, M. A. & Brinkley, H. J. Testosterone and sex-related behavior and morphology: relationship during maturation and in the adult Japanese quail. Hormones and Behavior 11 , 175-182, doi:10.1016/0018-506X(78)90046-6 (1978). Ottinger, M. A. & Brinkley, H. J. Testosterone and sex related physical characteristics during the maturation of the male Japanese quail (Coturnix coturnix japonica). Biology of reproduction 20 , 905-909, doi:10.1095/biolreprod20.4.905 (1979). Panzica, G. C. et al. Sexual differentiation and hormonal control of the sexually dimorphic medial preoptic nucleus in the quail. Brain Research 416 , 59-68, doi:10.1016/0006-8993(87)91496-X (1987). Calandreau, L. et al. Higher inherent fearfulness potentiates the effects of chronic stress in the Japanese quail. Behavioural Brain Research 225 , 505-510, doi:10.1016/j.bbr.2011.08.010 (2011). Jones, R. B. The tonic immobility reaction of the domestic fowl: a review. World's Poultry Science Journal 42 , 82-96, doi:10.1079/WPS19860008 (1986). Gallup, G. G. Tonic immobility: The role of fear and predation. The Psychological Record 27 , 41-61 (1977). Jones, R. B., Mills, A. D. & Faure, J. M. Genetic and experiential manipulation of fear-related behavior in Japanese quail chicks (Coturnix coturnix japonica). Journal of Comparative Psychology 105 , 15-24, doi:10.1037/0735-7036.105.1.15 (1991). Additional Declarations No competing interests reported. Supplementary Files CourtquailSupplementaryfigureslegends.docx SupplementaryFigureS1.tif SupplementaryFigureS2.tif SupplementaryFigureS3.tif SupplementaryFigureS4.tif SupplementaryTableS1morphology.xlsx SupplementaryTableS2brain.xlsx SupplementaryTableS3behavior.xlsx Cite Share Download PDF Status: Published Journal Publication published 09 Oct, 2024 Read the published version in Scientific Reports → Version 1 posted Editorial decision: Revision requested 08 Aug, 2024 Reviews received at journal 07 Aug, 2024 Reviews received at journal 12 Jul, 2024 Reviewers agreed at journal 25 Jun, 2024 Reviewers agreed at journal 24 Jun, 2024 Reviewers invited by journal 24 Jun, 2024 Editor assigned by journal 22 Jun, 2024 Editor invited by journal 10 Jun, 2024 Submission checks completed at journal 05 Jun, 2024 First submitted to journal 03 Jun, 2024 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4521069","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":314963234,"identity":"6e27f75a-01dc-45f2-97dc-9e047228a3cb","order_by":0,"name":"Lucas Court","email":"","orcid":"","institution":"National Research Institute for Agriculture, Food and Environment","correspondingAuthor":false,"prefix":"","firstName":"Lucas","middleName":"","lastName":"Court","suffix":""},{"id":314963235,"identity":"393130c9-8bdf-4de6-b463-386549f64855","order_by":1,"name":"Laura Talbottier","email":"","orcid":"","institution":"National Research Institute for Agriculture, Food and Environment","correspondingAuthor":false,"prefix":"","firstName":"Laura","middleName":"","lastName":"Talbottier","suffix":""},{"id":314963236,"identity":"26e27ca5-7778-438a-9e97-e9a52b3c0c8e","order_by":2,"name":"Julie Lemarchand","email":"","orcid":"","institution":"National Research Institute for Agriculture, Food and Environment","correspondingAuthor":false,"prefix":"","firstName":"Julie","middleName":"","lastName":"Lemarchand","suffix":""},{"id":314963237,"identity":"497407a8-c08b-4174-a006-53e6c25559ec","order_by":3,"name":"Fabien Cornilleau","email":"","orcid":"","institution":"National Research Institute for Agriculture, Food and Environment","correspondingAuthor":false,"prefix":"","firstName":"Fabien","middleName":"","lastName":"Cornilleau","suffix":""},{"id":314963238,"identity":"04d1cf88-d126-4b98-9e48-15352aa2a053","order_by":4,"name":"Emmanuel Pecnard","email":"","orcid":"","institution":"National Research Institute for Agriculture, Food and Environment","correspondingAuthor":false,"prefix":"","firstName":"Emmanuel","middleName":"","lastName":"Pecnard","suffix":""},{"id":314963239,"identity":"4f53d5be-6b72-4ad5-9c53-a5cdad593df6","order_by":5,"name":"Marie-Claire Blache","email":"","orcid":"","institution":"National Research Institute for Agriculture, Food and Environment","correspondingAuthor":false,"prefix":"","firstName":"Marie-Claire","middleName":"","lastName":"Blache","suffix":""},{"id":314963240,"identity":"6ba3f94a-4d8a-43cd-b184-1e5d79988e3c","order_by":6,"name":"Jacques Balthazart","email":"","orcid":"","institution":"University of Liège","correspondingAuthor":false,"prefix":"","firstName":"Jacques","middleName":"","lastName":"Balthazart","suffix":""},{"id":314963241,"identity":"c152fabd-3d59-4eba-b3f3-ca6344259edd","order_by":7,"name":"Charlotte Anne Cornil","email":"","orcid":"","institution":"University of Liège","correspondingAuthor":false,"prefix":"","firstName":"Charlotte","middleName":"Anne","lastName":"Cornil","suffix":""},{"id":314963242,"identity":"830fb189-8b3f-4c5e-94fe-cceb573b47f4","order_by":8,"name":"Matthieu Keller","email":"","orcid":"","institution":"National Research Institute for Agriculture, Food and Environment","correspondingAuthor":false,"prefix":"","firstName":"Matthieu","middleName":"","lastName":"Keller","suffix":""},{"id":314963243,"identity":"868c253a-bdc1-4ae1-b15b-23100bc481e7","order_by":9,"name":"Ludovic Calandreau","email":"","orcid":"","institution":"National Research Institute for Agriculture, Food and Environment","correspondingAuthor":false,"prefix":"","firstName":"Ludovic","middleName":"","lastName":"Calandreau","suffix":""},{"id":314963244,"identity":"88ef742d-1a19-4e3b-a8ca-18b5f2e13f1d","order_by":10,"name":"Lucie Pellissier","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAABCElEQVRIie2SvWrDMBRGPyOwFzlaFRraV7hF0FIIeZYWQ6ZCO3YIrcHgLoGs6Yt0VhAkSx4hSwl46pBsHtIftXKgQ+WsGXRAcOFyxHevBAQCR0jHHv1bJFEOECCY6wifEu+VmDVKt3Cdbt6iuKK5HKQPKTJ70xusTmPGim19379Ti3S+qR8gLz1OLIc0m6JSNlh5wml49Wo62ct4CdnTPuUWhsPclFZhIEMXhiukJR6lN5hVdjBPVrHB6ItUwVX08QnZqgDm+mdjkpMmYlyxNG9ReEWzMVXnpZslI2mDsd5cehWRZGu7n9WZSJ7X23o3IDFZquh91Pcqjv1b/KFdQPMBAoFAIPA/3xrlSl9dHWAxAAAAAElFTkSuQmCC","orcid":"","institution":"National Research Institute for Agriculture, Food and Environment","correspondingAuthor":true,"prefix":"","firstName":"Lucie","middleName":"","lastName":"Pellissier","suffix":""}],"badges":[],"createdAt":"2024-06-03 10:09:10","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4521069/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4521069/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1038/s41598-024-74906-3","type":"published","date":"2024-10-09T15:57:57+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":58633830,"identity":"027e66e5-60d9-4e6f-9b18-0fb381fc4fbb","added_by":"auto","created_at":"2024-06-19 06:17:43","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":304292,"visible":true,"origin":"","legend":"\u003cp\u003eMorphological data and brain measurements in adult quail lines selected for high versus low levels of social motivation (S+ vs. S-) along with the parental control quail line (CTL). \u003cstrong\u003eA.\u003c/strong\u003e Body mass. \u003cstrong\u003eB.\u003c/strong\u003e Brain mass. \u003cstrong\u003eC. \u003c/strong\u003eBrain measurements corresponding to the length of PVN, MPN, BNST and LS brain structures. \u003cstrong\u003eD.\u003c/strong\u003e Cloacal gland area and cloacal opening normalized by body mass. \u003cstrong\u003eE.\u003c/strong\u003e Testes and ovarian follicle masses normalized by body mass. Sample size includes 12 S+, 9 CTL and 11 S- male quail and 16 S+, 10 CTL, 17 S- female quail (batch 1; \u003cstrong\u003eA, B, D, E\u003c/strong\u003e) as well as 16 S+, 15 S- male and 15 S+, 15 S- female quail (batches 1 and 2; \u003cstrong\u003eC\u003c/strong\u003e). Morphological data were analyzed using two-way ANOVAs or one-way ANOVAs followed by Tukey’s (\u003cstrong\u003eA-C\u003c/strong\u003e) or Holm-Sidak's (\u003cstrong\u003eD-E\u003c/strong\u003e) post-hoc tests when the interaction was significant. *p \u0026lt; 0.5, **p \u0026lt; 0.01, ***p \u0026lt; 0.001. \u003cu\u003eAbbreviations\u003c/u\u003e: \u003cstrong\u003eBNST\u003c/strong\u003e: bed nucleus of the stria terminalis, \u003cstrong\u003eLS\u003c/strong\u003e: lateral septum, \u003cstrong\u003eMPN\u003c/strong\u003e: medial preoptic nucleus, \u003cstrong\u003ePVN\u003c/strong\u003e: paraventricular nucleus of the hypothalamus.\u003c/p\u003e","description":"","filename":"Figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-4521069/v1/8288af6825dff4c7be961ab2.png"},{"id":58632614,"identity":"6b528d12-a522-4c2f-a283-1fccffb25218","added_by":"auto","created_at":"2024-06-19 06:01:43","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":4124996,"visible":true,"origin":"","legend":"\u003cp\u003eSex- and brain region- specific expression of mesotocin (MT) in divergent quail lines selected for sociability. \u003cstrong\u003eA\u003c/strong\u003e. Normalized number of MT-immunoreactive (-ir) cells in the paraventricular nucleus (PVN) of adult male and female quail selected for a low level of sociability (S-; n = 8 by sex) compared to age- and sex-matched quail selected for a high level of sociability (S+; n = 8 by sex; batch 1). \u003cstrong\u003eB-D\u003c/strong\u003e. Percentage of area covered by MT-ir fibers in the medial preoptic nucleus (MPN; \u003cstrong\u003eB\u003c/strong\u003e), bed nucleus of the stria terminalis (BNST; \u003cstrong\u003eC\u003c/strong\u003e) and the lateral septum (LS; \u003cstrong\u003eD\u003c/strong\u003e). Photomicrographs illustrate the MT-ir cells and fibers in the PVN, MPN, BNST and LS of male (M) and female (F) quail from S+ or S- phenotypes. Data were analyzed using separate two-way ANOVAs for each brain nucleus followed by Sidak’s post-hoc test when the interaction was significant. **p \u0026lt; 0.01, ***p \u0026lt; 0.001, ns = not significant. \u003cu\u003eAbbreviations\u003c/u\u003e: \u003cstrong\u003eDSD\u003c/strong\u003e: dorsal supraoptic decussation, \u003cstrong\u003eLV\u003c/strong\u003e: lateral ventricle, \u003cstrong\u003eOC\u003c/strong\u003e: optic chiasm, \u003cstrong\u003epc\u003c/strong\u003e: pallial commissure, \u003cstrong\u003eVIII\u003c/strong\u003e: third ventricle.\u003c/p\u003e","description":"","filename":"Figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-4521069/v1/5284bd85fe5bcde23ad24e3e.png"},{"id":58632618,"identity":"7d2c8050-630f-47aa-b5e4-8b4666e8df72","added_by":"auto","created_at":"2024-06-19 06:01:43","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":4693118,"visible":true,"origin":"","legend":"\u003cp\u003eSex- and brain region- specific expression of vasotocin (VT) in divergent quail lines selected for sociability. \u003cstrong\u003eA\u003c/strong\u003e. Normalized number of VT-immunoreactive (-ir) cells in the paraventricular nucleus (PVN) of adult male and female quail selected for a low level of sociability (S-; n = 7 by sex) compared to age- and sex-matched quail selected for a high level of sociability (S+; n = 8 male and 7 female quail, batch 2) \u003cstrong\u003eB-D\u003c/strong\u003e. Percentage of area covered by VT-ir fibers in the medial preoptic nucleus (MPN; \u003cstrong\u003eB\u003c/strong\u003e), bed nucleus of the stria terminalis (BNST; \u003cstrong\u003eC\u003c/strong\u003e) and the lateral septum (LS; \u003cstrong\u003eD\u003c/strong\u003e). Photomicrographs illustrate the VT-ir cells and fibers in the PVN, MPN, BNST and LS of male (M) and female (F) quail from S+ or S- phenotypes. Data were analyzed using separate two-way ANOVAs for each brain nucleus. *p \u0026lt; 0.05, **p \u0026lt; 0.01, ***p \u0026lt; 0.001. \u003cu\u003eAbbreviations\u003c/u\u003e: \u003cstrong\u003eDSD\u003c/strong\u003e: dorsal supraoptic decussation, \u003cstrong\u003eLV\u003c/strong\u003e: lateral ventricle, \u003cstrong\u003eOC\u003c/strong\u003e: optic chiasm, \u003cstrong\u003epc\u003c/strong\u003e: pallial commissure, \u003cstrong\u003eVIII\u003c/strong\u003e: third ventricle.\u003c/p\u003e","description":"","filename":"Figure3.png","url":"https://assets-eu.researchsquare.com/files/rs-4521069/v1/b5fd577e81873d6ba270498a.png"},{"id":58633247,"identity":"3e274fa7-2396-4c9f-9cd3-f8f749b02c54","added_by":"auto","created_at":"2024-06-19 06:09:43","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":5645648,"visible":true,"origin":"","legend":"\u003cp\u003eSex- and brain region- specific expression of aromatase (ARO) in divergent quail lines selected for sociability. Normalized number of ARO-ir cells in the medial preoptic nucleus (MPN; \u003cstrong\u003eA\u003c/strong\u003e), bed nucleus of the stria terminalis (BNST; \u003cstrong\u003eB\u003c/strong\u003e) and the ventromedial nucleus of the hypothalamus (VMN; \u003cstrong\u003eC\u003c/strong\u003e) of adult male and female quail selected for a low level of sociability (S-; n = 7 by sex) compared to age- and sex-matched quail selected for a high level of sociability (S+; n = 8 male and 7 female quail, batch 2). Photomicrographs illustrate the ARO-ir cells in the left part of the MPN, BNST and VMN of male (M) and female (F) quail from S+ or S- phenotypes. Data were analyzed using separate two-way ANOVAs for each brain nucleus. (*)p \u0026lt; 0.10, *p \u0026lt; 0.05, **p \u0026lt; 0.01.\u003c/p\u003e","description":"","filename":"Figure4.png","url":"https://assets-eu.researchsquare.com/files/rs-4521069/v1/ba7275071407d7b830941adb.png"},{"id":58632620,"identity":"9371599d-6a30-4585-8ceb-cdf921078f3e","added_by":"auto","created_at":"2024-06-19 06:01:43","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":419161,"visible":true,"origin":"","legend":"\u003cp\u003eInfluence of early social environment on distinct social responses in adolescent quail selected for sociability. \u003cstrong\u003eA. \u003c/strong\u003eSchema of the experimental design consisting in 3 groups of experimental quail (32 males + 32 females) segregated by sex from hatching. Among those, we selected for behavioral studies a subgroup of quail selected on a high level of sociability (S+; n = 10 by sex), a subgroup of quail selected on a low level of sociability (S-; n = 10 by sex) or two mixed subgroups: S+(-) (n = 13 male and 15 female quail) and S-(+) (n = 17 male and 14 female quail; batch 3), each containing 50% S+ and 50% S- quail. \u003cstrong\u003eB.\u003c/strong\u003e Experimental timeline. \u003cstrong\u003eC-D. \u003c/strong\u003eTime spent in (\u003cstrong\u003eC\u003c/strong\u003e) and latency to reach (\u003cstrong\u003eD\u003c/strong\u003e) the compartment adjacent to a group of 5 age- and sex -matched control quail from the parental line by the experimental male or female quail during the social motivation test. \u003cstrong\u003eE. \u003c/strong\u003ePercentage of male quail able to reach the compartment adjacent to the group, at least once, during the social motivation test. Data were analyzed using two-way ANOVAs followed when significant by Tukey's post hoc tests (\u003cstrong\u003eC-D\u003c/strong\u003e) or by Fisher's exact probability test (\u003cstrong\u003eE\u003c/strong\u003e). *p \u0026lt; 0.05, **p \u0026lt; 0.01, ***p \u0026lt; 0.001, ns = not significant.\u003c/p\u003e","description":"","filename":"Figure5.png","url":"https://assets-eu.researchsquare.com/files/rs-4521069/v1/56f2c7543f44e0cfe9796fbf.png"},{"id":58632628,"identity":"78cc91d8-b06f-4883-8fcd-05dc39b7b6c1","added_by":"auto","created_at":"2024-06-19 06:01:44","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":257946,"visible":true,"origin":"","legend":"\u003cp\u003eInfluence of early social environment on non-social behaviors in adolescent quail selected for sociability. Tests were performed in 3 groups of experimental quail (32 males + 32 females) segregated by sex from hatching. Among those, we selected for behavioral studies a subgroup of quail selected on a high level of sociability (S+; n = 10 by sex), a subgroup of quail selected on a low level of sociability (S-; n = 10 by sex) or two mixed subgroups: S+(-) (n = 13 male and 15 female quail) and S-(+) (n = 17 male and 14 female quail; batch 3), each containing 50% S+ and 50% S- quail. \u003cstrong\u003eA.\u003c/strong\u003e Total distance traveled used as a measure of locomotor activity and social response induced by short-term isolation in the open field. \u003cstrong\u003eB.\u003c/strong\u003e Time spent in a central area of the open field as a measure of anxious-like behavior. \u003cstrong\u003eC.\u003c/strong\u003e Duration of immobility quantified by the time spent by an experimental quail on its back in the tonic immobility test, used as a measure of innate behavioral response to predators. Data were analyzed using two-way ANOVAs followed when significant by Tukey’s post-hoc tests. ***p \u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"Figure6.png","url":"https://assets-eu.researchsquare.com/files/rs-4521069/v1/b9421363fa14ddeba7b10f69.png"},{"id":66597881,"identity":"c1b2ed9b-0713-46ab-8479-4a59207f5e53","added_by":"auto","created_at":"2024-10-14 16:11:36","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":19134553,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4521069/v1/ddf72daf-6eb6-4d50-8048-7b2e157d108f.pdf"},{"id":58632617,"identity":"209a0710-b9b3-4920-8767-2a37c7f9efc0","added_by":"auto","created_at":"2024-06-19 06:01:43","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":5446316,"visible":true,"origin":"","legend":"","description":"","filename":"CourtquailSupplementaryfigureslegends.docx","url":"https://assets-eu.researchsquare.com/files/rs-4521069/v1/c8ddba22ca7036784a96906b.docx"},{"id":58633245,"identity":"07c02a24-bfed-422a-b368-b7024182a214","added_by":"auto","created_at":"2024-06-19 06:09:43","extension":"tif","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":738926,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryFigureS1.tif","url":"https://assets-eu.researchsquare.com/files/rs-4521069/v1/94cdfe4dfeb22e96d6e1b401.tif"},{"id":58632621,"identity":"03539599-7741-431e-a6f2-51f754703894","added_by":"auto","created_at":"2024-06-19 06:01:43","extension":"tif","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":6372896,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryFigureS2.tif","url":"https://assets-eu.researchsquare.com/files/rs-4521069/v1/d97c2b2171f8df6d08f9d331.tif"},{"id":58633248,"identity":"024b8b5a-23a1-4d9f-9bd5-7c6b157f0d11","added_by":"auto","created_at":"2024-06-19 06:09:43","extension":"tif","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":13483798,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryFigureS3.tif","url":"https://assets-eu.researchsquare.com/files/rs-4521069/v1/8b8be268ec05a011c39f5c43.tif"},{"id":58632625,"identity":"313585b9-0f5a-4941-a28a-5e4e9c8b5408","added_by":"auto","created_at":"2024-06-19 06:01:43","extension":"tif","order_by":5,"title":"","display":"","copyAsset":false,"role":"supplement","size":629890,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryFigureS4.tif","url":"https://assets-eu.researchsquare.com/files/rs-4521069/v1/3dceca7f308e5f76eb8177ac.tif"},{"id":58632624,"identity":"affda108-3971-44a7-889a-e501d59622b5","added_by":"auto","created_at":"2024-06-19 06:01:43","extension":"xlsx","order_by":6,"title":"","display":"","copyAsset":false,"role":"supplement","size":35778,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryTableS1morphology.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-4521069/v1/0ec569c179ae0dc8453f63ca.xlsx"},{"id":58632626,"identity":"1fb5f731-c711-4a6b-a923-294ca1f238b7","added_by":"auto","created_at":"2024-06-19 06:01:43","extension":"xlsx","order_by":7,"title":"","display":"","copyAsset":false,"role":"supplement","size":48891,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryTableS2brain.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-4521069/v1/47200084a80e8bcdc08b7f7a.xlsx"},{"id":58632627,"identity":"c0009a7b-ee08-4aa2-9f4e-873c854b250b","added_by":"auto","created_at":"2024-06-19 06:01:44","extension":"xlsx","order_by":8,"title":"","display":"","copyAsset":false,"role":"supplement","size":49327,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryTableS3behavior.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-4521069/v1/9972d75397f6eb80761e09dd.xlsx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Exploring neuronal markers and early social environment influence in divergent quail lines selected for social motivation","fulltext":[{"header":"Introduction","content":"\u003cp\u003eIn vertebrates, social behaviors \u0026ndash; the interactions between at least two conspecifics \u0026ndash; encompass multiple forms of behavior, including sexual, aggressive and parental behaviors. They play a crucial role for the adaptation and survival of most social species, including humans\u003csup\u003e1-4\u003c/sup\u003e. Conversely, some neurological disorders including autism spectrum disorder (ASD) are characterized by social impairments\u003csup\u003e5,6\u003c/sup\u003e, leading to difficulties in adapting to the social environment. Social experience shapes neural circuits during development and influences social abilities in adults\u003csup\u003e7,8\u003c/sup\u003e. For example, social deprivation alters sociability while enriched social environment improves social skills in models of ASD\u003csup\u003e9-13\u003c/sup\u003e. However, few studies have examined the influence of early social environment and genetic factors and their interplay on sociability in the context of artificial selection.\u003c/p\u003e\n\u003cp\u003eIn vertebrates, social behaviors are regulated by an evolutionary conserved set of interconnected brain nuclei forming the social behavior network\u003csup\u003e14-18\u003c/sup\u003e. Oxytocin and vasopressin neuropeptides, as well as mesotocin (MT) and vasotocin (VT) homologs in birds, are primarily synthesized in neurons located in the paraventricular (PVN) and the supraoptic nuclei of the hypothalamus\u003csup\u003e19-21\u003c/sup\u003e. These neurons project to key brain structures within this network, including medial preoptic nucleus (MPN), bed nucleus of the stria terminalis (BNST), lateral septum (LS) and ventromedial nucleus of the hypothalamus (VMN)\u003csup\u003e22-25\u003c/sup\u003e. Moreover, receptors for these neuropeptides, as well as aromatase (ARO) that converts androgens into estrogens, and estrogen receptors are highly conserved and expressed in most brain nuclei within this network, thereby modulating social and socio-sexual behaviors\u003csup\u003e7,26-37\u003c/sup\u003e. Thus, MT, VT and ARO are valuable markers to study sociability.\u003c/p\u003e\n\u003cp\u003eBirds exhibit a wide range of social behaviors that can be modulated through \u003cem\u003ein ovo\u003c/em\u003e manipulations\u003csup\u003e38,39\u003c/sup\u003e or genetic selection\u003csup\u003e40\u003c/sup\u003e. They are useful models to study brain and neural mechanisms, as well as environmental and genetic factors affecting social behaviors. Divergent lines of Japanese quail (\u003cem\u003eCoturnix japonica\u003c/em\u003e) have been selected over 45 generations for either low or high motivation to approach a group of conspecifics and designated as S- and S+, respectively\u003csup\u003e40\u003c/sup\u003e. Subsequent studies have confirmed the social divergence in their approach towards the group while mixed effects were also observed in their expression of sexual behavior, aggressiveness and reaction to humans\u003csup\u003e41-47\u003c/sup\u003e. Notably, S+ quail exhibit increased locomotor activity in response to social isolation\u003csup\u003e45,48,49\u003c/sup\u003e. These lines serve as valuable genetic models to investigate sociability and the influence of distinct social environments. However, the putative neuronal markers of sociability in Japanese quail, particularly in these divergent lines, remain to be tested.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eIn this study, we explore the effects of quail genetic selection on three neurochemical markers that have been associated with sociality:\u0026nbsp;MT, VT and ARO. Results reveal a differential expression of these markers between S+ and S- quail lines, particularly in the PVN and MPN. In addition, we tested the influence of early social environment on social abilities by mixing S+ and S- quail lines. S- male quail exposed to S+ males showed a partial improvement in social motivation when the percentage of birds approaching a social group was examined. These results suggest that divergent quail lines selected for sociability offer a valuable animal model for investigating the interplay between social environment and decades of genetic selection.\u003cstrong\u003e\u003cbr\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cstrong\u003eMorphological differences between S+ and S- quail\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFrom hatching to adulthood, S+ quail exhibited a smaller body mass than their S- counterparts and their control (CTL) parental line in 7-month-old quail (Fig. 1A and Supplementary Fig. S1A-B and Supplementary Table S1). Similarly, S+ brain mass was decreased compared to S- adult quail (Fig. 1B). In parallel, all brain measurements collected in the PVN, MPN, BNST, and LS were significantly smaller in S+ than in S- brains (Fig. 1C). Therefore, subsequent quantifications were normalized based on body mass or brain measurements.\u003c/p\u003e\n\u003cp\u003eIn 7-month-old quail, S+ males exhibited smaller testes than S- but no line-related change was found in the cloacal gland area of males or of ovarian follicle mass and cloacal opening of female quail after correction for body mass (Fig. 1D-E; no difference was similarly present without correction, see Supplementary Table S1). At the age of 3 weeks, the cloacal gland area or opening was significantly larger in S- birds but these differences disappeared after correction for body mass (Supplementary Fig. S1C-D and Supplementary Table S1).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eBrain region- and sex- specific expression of MT, VT and ARO social markers in divergent quail lines\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eS+ and S- quail lines were compared to determine whether MT, VT, and ARO expression patterns had been impacted by the genetic selection.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eMT-immunoreactivity (-ir) somas were mainly located in the PVN while the highest density of MT-ir projecting fibers was observed in the MPN, BNST and LS (Fig. 2, Supplementary Fig. S2 and S3). Following the neuroanatomical nomenclature provided by Puelles and colleagues\u003csup\u003e50\u003c/sup\u003e, MT-ir somas and fibers were also identified in regions corresponding to the prethalamus (plates 14-15 of his atlas), the intermediate nucleus of the supraoptic decussation (plate 22), the tectothalamic tract (plate 22), the pregeniculate nucleus (plate 15), the arcuate hypothalamic nucleus (plate 27), the periventricular stratum (plate 27), the intercollicular area (plate 27), the periaqueductal gray (plate 27) and the parvicellular or magnocellular nucleus of the posterior commissure (plate 28) (Supplementary Fig. S3).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eQuantification revealed a lower absolute number or normalized number of MT-ir neurons in the PVN of S+ compared to S- quail, as well as a lower density of MT-ir fibers in the MPN of S+ males (Fig. 2A, Supplementary Fig S4A and Table S2). No difference in MT-ir was found in the BNST and LS. Although sex differences in oxytocin expression have rarely been described in mammals and birds\u003csup\u003e51,52\u003c/sup\u003e, we observed, for the first time in quail, a higher density of MT-ir fibers in MPN, BNST, and LS in males compared to females, while no sex difference was found in the number of neurons in the PVN (Fig. 2 and Supplementary Table S2).\u003c/p\u003e\n\u003cp\u003eWe observed a difference in the number of VT-ir neurons in the PVN but this difference disappeared after normalization (Fig. 3, Supplementary Figure S4B and Table S2). A lower density of VT-ir fibers was also observed in the MPN and BNST of S+ quail compared to S- but not in the LS. In line with previous studies\u003csup\u003e53-55\u003c/sup\u003e, large sex differences were observed in the density of VT-ir fibers, with higher densities of VT fibers being present in the MPN, BNST and LS of male quail compared to females (Fig. 3 and Supplementary Table S2).\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;Finally, a lower absolute number or normalized number of ARO-ir cells was detected in the MPN of S+ quail compared to S-. A difference between lines was also found in the BNST, but this difference became only a statistical tendency following normalization. No difference between lines was observed in the VMN (Fig. 4, Supplementary Figure S4C and Supplementary Table S2). Contrary to previous studies\u003csup\u003e56,57\u003c/sup\u003e, we found no sex differences in the MPN nor in the BNST. This discrepancy might be due to the higher sociability displayed by the female quail in our study, unlike previous studies that used different quail strains, where most females remained immobile when given the choice to engage in social interaction\u003csup\u003e58\u003c/sup\u003e. A higher number of ARO-ir cells was however observed in the VMN of males compared to females (Fig. 4 and Supplementary Table S2).\u003c/p\u003e\n\u003cp\u003eIn conclusion, our results provide evidence that the three neuronal markers, MT, VT and ARO, are differentially expressed between the two divergent lines, thus validating the social divergence of these quail lines. \u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEarly social environment improved social ability in some S- males\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAfter the immunohistochemical studies confirming that these neuronal markers were differentially expressed between S+ and S- lines, we assessed the influence of distinct social environments from hatching to 3-4 weeks of age on social and non-social (not targeted) behaviors in adolescent males and females across 3 social groups (Fig. 5A-B). The first group exclusively consisted of S+ quail [S+], the second group comprised only S- quail [S-] (representing a low social environment for the S- quail), and the third group included quail raised with in a mix of 50% of S+ [S+(-)] and 50% of S- [S-(+)] (representing an enriched social environment for the S- quail).\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;Given the observed differences in locomotion\u003csup\u003e45,48\u003c/sup\u003e, which could potentially influence sociability, the social motivation test was performed without activating the treadmill, unlike the methods used for the selection process\u003csup\u003e40\u003c/sup\u003e. In this adapted behavioral test, S- male quail exhibited a reduced time in the compartment adjacent to the group of conspecifics and a longer latency to reach the group compared to S+ males (Fig. 5C-D and Supplementary Table S3). Furthermore, only 30% of the S- males reached the compartment adjacent to the social group, while 100% of the S+ males rapidly joined the group (Fig. 5E). \u0026nbsp;Although no significant difference was observed between S+ and S- female quail, likely due the high inter-individual variability, the social divergence was confirmed in both sexes when considering the social line in the statistical analysis (Supplementary Table S3). These results, particularly in males, confirmed the divergence in sociability between the two quail lines independently of treadmill use.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eIn the group composed of a mix of S+ and S- quail, S+(-) male quail were not affected by the presence of S- males, while S-(+) inter-individual variability increased (Fig. 5C-D). Indeed, 70% of S-(+) male quail raised with S+ males approached the social group while only 30% S- males did (Fig. 5E). Notably, the proportion of S-(+) males reaching the group was no longer different than S+ and S+(-), suggesting that an early enriched social environment with S+ partially restored S- social abilities.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eSubsequently, we examined the influence of these distinct early social environments in the open field and in the tonic immobility tests. In the open field, S+ male and female quail traveled a longer distance compared to the S- quail (Fig. 6A), and S-(+) performed similarly to S- quail, despite a high inter-individual variability. Furthermore, irrespective of the social environment they had been exposed, S+ and S- quail lines spent similar time in the center of the open field (Fig. 6B) or immobile (Fig. 6C, Supplementary Table S3), two measures considered as an index of anxious-like behaviors or of an innate behavioral response to predators.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eIn conclusion, our results confirm the social divergence between the S+ and S- lines, particularly in male quail. Furthermore, an early enriched social environment with S+ partially restored S- social abilities in some male quail without affecting non targeted behaviors.\u003cstrong\u003e\u003c/strong\u003e\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eIn this study, we found a reduction in body and brain masses in the S+ quail line relative to S-, along with similar differences in the size of the male gonads and brain structures. This difference in body mass was previously reported\u003csup\u003e45\u003c/sup\u003e and might be partly explained by the genetic selection using a treadmill. Indeed, quail lines exhibiting low and high levels of social motivation were selected based on the speed and traveled distance to rejoin the social group\u003csup\u003e40\u003c/sup\u003e. Thus, the observed morphological differences might result from a selection of multiple phenotypic traits including social motivation, but also locomotor performance in the treadmill.\u0026nbsp;It should also be noted that\u0026nbsp;only the S+ line displayed a difference in body mass compared to the parental (S- were not different), in agreement with the idea that the genetic selection possibly concerned locomotor ability. Co-selection of multiple factors occurs during genetic selection and has been described in multiple species\u003csup\u003e59\u003c/sup\u003e. In addition, as\u0026nbsp;these differences were present from hatching and persisted throughout life, we speculate that the genetic selection targeted genes that might be involved in developmental processes.\u0026nbsp;In an attempt to compensate for these size differences, cell numbers were presented as absolute values and after normalization by the length of each brain structure. Note however that differences in immunoreactive cell numbers or fiber densities\u0026nbsp;were not found in all brain regions of the social behavior network (e.g., LS or VMN), suggesting that their differential expression did not systematically mirror the differences in body mass.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eIn the present study, we showed that the length of several brain structures, the number of MT neurons in the PVN, as well as the density of MT or VT fibers and the number of ARO cells in the MPN are reduced in the S+ quail line compared to the S- line.\u0026nbsp;These differences clearly indicate that the genetic selection for high or low sociability had a major impact on the expression of several neurochemical markers in the social behavior network. The direction of the observed changes is however counterintuitive: \u003cem\u003ea priori\u0026nbsp;\u003c/em\u003eone would expect that the more social line would display higher densities of markers such as VT or MT (avian homolog of the mammalian OT and VP) that have been shown to be associated with social behavior. Two obvious reasons might explain this apparent discrepancy. First, it must be recalled that the S+ line also displayed a marked decrease in testis size and previous research has demonstrated that in mammals and birds, including quail, the expression of MT/OT, VT/VP and ARO is activated by testosterone (e.g.,\u0026nbsp;\u003csup\u003e27,29,33,53,56,60\u003c/sup\u003e). If as suspected the decreased testis mass was associated with a lower testosterone circulating concentration, this could explain the decreased density of markers in males of the S+ line. This mechanism could also potentially apply to females even if the difference in gonadal development is less obvious in this sex. The second reason that could explain the unexpected changes in neurochemical markers is that the density of an immunocytochemical marker reflects both its production (synthesis) turnover and its rate of release and use by the brain. A lower density of the immunocytochemical signal might thus reflect a decreased synthesis and/or a faster release of the corresponding peptide (MT, VT) or protein (ARO). It is indeed well-known that some neuropeptides are very difficult to reveal by immunocytochemistry if one does not previously block their release by a drug such as colchicine. The lower density of fibers observed here in the S+ quail might thus reflect\u0026nbsp;a higher activity of MT, VT and ARO neurons resulting in a more intensive release. Additional work collecting information on the rate of synthesis of these markers, using quantitative \u003cem\u003ein situ\u003c/em\u003e hybridization, quantitative PCR for the corresponding mRNAs or MT/VT peptide dosage, would be needed to test this interpretation.\u003c/p\u003e\n\u003cp\u003ePrevious\u0026nbsp;studies using\u0026nbsp;optogenetic or neurochemical manipulations\u0026nbsp;have indeed highlighted the functional role of OT, AVP and ARO on social behaviors across various species. In male mice, inhibiting or activating PVN-OT neurons respectively impaired or facilitated social behavior\u003csup\u003e61,62\u003c/sup\u003e. Similarly, in male rats, activating the magnocellular OT neurons of the PVN, supraoptic and accessory nuclei of the hypothalamus, resulted in a higher social motivation towards an opponent in a social defeat paradigm\u003csup\u003e63\u003c/sup\u003e. Moreover, in zebra finches, knockdown of OT and VT synthesis in the PVN resulted in sex-specific effects on gregariousness and aggressiveness\u003csup\u003e64\u003c/sup\u003e. Similarly in mice, deleting AVP expressing cells in the PVN increased social investigation in females\u003csup\u003e65\u003c/sup\u003e while\u0026nbsp;knockdown of AVP expression in BNST reduced social investigation, sexual and aggressive behaviors in males\u003csup\u003e66\u003c/sup\u003e. Similar approaches should be used\u0026nbsp;to assess MT and VT activity and peptide release in these divergent quail lines.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eFinally, the role of brain aromatase in the control of social behavior has been poorly investigated. In quail, chronic intracerebroventricular administration of an aromatase inhibitor affected social motivation depending on the sex of the stimuli used in the group\u003csup\u003e33\u003c/sup\u003e. In addition, site-specific inhibition of ARO in the male quail MPN or BNST reduced the expression of sexual behavior\u003csup\u003e67,68\u003c/sup\u003e. Optogenetic or chemogenetic manipulations of aromatase-expressing neurons similarly affect aggressiveness and some components of sexual behavior in mice\u003csup\u003e69\u003c/sup\u003e.\u0026nbsp;However a study examining sexual or aggressive behaviors in the divergent quail lines found no differences between S+ and S- quail at the age of 7 weeks\u003csup\u003e45\u003c/sup\u003e. The present results demonstrating differences in ARO cell number between S+ and S- quail would then suggest that this neuronal marker could be implicated in social motivation besides its potential role in sexual behavior. Overall, further pharmacological or genetic manipulations to target PVN or MPN would be needed to confirm a differential functional role of MT, VT and ARO in the expression of social motivation or in other forms of social behavior.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eAlthough the treadmill was not used in the present study and the sociability tests were performed in 6-week-old quail rather than quail chicks, the social divergence between S+ and S- males was confirmed without any associated effect on anxious-like behaviors or on a presumed innate behavioral response to predators. These findings confirm previous studies\u003csup\u003e40,45,48\u003c/sup\u003e and support the idea that the differential expression in MT and VT observed here are specific to social abilities and do not relate to anxious-like behaviors or stress. These neuropeptides are indeed implicated in both processes (review in\u0026nbsp;\u003csup\u003e70\u003c/sup\u003e). Interestingly, the early enriched social environment mixing S+ and S- male quail\u0026nbsp;led to an intermediary profile of sociability in S-(+) males relative to S+ and S- counterparts, without affecting other behaviors. This suggests that a subset of S-(+) males exhibit behavioral flexibility and can be influenced by their conspecifics.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThis study thus provides original information on the influence of the early social environment in divergent lines of animals selected for sociability. More generally, few studies have been conducted on the influence of early social environment on genetic models. Consistent with our results, improved social interactions were found in a mouse model of social deficits, the inbred BTBR T1tf/J mouse line or in mice exposed \u003cem\u003ein utero\u003c/em\u003e to valproic acid, when reared with control cage mates\u003csup\u003e11,12\u003c/sup\u003e. Furthermore, \u003cem\u003eShank3\u003c/em\u003e knock-out mice also display differential social skills when exposed to low or enriched early social environment\u003csup\u003e13\u003c/sup\u003e. Overall, the divergent quail lines selected for social motivation are a valuable model to study the genetic, neural, and behavioral aspects underlying social skills and provide insights into potential behavioral strategies to optimize animal welfare and human conditions associated with social deficits.\u003c/p\u003e"},{"header":"Methods","content":"\u003cp\u003e\u003cstrong\u003eAnimals\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eJapanese quail chicks (\u003cem\u003eCoturnix japonica\u003c/em\u003e) were\u0026nbsp;selected on their high versus low levels of motivation to approach a group of conspecifics (S+ and S-)\u0026nbsp;while ensuring no difference in the tonic immobility test\u003csup\u003e40\u003c/sup\u003e over 45 generations. Independent batches of 7-month-old male and female quail were used to characterize potential morphological differences between the divergent quail lines (batch 1 only) and to perform brain measurements and immunohistochemistry for neuronal markers (batches 1 and 2). Male and female quail were raised separately in groups on the ground floor until they reached sexual maturity and then, they were transferred in cages of one male and two females (batches 1 and 2), with colored balls provided as enrichment.\u003c/p\u003e\n\u003cp\u003eA third batch of birds was used to investigate the influence of early social environment on the later sociability of the divergent lines in adolescent quail (batch 3). During the period covering the experiment, quail were housed on the ground floor that was covered with wood chips and enrichments were composed of tunnels made with plastic mesh.\u003c/p\u003e\n\u003cp\u003eIn all batches, adolescent or adult quail were maintained under a 12 h light and 12 h dark photoperiod. Food and water were provided \u003cem\u003ead libitum\u003c/em\u003e. We confirm that the experimental protocol was approved by Ethics Committee CNREEA Val de Loire N\u0026deg;19 animal care committee\u0026rsquo;s regulations and the French and European Directives (Advisory CE 19-2022-2505-1). The experimental procedures were performed following the ARRIVE guidelines. All animal procedures were performed in accordance with the Ethics Committee CNREEA Val de Loire N\u0026deg;19 animal care committee\u0026rsquo;s regulations, with relevant named guidelines, and French and European Directives (Advisory CE 19-2022-2505-1).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eIncubation\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eEggs were selected and gathered by quail lines for incubation at 37.5\u0026deg;C and 55% of relative humidity over 15 days (Egg Incubator Zundel). On incubation day 15, eggs were candled and if considered as developed, they were gathered in stainless steel hatching baskets (93 x 32 x 8 cm) to prevent mixing of chicks from distinct quail lines. Humidity was increased to 75% to facilitate hatching. On incubation day 19, newly hatched quail chicks were labelled with a ring placed on the right wing. Chicks were then placed in a brooder at a temperature of 38-40\u0026deg;C that was gradually decreased over two weeks to reach room temperature (about 22\u0026deg;C).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSex genotyping\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSex identification of newly hatched quail was adapted from a previous study\u003csup\u003e71\u003c/sup\u003e. Five down feathers were collected behind the neck of the quail chicks with a pair of forceps washed with 1% liquid detergent RBS followed by distilled water between each bird. Feathers were placed into a 1.5 mL Eppendorf tube and stored at room temperature. On the same day, genomic DNA extraction was performed by incubating feathers in 200 \u0026micro;l of 10% Chelex\u0026reg; 100 Resin (Bio-rad, 142-1253) in distilled water and 2 \u0026micro;l of proteinase K (20mg/ml; Qiagen, RP103B) for 1 hour at 55\u0026deg;C in a heating-shaking dry bath, followed by centrifugation at 1000 rpm and supernatant collection. PCR was performed using 2 \u0026micro;l of 10X PCR DNA-free MgCl\u003csub\u003e2\u003c/sub\u003e buffer, 1 \u0026micro;l of 50mM DNA free MgCl\u003csub\u003e2\u003c/sub\u003e, 0.4 \u0026micro;l of 10 mM dNTP, 0.2 \u0026micro;l of Invitrogen Platinum Taq DNA polymerase (Thermo Fisher Scientific, 10966034), 1 \u0026micro;l of sample and 0.4 \u0026micro;l of the three primers (ZF, WF and P2) mixed in a total reaction volume of 20 \u0026micro;l of nuclease-free water. Primers for sex determination were as followed ZF (5ʹ-CTCTGGGTTTTGACTGTATTG-3ʹ) and WF (5ʹ-CATCTGTTTTCCCCCCCAAA-3ʹ) forward primers for the Z and W allele, respectively, as well as P2 common reverse primer (5ʹ-TCTGCATCGCTAAATCCTTT-3ʹ). The PCR program was set at 95\u0026deg;C for 5 min followed by 30 cycles of 45 s for each step at 95\u0026deg;C, 58\u0026deg;C and 72\u0026deg;C and a final extension at 72\u0026deg;C for 10 min. 5 \u0026micro;L of PCR volume were migrated on a 2% agarose gel stained with GelRed (Biotium). Based on PCR results, the 2-day-old quail chicks were separated by sex.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMorphology and brain measurements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTotal body mass (g) was measured at three distinct time points: hatching, 3 weeks at the end of the social motivation test (batch 3), and at 7 months (batch 1). The cloacal gland area (length x width) and the cloacal opening (width) considered as indirect markers of circulating concentrations of androgens\u003csup\u003e72-74\u003c/sup\u003e and estrogens\u003csup\u003e75-77\u003c/sup\u003e respectively were also assessed at 3 weeks (batch 1) and 7 months (batch 3). Additional measures of the brain mass, the mass of the two testes and the mass of the largest ovarian follicle (g) were performed in the S+, S- and CTL phenotypes (batch 1).\u003c/p\u003e\n\u003cp\u003eBrain measurements were obtained using neuroanatomical markers specific to the brain sections containing the PVN, MPN, BNST, LS and VMN (batch 1 and 2). The rostral part of the PVN is\u0026nbsp;located at the level of the hippocampal pallial commissure and the beginning of the supraoptic decussation, corresponding to plates 15 and 16 of the chick brain atlas\u003csup\u003e50\u003c/sup\u003e. This atlas provides descriptions that are relatively closer, more detailed and updated than the earlier quail brain atlas version\u003csup\u003e78\u003c/sup\u003e. In the brain section containing the PVN,\u0026nbsp;the length between the top of the pallial commissure to the top of the\u0026nbsp;dorsal supraoptic decussation was measured within the medial part of the brain section,\u0026nbsp;at the level of the third ventricle. The left and right caudal MPN, rostral BNST and medial LS are located\u0026nbsp;in brain sections corresponding to plate 14\u003csup\u003e50\u003c/sup\u003e where the anterior commissure reaches its largest extension. In the brain section containing MPN, the length was measured between the bottom of the anterior commissure to the top of the periventricular fibers at the level of the third ventricle (for MT- or VT-immunostained brain sections) or between the bottom of the anterior commissure to the top of the optic chiasm (for the ARO-immunostained brain sections) at the level of the third ventricle. In the same brain section containing the BNST and LS, the length was measured between the internal edge of the left and right lateral ventricles. Additional measures of the area of MPN, BNST and VMN containing ARO-ir cells were obtained by drawing a polygon shape around the population of these immunoreactive cells. Finally, the rostral VMN was located at the beginning of the median eminence when the optic tectum is not fully developed corresponding approximatively to plates 22 to 23\u003csup\u003e50\u003c/sup\u003e. At the VMN level, the length used for standardization was measured between the two central external parts of each hemisphere.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eImmunohistochemistry\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSeven-month-old birds were transcardially perfused with 4% formaldehyde (VWR Chemicals BDH, 20910.363) in 0.01 M phosphate-buffered saline (PBS, pH = 7.2) following anesthesia with sodium pentobarbital (Exagon\u003csup\u003e\u0026nbsp;\u003c/sup\u003e400mg/ml; 150 \u0026micro;l per quail) and a transcardiac injection of 100 \u0026micro;l heparine (25 000 UI/5 ml; Cheplapharm France). Brains were removed from the skull, post-fixed in formaldehyde 4% overnight and then rinsed three times in PBS (0.01 M, pH 7.4). Brains were then immersed in 30% sucrose with 0.5% sodium azide (Sigma-Aldrich, 8.22335) until they sank (approximately two nights), frozen on dry ice and stored at -70\u0026deg;C until further use. Seven to eight brains of S+ and S- male and female quail (MT, batch 1; VT and ARO, batch 2) were cryosectioned in four series of 30 \u0026mu;m thick coronal slices\u0026nbsp;from the septopalliomesencephalic tract, which marks the rostral end of the medial preoptic nucleus, to the third nerve indicating the caudal end of the hypothalamus and\u0026nbsp;stored in antifreeze at -20\u0026deg;C until further use. Antifreeze was prepared using 40% v/v PBS 0.01M, 1% polyvinylpyrrolidone (Sigma-Aldrich, P5288), 30% saccharose (VWR Chemicals BDH, 27478.296), 30% v/v ethylene glycol (VWR Chemicals BDH, 24041.297), which was adjusted with PBS to achieve the final volume. Brain sections were immunostained for each neuronal marker using validated antibodies against MT, VT and ARO according to previous studies\u003csup\u003e37,51,55\u003c/sup\u003e.\u0026nbsp;Sections were first rinsed three times in 0.01 M tris-buffered saline (TBS, pH 7.6) for 5 minutes each (same duration applied for all following rinses). For ARO and MT, peroxidase activity was blocked with 0.6% of hydrogen peroxide (H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2;\u0026nbsp;\u003c/sub\u003eMerck Millipore, 1072090250) in TBS at room temperature (RT) for 20 or 30 minutes, respectively. After rinses with TBS (ARO) or TBS containing 0.1% Triton X-100 (TBST; MT), sections were blocked and permeabilized for 1 hur in 5% (ARO) or 20% (MT) normal goat serum (NGS; ARO: Merck Millipore, S26, MT: Cell signaling technology, 5425S) in TBST. For VT, peroxidase activity was blocked with 1% of H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e at RT for 30 minutes with 10% NGS (Merck Millipore, S26) in TBST. Sections were then incubated with a rabbit anti-oxytocin primary antibody (1/10000, Immunostar, Ref: 20068), rabbit anti-arginine vasopressin primary antibody (1/5000, Merck Millipore, Ref: AB1565) or a rabbit anti-aromatase primary antibody (1/3000, Harada QR 02/05) with 2% (MT and VT) or 5% (ARO) NGS in TBST for two nights at 4\u0026deg;C. Sections were rinsed in TBS (VT and ARO) or TBST (MT) and incubated for 2 hours at RT with a goat anti-rabbit biotinylated secondary antibody (1/250 for MT and 1/400 for VT and ARO respectively, Jackson ImmunoResearch, Ref: 111-065-003) in TBST. Sections were washed in TBS (VT and ARO) or TBST (MT) and incubated for one hour and a half in the vectastain Elite ABC-HRP Kit, Peroxidase in TBST (1/200, 1/1000, 1/800 of each reagent for MT, VT and ARO, respectively; Vector Laboratories, PK-6100) at RT, then rinsed in TBS (VT and ARO) or TBST (MT). The peroxidase was visualized with 0.04% of 3,3\u0026rsquo;-diaminobenzidine tetrahydrochloride hydrate (Sigma-Aldrich, D537) used as chromogen along with 0.012% of H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e in TBS at RT. Sections were finally rinsed, mounted on slides, dried overnight, left 10 min in xylene and cover-slipped using Eukitt (Sigma-Aldrich, 03989).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eImage acquisition and analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003ePhotographs of\u0026nbsp;brain sections were acquired using a microscope slide scanner (Zeiss, Axio Scan.Z1) with an objective 20x/0.8 and a HV-F202SCL Hitachi camera. High-resolution images were generated by the Zen Blue 3.1 software (Carl Zeiss, Oberkochen, Germany) and visualized with QuPath software, version 0.5.0\u003csup\u003e79\u003c/sup\u003e. Specific brain regions were delimitated by rectangular quantification fields of identical dimensions\u0026nbsp;across social lines and sexes in the left and right hemispheres for each brain region of interest\u0026nbsp;using QuPath. These rectangular fields were then exported for further analyses in ImageJ 1.54f\u003csup\u003e80\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eFor the MPN, the rectangular quantification field was placed in the corner formed by the ventral edge of the anterior commissure and the lateral edge of the third ventricle. For MT and VT staining, small anatomical inter-individual differences prevented the positioning of a standardized quantification field covering the entire immunoreactive (ir) fibers of the MPN, without including magnocellular MT- or VT-ir cells and fibers from the periventricular region. Fiber density was thus quantified in a rectangular field covering the dorsal portion of the MPN, which remained identical across all brain sections regardless of social line and sex. For the ARO staining, the quantification field included the entire MPN, as no interfering signal was present in adjacent regions. In the BNST, LS and VMN, the quantification fields were placed to cover the entire immunoreactive part of the brain nucleus located dorsally to the most lateral edge of the anterior commissure, approximately under the lateral ventricle (BNST), adjacent to the lateral ventricle (LS), or in the corner formed by the lateral edge of the third ventricle and the ventral limit of the brain (VMN).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe number of MT- and VT-ir cells within the rostral PVN was determined manually using the cell counter plugin in ImageJ.\u0026nbsp;The number of ARO-ir cells in the left and right part of the caudal MPN, rostral BNST and rostral VMN was determined by an automatic method using the cyto2 model from the Cellpose deep learning-based segmentation method\u003csup\u003e81\u003c/sup\u003e in ImageJ.\u003c/p\u003e\n\u003cp\u003eThe percentage of area covered by MT- or VT-ir fibers in the left and right part of the caudal MPN, rostral BNST and medial LS was determined by a semi-automatic method using the threshold method in ImageJ where threshold was manually adjusted for each subject. The values from the left and right quantification field were averaged to obtain one single value for each brain region. The number of cells was normalized based on the length between neuroanatomical markers identified in the brain sections containing each brain nucleus of interest (detailed in the Morphology and brain measurements section).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eFor all measurements and analyses, the investigator was blind to the social line or the sex of the birds. \u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEarly social environment\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTwo-day-old quail chicks were segregated by sex from hatching to the age of 6 week in 6 social groups of 32 quail, generating 8 statistical groups:\u0026nbsp;two groups (males and females) comprised solely S+ quail [S+], two groups only S- quail [S-] and two groups contained a ratio of 50% of S+ [S+(-)] and 50% of S- [S-(+)] quail. Nineteen days after hatching, quail were transferred in larger spaces comprising two rooms of 5.6 x 3.6 meters (one for each sex) divided in three equal compartments to receive the three distinct social groups (S+, S- and S+(-) with S-(+) respectively). Compartments were separated by wooden board at the bottom and wire mesh at the top to prevent visual contacts with the other groups. A third room was dedicated to the parental quail line (N= 25 quail of each sex) whose individuals were used as stimuli in subsequent behavioral tests.\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eBehaviors\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThree- to four-week-old adolescent quail were used to conduct the behavioral tests in order to avoid the surge of testosterone production and the activation of sexual behaviors\u003csup\u003e82-84\u003c/sup\u003e. Ten to seventeen birds from each social group and sex were tested for social motivation, open field activity and tonic immobility with three and four resting days between each test. \u0026nbsp;Birds were carried individually to the testing room in a transport box (15 [length] x 15 [width] x 20 [height] cm) made of opaque polycarbonate, alternating between sex and social group. The testing apparatus was cleaned using water and paper towels between subjects. Behavioral scoring and analyses were carried out by the investigator who was unaware of the social group to which the birds belonged.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eSocial motivation test\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eThree-week-old experimental quail placed at one end of a long corridor (245 [length] x 30 [width] x 30 [height] cm) was offered the choice to approach and stay close to a group of 5 age- and sex-matched quail from the parental line located at the other end. The corridor made with wire mesh was divided into five virtual compartments (C1-5) of equal length. The experimental quail was placed for 10 seconds for habituation in C1 defined by a transparent Plexiglas. Then, the partition was removed, and the experimental quail could freely move for 5 minutes within compartments C1 to C4 and get closer visual access to the group confined in C5 at the opposite end of the corridor. The full test was recorded. The time spent in each compartment was measured, as well as the latency to reach the compartment adjacent to the social group (C4).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eOpen field test\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eFour-week-old experimental quail were individually placed for 5 min in the center of a square arena\u0026nbsp;(80 [length] x 80 [width] x 50 [height] cm) made of wood\u0026nbsp;with the floor covered by a plastic surface and\u0026nbsp;surrounded by an opaque green curtain. A light and a camera positioned above the center of the arena was used to record the behavior. Locomotor activity, also used in previous studies as a proxy for social response induced by short-term isolation\u003csup\u003e48,85\u003c/sup\u003e and anxious-like behaviors were assessed by quantifying the mean distance traveled and the time spent in the squared central zone, respectively. These measures were automatically measured using the EthoVision\u003csup\u003e\u0026reg;\u003c/sup\u003eXT tracking software.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eTonic immobility test\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eTonic immobility is an innate behavioral response to predators in birds\u003csup\u003e86-88\u003c/sup\u003e, which has been counter-selected during the divergence process in these two lines. A quail was placed and maintained on its back by the experimenter\u0026rsquo;s hand for 10 seconds (induction) in a plastic U-shaped cradle (20 [length] x 10 [width] x 10 [height] cm) covered with a cloth. Then, the experimenter\u0026rsquo;s hand was gently removed from the bird and the time spent by the bird to stand up was quantified (tonic immobility duration \u003cem\u003eper se\u003c/em\u003e). When the induction phase failed, the attempt was repeated. After 5 attempts, the tonic immobility duration was scored as 0 second. Conversely, a score of 300 seconds was attributed when a quail failed to stand up after the maximal duration of the test set at 5 minutes. The tonic immobility duration and the number of induction attempts were quantified in this test. \u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eStatistics and writing\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eConsidering social line and sex as independent factors, morphological and behavioral data were analyzed by two-way ANOVAs followed by Sidak\u0026rsquo;s or Tukey\u0026rsquo;s post-hoc tests when significant. The data from the cloacal gland area, the cloacal opening and the testes or ovarian follicle masses were analyzed by ordinary one-way ANOVAs followed by Holm-Sidak\u0026rsquo;s post-hoc tests when significant. The percentage of male quail reaching the compartment adjacent to the social group in the social motivation test were compared using the Fisher\u0026rsquo;s exact probability test followed by a Bonferroni correction for multiple comparisons and thus, all p values were multiplied by the number of comparisons (adjusted p or p\u003csub\u003eadj\u003c/sub\u003e). All analyses were performed with GraphPad Prism version 8.0.2. Results were considered significant for p value \u0026lt;0.05 and were presented by boxplots with individual data points, median, first and third quartiles and whiskers indicating the smallest and largest value. English language editing was partly provided by ChatGPT 3.5 developed by OpenAI.\u0026nbsp;\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by INRAE \u0026ldquo;SOCIALOME\u0026rdquo; project (PAF_29). Quail breeding and care was conducted in Poultry Experimental Facility (PEAT) from the INRAE experimental unit 1295\u0026nbsp;(F-37380 Nouzilly, France, DOI: 10.15454/1.5572326250887292E12).\u0026nbsp;Microscopy was performed through the facilities and expertise of the \u0026quot;Plateforme d\u0026apos;Imagerie Cellulaire\u0026quot; (PIC) of the UMR PRC, INRAE. The authors thank Dr. Nobuhiro Harada for providing the aromatase antibodies.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor Contributions statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eLucas Court (LC), JL, FC, JB, CAC, MK, Ludovic Calandreau (LCC) and LP conceived the experiments; LC, LT, JL, EP, FC conducted the experiments; LC, JL, MK, LCC and LP analyzed the results; LC and LP wrote the paper; MCB contributed analytic tools. All authors reviewed the manuscript.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAdditional Information\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll data generated and analyzed during this study are included in the Supplementary Tables 1 to 3.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eLee, V. E., Arnott, G. \u0026amp; Turner, S. P. Social behavior in farm animals: Applying fundamental theory to improve animal welfare. \u003cem\u003eFrontiers in Veterinary Science\u003c/em\u003e \u003cstrong\u003e9\u003c/strong\u003e, doi:10.3389/fvets.2022.932217 (2022).\u003c/li\u003e\n\u003cli\u003eRegan, A., Rado\u0026scaron;ić, N. \u0026amp; Lyubomirsky, S. Experimental effects of social behavior on well-being. \u003cem\u003eTrends in Cognitive Sciences\u003c/em\u003e \u003cstrong\u003e26\u003c/strong\u003e, 987-998, doi:10.1016/j.tics.2022.08.006 (2022).\u003c/li\u003e\n\u003cli\u003eFerrara, N. C.\u003cem\u003e et al.\u003c/em\u003e Neural Circuit Transitions Supporting Developmentally Specific Social Behavior. \u003cem\u003eThe Journal of neuroscience\u003c/em\u003e \u003cstrong\u003e43\u003c/strong\u003e, 7456-7462, doi:10.1523/jneurosci.1377-23.2023 (2023).\u003c/li\u003e\n\u003cli\u003eChen, P. \u0026amp; Hong, W. Neural Circuit Mechanisms of Social Behavior. \u003cem\u003eNeuron\u003c/em\u003e \u003cstrong\u003e98\u003c/strong\u003e, 16-30, doi:10.1016/j.neuron.2018.02.026 (2018).\u003c/li\u003e\n\u003cli\u003eKennedy, D. P. \u0026amp; Adolphs, R. The social brain in psychiatric and neurological disorders. \u003cem\u003eTrends in Cognitive Sciences\u003c/em\u003e \u003cstrong\u003e16\u003c/strong\u003e, 559-572, doi:10.1016/j.tics.2012.09.006 (2012).\u003c/li\u003e\n\u003cli\u003eBarak, B. \u0026amp; Feng, G. Neurobiology of social behavior abnormalities in autism and Williams syndrome. \u003cem\u003eNature Neuroscience\u003c/em\u003e \u003cstrong\u003e19\u003c/strong\u003e, 647-655, doi:10.1038/nn.4276 (2016).\u003c/li\u003e\n\u003cli\u003eCushing, B. S. \u0026amp; Kramer, K. M. Mechanisms underlying epigenetic effects of early social experience: the role of neuropeptides and steroids. \u003cem\u003eNeuroscience and biobehavioral reviews\u003c/em\u003e \u003cstrong\u003e29\u003c/strong\u003e, 1089-1105, doi:10.1016/j.neubiorev.2005.04.001 (2005).\u003c/li\u003e\n\u003cli\u003eVeenema, A. H. Toward understanding how early-life social experiences alter oxytocin- and vasopressin-regulated social behaviors. \u003cem\u003eHormones and Behavior\u003c/em\u003e \u003cstrong\u003e61\u003c/strong\u003e, 304-312, doi:10.1016/j.yhbeh.2011.12.002 (2012).\u003c/li\u003e\n\u003cli\u003eBurke, A. R., McCormick, C. M., Pellis, S. M. \u0026amp; Lukkes, J. L. Impact of adolescent social experiences on behavior and neural circuits implicated in mental illnesses. \u003cem\u003eNeuroscience and biobehavioral reviews\u003c/em\u003e \u003cstrong\u003e76\u003c/strong\u003e, 280-300, doi:10.1016/j.neubiorev.2017.01.018 (2017).\u003c/li\u003e\n\u003cli\u003eStrathearn, L. The elusive etiology of autism: nature and nurture? \u003cem\u003eFrontiers in Behavioral Neuroscience\u003c/em\u003e \u003cstrong\u003e3\u003c/strong\u003e, 11, doi:10.3389/neuro.08.011.2009 (2009).\u003c/li\u003e\n\u003cli\u003eYang, M., Perry, K., Weber, M. D., Katz, A. M. \u0026amp; Crawley, J. N. Social peers rescue autism-relevant sociability deficits in adolescent mice. \u003cem\u003eAutism research : official journal of the International Society for Autism Research\u003c/em\u003e \u003cstrong\u003e4\u003c/strong\u003e, 17-27, doi:10.1002/aur.163 (2011).\u003c/li\u003e\n\u003cli\u003eCampolongo, M.\u003cem\u003e et al.\u003c/em\u003e Sociability deficits after prenatal exposure to valproic acid are rescued by early social enrichment. \u003cem\u003eMolecular Autism\u003c/em\u003e \u003cstrong\u003e9\u003c/strong\u003e, 36, doi:10.1186/s13229-018-0221-9 (2018).\u003c/li\u003e\n\u003cli\u003eGora, C.\u003cem\u003e et al.\u003c/em\u003e Effect of the social environment on olfaction and social skills in WT and a mouse model of autism. \u003cem\u003eTranslational Psychiatry.\u003c/em\u003e, doi:10.21203/rs.3.rs-3759429/v1 (2024).\u003c/li\u003e\n\u003cli\u003eO\u0026apos;Connell, L. A. \u0026amp; Hofmann, H. A. The vertebrate mesolimbic reward system and social behavior network: a comparative synthesis. \u003cem\u003eThe Journal of comparative neurology\u003c/em\u003e \u003cstrong\u003e519\u003c/strong\u003e, 3599-3639, doi:10.1002/cne.22735 (2011).\u003c/li\u003e\n\u003cli\u003eO\u0026apos;Connell, L. A. \u0026amp; Hofmann, H. A. Evolution of a vertebrate social decision-making network. \u003cem\u003eScience\u003c/em\u003e \u003cstrong\u003e336\u003c/strong\u003e, 1154-1157, doi:10.1126/science.1218889 (2012).\u003c/li\u003e\n\u003cli\u003eGoodson, J. L. The vertebrate social behavior network: Evolutionary themes and variations. \u003cem\u003eHormones and behavior\u003c/em\u003e \u003cstrong\u003e48\u003c/strong\u003e, 11-22, doi:10.1016/j.yhbeh.2005.02.003 (2005).\u003c/li\u003e\n\u003cli\u003eGoodson, J. L. \u0026amp; Kabelik, D. Dynamic limbic networks and social diversity in vertebrates: From neural context to neuromodulatory patterning. \u003cem\u003eFrontiers in Neuroendocrinology\u003c/em\u003e \u003cstrong\u003e30\u003c/strong\u003e, 429-441, doi:10.1016/j.yfrne.2009.05.007 (2009).\u003c/li\u003e\n\u003cli\u003eRobinson, G. E., Fernald, R. D. \u0026amp; Clayton, D. F. Genes and Social Behavior. \u003cem\u003eScience\u003c/em\u003e \u003cstrong\u003e322\u003c/strong\u003e, 896-900, doi:10.1126/science.1159277 (2008).\u003c/li\u003e\n\u003cli\u003eDonaldson, Z. R. \u0026amp; Young, L. J. Oxytocin, vasopressin, and the neurogenetics of sociality. \u003cem\u003eScience\u003c/em\u003e \u003cstrong\u003e322\u003c/strong\u003e, 900-904, doi:10.1126/science.1158668 (2008).\u003c/li\u003e\n\u003cli\u003eLiutkeviciute, Z., Koehbach, J., Eder, T., Gil-Mansilla, E. \u0026amp; Gruber, C. W. Global map of oxytocin/vasopressin-like neuropeptide signalling in insects. \u003cem\u003eScientific Reports\u003c/em\u003e \u003cstrong\u003e6\u003c/strong\u003e, 39177, doi:10.1038/srep39177 (2016).\u003c/li\u003e\n\u003cli\u003eGoodson, J. L., Kelly, A. M. \u0026amp; Kingsbury, M. A. Evolving nonapeptide mechanisms of gregariousness and social diversity in birds. \u003cem\u003eHorm Behav\u003c/em\u003e \u003cstrong\u003e61\u003c/strong\u003e, 239-250, doi:10.1016/j.yhbeh.2012.01.005 (2012).\u003c/li\u003e\n\u003cli\u003eCaldwell, H. K. Oxytocin and Vasopressin: Powerful Regulators of Social Behavior. \u003cem\u003eThe Neuroscientist : a review journal bringing neurobiology, neurology and psychiatry\u003c/em\u003e \u003cstrong\u003e23\u003c/strong\u003e, 517-528, doi:10.1177/1073858417708284 (2017).\u003c/li\u003e\n\u003cli\u003eMarlin, B. J. \u0026amp; Froemke, R. C. Oxytocin modulation of neural circuits for social behavior. \u003cem\u003eDevelopmental Neurobiology\u003c/em\u003e \u003cstrong\u003e77\u003c/strong\u003e, 169-189, doi:10.1002/dneu.22452 (2017).\u003c/li\u003e\n\u003cli\u003eRigney, N., de Vries, G. J., Petrulis, A. \u0026amp; Young, L. J. Oxytocin, Vasopressin, and Social Behavior: From Neural Circuits to Clinical Opportunities. \u003cem\u003eEndocrinology\u003c/em\u003e \u003cstrong\u003e163\u003c/strong\u003e, doi:10.1210/endocr/bqac111 (2022).\u003c/li\u003e\n\u003cli\u003eRigney, N., de Vries, G. J. \u0026amp; Petrulis, A. Modulation of social behavior by distinct vasopressin sources. \u003cem\u003eFrontiers in Endocrinology\u003c/em\u003e \u003cstrong\u003e14\u003c/strong\u003e, 1127792, doi:10.3389/fendo.2023.1127792 (2023).\u003c/li\u003e\n\u003cli\u003eBons, N. The topography of mesotocin and vasotocin systems in the brain of the domestic mallard and japanese quail: Immunocytochemical identification. \u003cem\u003eCell and tissue research\u003c/em\u003e \u003cstrong\u003e213\u003c/strong\u003e, 37-51, doi:10.1007/BF00236919 (1980).\u003c/li\u003e\n\u003cli\u003ePanzica, G., Aste, N., Castagna, C., Viglietti-Panzica, C. \u0026amp; Balthazart, J. Steroid-induced plasticity in the sexually dimorphic vasotocinergic innervation of the avian brain: behavioral implications. \u003cem\u003eBrain research reviews\u003c/em\u003e \u003cstrong\u003e37\u003c/strong\u003e, 178-200, doi:10.1016/s0165-0173(01)00118-7 (2001).\u003c/li\u003e\n\u003cli\u003eBalthazart, J., Absil, P., Viglietti-Panzica, C. \u0026amp; Panzica, G. C. Vasotocinergic innervation of areas containing aromatase-immunoreactive cells in the quail forebrain. \u003cem\u003eJournal of Neurobiology\u003c/em\u003e \u003cstrong\u003e33\u003c/strong\u003e, 45-60 (1997).\u003c/li\u003e\n\u003cli\u003eAbsil, P., Baillien, M., Ball, G. F., Panzica, G. C. \u0026amp; Balthazart, J. The control of preoptic aromatase activity by afferent inputs in Japanese quail. \u003cem\u003eBrain research. Brain research reviews\u003c/em\u003e \u003cstrong\u003e37\u003c/strong\u003e, 38-58, doi:10.1016/s0165-0173(01)00122-9 (2001).\u003c/li\u003e\n\u003cli\u003eStanić, D.\u003cem\u003e et al.\u003c/em\u003e Characterization of aromatase expression in the adult male and female mouse brain. I. Coexistence with oestrogen receptors \u0026alpha; and \u0026beta;, and androgen receptors. \u003cem\u003ePloS one\u003c/em\u003e \u003cstrong\u003e9\u003c/strong\u003e, e90451-e90451, doi:10.1371/journal.pone.0090451 (2014).\u003c/li\u003e\n\u003cli\u003eSoumier, A., Habart, M., Lio, G., Demily, C. \u0026amp; Sirigu, A. Differential fate between oxytocin and vasopressin cells in the developing mouse brain. \u003cem\u003eiScience\u003c/em\u003e \u003cstrong\u003e25\u003c/strong\u003e, 103655, doi:10.1016/j.isci.2021.103655 (2022).\u003c/li\u003e\n\u003cli\u003eGoodson, J. L., Schrock, S. E., Klatt, J. D., Kabelik, D. \u0026amp; Kingsbury, M. A. Mesotocin and Nonapeptide Receptors Promote Estrildid Flocking Behavior. \u003cem\u003eScience\u003c/em\u003e \u003cstrong\u003e325\u003c/strong\u003e, 862-866 (2009).\u003c/li\u003e\n\u003cli\u003eCourt, L., Balthazart, J., Ball, G. F. \u0026amp; Cornil, C. A. Effect of chronic intracerebroventricular administration of an aromatase inhibitor on the expression of socio-sexual behaviors in male Japanese quail. \u003cem\u003eBehavioural Brain Research\u003c/em\u003e, 113315, doi:10.1016/j.bbr.2021.113315 (2021).\u003c/li\u003e\n\u003cli\u003eAlbers, H. E. Species, sex and individual differences in the vasotocin/vasopressin system: Relationship to neurochemical signaling in the social behavior neural network. \u003cem\u003eFrontiers in Neuroendocrinology\u003c/em\u003e \u003cstrong\u003e36\u003c/strong\u003e, 49-71, doi:10.1016/j.yfrne.2014.07.001 (2015).\u003c/li\u003e\n\u003cli\u003eDhakar, M. B., Stevenson, E. L. \u0026amp; Caldwell, H. K. in \u003cem\u003eOxytocin, Vasopressin and Related Peptides in the Regulation of Behavior\u003c/em\u003e (eds Donald Pfaff, Elena Choleris, \u0026amp; Martin Kavaliers) Ch. 1, 3 - 26 (Cambridge University Press, 2013).\u003c/li\u003e\n\u003cli\u003eWu, M. V.\u003cem\u003e et al.\u003c/em\u003e Estrogen Masculinizes Neural Pathways and Sex-Specific Behaviors. \u003cem\u003eCell\u003c/em\u003e \u003cstrong\u003e139\u003c/strong\u003e, 61-72 (2009).\u003c/li\u003e\n\u003cli\u003eFoidart, A.\u003cem\u003e et al.\u003c/em\u003e Critical re-examination of the distribution of aromatase-immunoreactive cells in the quail forebrain using antibodies raised against human placental aromatase and against the recombinant quail, mouse or human enzyme. \u003cem\u003eJournal of chemical neuroanatomy\u003c/em\u003e \u003cstrong\u003e8\u003c/strong\u003e, 267-282, doi:10.1016/0891-0618(95)00054-b (1995).\u003c/li\u003e\n\u003cli\u003eCsillag, A., \u0026Aacute;d\u0026aacute;m, \u0026Aacute;. \u0026amp; Zachar, G. Avian models for brain mechanisms underlying altered social behavior in autism. \u003cem\u003eFrontiers in physiology\u003c/em\u003e \u003cstrong\u003e13\u003c/strong\u003e, 1032046, doi:10.3389/fphys.2022.1032046 (2022).\u003c/li\u003e\n\u003cli\u003eBalthazart, J. \u0026amp; Ball, G. F. The Japanese quail as a model system for the investigation of steroid-catecholamine interactions mediating appetitive and consummatory aspects of male sexual behavior. \u003cem\u003eAnnual Review of Sex Research\u003c/em\u003e \u003cstrong\u003e9\u003c/strong\u003e, 96-176 (1998).\u003c/li\u003e\n\u003cli\u003eMills, A. D. \u0026amp; Faure, J. M. Divergent selection for duration of tonic immobility and social reinstatement behavior in Japanese quail (Coturnix coturnix japonica) chicks. \u003cem\u003eJournal of Comparative Psychology\u003c/em\u003e \u003cstrong\u003e105\u003c/strong\u003e, 25-38, doi:10.1037/0735-7036.105.1.25 (1991).\u003c/li\u003e\n\u003cli\u003eLaunay, F., Mills, A. D. \u0026amp; Faure, J. M. Social motivation in Japanese quail coturnix coturnix japonica chicks selected for high or low levels of treadmill behaviour. \u003cem\u003eBehavioural Processes\u003c/em\u003e \u003cstrong\u003e24\u003c/strong\u003e, 95-110, doi:10.1016/0376-6357(91)90002-H (1991).\u003c/li\u003e\n\u003cli\u003eLaunay, F., Mills, A. D. \u0026amp; Faure, J. M. Effects of test age, line and sex on tonic immobility responses and social reinstatement behaviour in Japanese quail Coturnix japonica. \u003cem\u003eBehavioural Processes\u003c/em\u003e \u003cstrong\u003e29\u003c/strong\u003e, 1-16, doi:10.1016/0376-6357(93)90023-k (1993).\u003c/li\u003e\n\u003cli\u003eMills, A. D., Jones, R. B. \u0026amp; Faure, J. M. Species specificity of social reinstatement in Japanese quail Coturnix japonica genetically selected for high or low levels of social reinstatement behaviour. \u003cem\u003eBehavioural Processes\u003c/em\u003e \u003cstrong\u003e34\u003c/strong\u003e, 13-22, doi:10.1016/0376-6357(94)00044-h (1995).\u003c/li\u003e\n\u003cli\u003eJones, R. B., Mills, A. D. \u0026amp; Faure, J. M. Social discrimination in Japanese quail Coturnix japonica chicks genetically selected for low or high social reinstatement motivation. \u003cem\u003eBehavioural Processes\u003c/em\u003e \u003cstrong\u003e36\u003c/strong\u003e, 117-124, doi:10.1016/0376-6357(95)00024-0 (1996).\u003c/li\u003e\n\u003cli\u003eRecoquillay, J.\u003cem\u003e et al.\u003c/em\u003e Evidence of phenotypic and genetic relationships between sociality, emotional reactivity and production traits in Japanese quail. \u003cem\u003ePloS one\u003c/em\u003e \u003cstrong\u003e8\u003c/strong\u003e, e82157, doi:10.1371/journal.pone.0082157 (2013).\u003c/li\u003e\n\u003cli\u003eBurns, M., Domjan, M. \u0026amp; Mills, A. D. Effects of genetic selection for fearfulness or social reinstatement behavior on adult social and sexual behavior in domestic quail (Coturnix japonica). \u003cem\u003ePsychobiology\u003c/em\u003e \u003cstrong\u003e26\u003c/strong\u003e, 249-257, doi:10.3758/BF03330613 (1998).\u003c/li\u003e\n\u003cli\u003eFran\u0026ccedil;ois, N., Decros, S., Picard, M., Faure, J. M. \u0026amp; Mills, A. D. Effect of group disruption on social behaviour in lines of Japanese quail (Coturnix japonica) selected for high or low levels of social reinstatement behaviour. \u003cem\u003eBehavioural Processes\u003c/em\u003e \u003cstrong\u003e48\u003c/strong\u003e, 171-181, doi:10.1016/S0376-6357(99)00081-9 (2000).\u003c/li\u003e\n\u003cli\u003eMills, A. D., Jones, R. B., Faure, J. M. \u0026amp; Williams, J. B. Responses to isolation in Japanese quail genetically selected for high or low sociality. \u003cem\u003ePhysiology \u0026amp; Behavior\u003c/em\u003e \u003cstrong\u003e53\u003c/strong\u003e, 183-189, doi:10.1016/0031-9384(93)90029-f (1993).\u003c/li\u003e\n\u003cli\u003eSchweitzer, C., Houdelier, C., Lumineau, S., L\u0026eacute;vy, F. \u0026amp; Arnould, C. Social motivation does not go hand in hand with social bonding between two familiar Japanese quail chicks, Coturnix japonica. \u003cem\u003eAnimal Behaviour\u003c/em\u003e \u003cstrong\u003e79\u003c/strong\u003e, 571-578, doi:10.1016/j.anbehav.2009.11.023 (2010).\u003c/li\u003e\n\u003cli\u003ePuelles, L., Martinez-de-la-Torre, M., Martinez, S., Watson, C. \u0026amp; Paxinos, G. \u003cem\u003eThe Chick Brain in Stereotaxic Coordinates and Alternate Stains: Featuring Neuromeric Divisions and Mammalian Homologies\u003c/em\u003e. 2nd Edition Featuring Neuromeric Divisions and Mammalian Homologies edn, (Academic Press, 2018).\u003c/li\u003e\n\u003cli\u003eHaakenson, C. M., Balthazart, J., Madison, F. N. \u0026amp; Ball, G. F. The neural distribution of the avian homologue of oxytocin, mesotocin, in two songbird species, the zebra finch and the canary: A potential role in song perception and production. \u003cem\u003eJournal of Comparative Neurology\u003c/em\u003e \u003cstrong\u003e530\u003c/strong\u003e, 2402-2414, doi:10.1002/cne.25338 (2022).\u003c/li\u003e\n\u003cli\u003eDumais, K. M. \u0026amp; Veenema, A. H. Vasopressin and oxytocin receptor systems in the brain: Sex differences and sex-specific regulation of social behavior. \u003cem\u003eFrontiers in Neuroendocrinology\u003c/em\u003e \u003cstrong\u003e40\u003c/strong\u003e, 1-23, doi:10.1016/j.yfrne.2015.04.003 (2016).\u003c/li\u003e\n\u003cli\u003eViglietti-Panzica, C., Anselmetti, G. C., Balthazart, J., Aste, N. \u0026amp; Panzica, G. C. Vasotocinergic innervation of the septal region in the Japanese quail: sexual differences and the influence of testosterone. \u003cem\u003eCell and tissue research\u003c/em\u003e \u003cstrong\u003e267\u003c/strong\u003e, 261-265, doi:10.1007/bf00302963 (1992).\u003c/li\u003e\n\u003cli\u003ePanzica, G. C.\u003cem\u003e et al.\u003c/em\u003e Organizational effects of estrogens on brain vasotocin and sexual behavior in quail. \u003cem\u003eDevelopmental Neurobiology\u003c/em\u003e \u003cstrong\u003e37\u003c/strong\u003e, 684-699, doi:10.1002/(sici)1097-4695(199812)37:4\u0026lt;684::aid-neu15\u0026gt;3.0.co;2-u (1998).\u003c/li\u003e\n\u003cli\u003eCourt, L., Vandries, L., Balthazart, J. \u0026amp; Cornil, C. A. Key role of estrogen receptor \u0026beta; in the organization of brain and behavior of the Japanese quail. \u003cem\u003eHormones and Behavior\u003c/em\u003e, 104827, doi:10.1016/j.yhbeh.2020.104827 (2020).\u003c/li\u003e\n\u003cli\u003eFoidart, A., de Clerck, A., Harada, N. \u0026amp; Balthazart, J. Aromatase-immunoreactive cells in the quail brain: effects of testosterone and sex dimorphism. \u003cem\u003ePhysiology \u0026amp; Behavior\u003c/em\u003e \u003cstrong\u003e55\u003c/strong\u003e, 453-464, doi:10.1016/0031-9384(94)90100-7 (1994).\u003c/li\u003e\n\u003cli\u003eBalthazart, J., Tlem\u0026ccedil;ani, O. \u0026amp; Harada, N. Localization of testosterone-sensitive and sexually dimorphic aromatase-immunoreactive cells in the quail preoptic area. \u003cem\u003eJournal of Chemical Neuroanatomy\u003c/em\u003e \u003cstrong\u003e11\u003c/strong\u003e, 147-171, doi:10.1016/0891-0618(96)00149-4 (1996).\u003c/li\u003e\n\u003cli\u003eCourt, L. \u003cem\u003eRole of neuroestrogens in the regulation of social behaviors\u003c/em\u003e Doctor thesis, ULi\u0026egrave;ge, (2022).\u003c/li\u003e\n\u003cli\u003eRauw, W. M., Kanis, E., Noordhuizen-Stassen, E. N. \u0026amp; Grommers, F. J. Undesirable side effects of selection for high production efficiency in farm animals: a review. \u003cem\u003eLivestock Production Science\u003c/em\u003e \u003cstrong\u003e56\u003c/strong\u003e, 15-33, doi:10.1016/S0301-6226(98)00147-X (1998).\u003c/li\u003e\n\u003cli\u003eZhou, L., Blaustein, J. D. \u0026amp; De Vries, G. J. Distribution of androgen receptor immunoreactivity in vasopressin- and oxytocin-immunoreactive neurons in the male rat brain. \u003cem\u003eEndocrinology\u003c/em\u003e \u003cstrong\u003e134\u003c/strong\u003e, 2622-2627, doi:10.1210/en.134.6.2622 (1994).\u003c/li\u003e\n\u003cli\u003eAnpilov, S.\u003cem\u003e et al.\u003c/em\u003e Wireless Optogenetic Stimulation of Oxytocin Neurons in a Semi-natural Setup Dynamically Elevates Both Pro-social and Agonistic Behaviors. \u003cem\u003eNeuron\u003c/em\u003e \u003cstrong\u003e107\u003c/strong\u003e, 644-655.e647, doi:10.1016/j.neuron.2020.05.028 (2020).\u003c/li\u003e\n\u003cli\u003eResendez, S. L.\u003cem\u003e et al.\u003c/em\u003e Social Stimuli Induce Activation of Oxytocin Neurons Within the Paraventricular Nucleus of the Hypothalamus to Promote Social Behavior in Male Mice. \u003cem\u003eJournal of Neuroscience\u003c/em\u003e \u003cstrong\u003e40\u003c/strong\u003e, 2282-2295, doi:10.1523/jneurosci.1515-18.2020 (2020).\u003c/li\u003e\n\u003cli\u003eGrund, T.\u003cem\u003e et al.\u003c/em\u003e Chemogenetic activation of oxytocin neurons: Temporal dynamics, hormonal release, and behavioral consequences. \u003cem\u003ePsychoneuroendocrinology\u003c/em\u003e \u003cstrong\u003e106\u003c/strong\u003e, 77-84, doi:10.1016/j.psyneuen.2019.03.019 (2019).\u003c/li\u003e\n\u003cli\u003eKelly, A. M. \u0026amp; Goodson, J. L. Hypothalamic oxytocin and vasopressin neurons exert sex-specific effects on pair bonding, gregariousness, and aggression in finches. \u003cem\u003eProceedings of the National Academy of Sciences\u003c/em\u003e \u003cstrong\u003e111\u003c/strong\u003e, 6069-6074, doi:10.1073/pnas.1322554111 (2014).\u003c/li\u003e\n\u003cli\u003eRigney, N., Whylings, J., de Vries, Geert J. \u0026amp; Petrulis, A. Sex Differences in the Control of Social Investigation and Anxiety by Vasopressin Cells of the Paraventricular Nucleus of the Hypothalamus. \u003cem\u003eNeuroendocrinology\u003c/em\u003e \u003cstrong\u003e111\u003c/strong\u003e, 521-535, doi:10.1159/000509421 (2020).\u003c/li\u003e\n\u003cli\u003eRigney, N., Zbib, A., de Vries, G. J. \u0026amp; Petrulis, A. Knockdown of sexually differentiated vasopressin expression in the bed nucleus of the stria terminalis reduces social and sexual behaviour in male, but not female, mice. \u003cem\u003eJournal of Neuroendocrinology\u003c/em\u003e \u003cstrong\u003e34\u003c/strong\u003e, e13083, doi:10.1111/jne.13083 (2022).\u003c/li\u003e\n\u003cli\u003eCourt, L., Balthazart, J., Ball, G. F. \u0026amp; Cornil, C. A. Role of aromatase in distinct brain nuclei of the social behaviour network in the expression of sexual behaviour in male Japanese quail. \u003cem\u003eJournal of Neuroendocrinology\u003c/em\u003e \u003cstrong\u003e34\u003c/strong\u003e, e13127, doi:10.1111/jne.13127 (2022).\u003c/li\u003e\n\u003cli\u003ede Bournonville, M. P., Vandries, L. M., Ball, G. F., Balthazart, J. \u0026amp; Cornil, C. A. Site-specific effects of aromatase inhibition on the activation of male sexual behavior in male Japanese quail (Coturnix japonica). \u003cem\u003eHormones and Behavior\u003c/em\u003e \u003cstrong\u003e108\u003c/strong\u003e, 42-49, doi:10.1016/j.yhbeh.2018.12.015 (2019).\u003c/li\u003e\n\u003cli\u003eBayless, D. W.\u003cem\u003e et al.\u003c/em\u003e Limbic Neurons Shape Sex Recognition and Social Behavior in Sexually Naive Males. \u003cem\u003eCell\u003c/em\u003e, doi:10.1016/j.cell.2018.12.041 (2019).\u003c/li\u003e\n\u003cli\u003eNeumann, I. D. \u0026amp; Landgraf, R. Balance of brain oxytocin and vasopressin: implications for anxiety, depression, and social behaviors. \u003cem\u003eTrends in Neurosciences\u003c/em\u003e \u003cstrong\u003e35\u003c/strong\u003e, 649-659, doi:10.1016/j.tins.2012.08.004 (2012).\u003c/li\u003e\n\u003cli\u003eCoustham, V., Godet, E. \u0026amp; Beauclair, L. A simple PCR method for sexing Japanese quail Coturnix japonica at hatching. \u003cem\u003eBritish poultry science\u003c/em\u003e \u003cstrong\u003e58\u003c/strong\u003e, 59-62, doi:10.1080/00071668.2016.1246708 (2017).\u003c/li\u003e\n\u003cli\u003eSachs, B. D. Photoperiodic Control of the Cloacal Gland of the Japanese Quail. \u003cem\u003eScience\u003c/em\u003e \u003cstrong\u003e157\u003c/strong\u003e, 201-203, doi:10.1126/science.157.3785.201 (1967).\u003c/li\u003e\n\u003cli\u003eBalthazart, J., Schumacher, M. \u0026amp; Ottinger, M. A. Sexual differences in the Japanese quail: behavior, morphology, and intracellular metabolism of testosterone. \u003cem\u003eGeneral and Comparative Endocrinology\u003c/em\u003e \u003cstrong\u003e51\u003c/strong\u003e, 191-207, doi:10.1016/0016-6480(83)90072-2 (1983).\u003c/li\u003e\n\u003cli\u003eBiswas, A., Ranganatha, O. S., Mohan, J. \u0026amp; Sastry, K. V. H. Relationship of cloacal gland with testes, testosterone and fertility in different lines of male Japanese quail. \u003cem\u003eAnimal reproduction science\u003c/em\u003e \u003cstrong\u003e97\u003c/strong\u003e, 94-102, doi:10.1016/j.anireprosci.2005.12.012 (2007).\u003c/li\u003e\n\u003cli\u003eNoble, R. The effects of estrogen and progesterone on copulation in female quail (Coturnix coturnix japonica) housed in continuous dark. \u003cem\u003eHormones and Behavior\u003c/em\u003e \u003cstrong\u003e3\u003c/strong\u003e, 199-204, doi:10.1016/0018-506X(72)90032-3 (1972).\u003c/li\u003e\n\u003cli\u003eNoble, R. Hormonal Control of Receptivity in Female Quail (Coturnix coturnix japonica). \u003cem\u003eHormones and Behavior\u003c/em\u003e \u003cstrong\u003e4\u003c/strong\u003e, 61-72, doi:10.1016/0018-506X(73)90017-2 (1973).\u003c/li\u003e\n\u003cli\u003eDelville, Y. \u0026amp; Balthazart, J. Hormonal Control of Female Sexual Behavior in the Japanese Quail. \u003cem\u003eHormones and behavior\u003c/em\u003e \u003cstrong\u003e21\u003c/strong\u003e, 288-309, doi:10.1016/0018-506x(87)90016-x (1987).\u003c/li\u003e\n\u003cli\u003eBayl\u0026eacute;, J.-D., Ramade, F. \u0026amp; Oliver, J. \u003cem\u003eStereotaxic topography of the brain of the quail (Coturnix coturnix japonica)\u003c/em\u003e. Vol. 68 219-241 (The Journal of Physiology, 1974).\u003c/li\u003e\n\u003cli\u003eBankhead, P.\u003cem\u003e et al.\u003c/em\u003e QuPath: Open source software for digital pathology image analysis. \u003cem\u003eScientific Reports\u003c/em\u003e \u003cstrong\u003e7\u003c/strong\u003e, 16878, doi:10.1038/s41598-017-17204-5 (2017).\u003c/li\u003e\n\u003cli\u003eSchindelin, J.\u003cem\u003e et al.\u003c/em\u003e Fiji: an open-source platform for biological-image analysis. \u003cem\u003eNature methods\u003c/em\u003e \u003cstrong\u003e9\u003c/strong\u003e, 676-682, doi:10.1038/nmeth.2019 (2012).\u003c/li\u003e\n\u003cli\u003eStringer, C., Wang, T., Michaelos, M. \u0026amp; Pachitariu, M. Cellpose: a generalist algorithm for cellular segmentation. \u003cem\u003eNature methods\u003c/em\u003e \u003cstrong\u003e18\u003c/strong\u003e, 100-106, doi:10.1038/s41592-020-01018-x (2021).\u003c/li\u003e\n\u003cli\u003eOttinger, M. A. \u0026amp; Brinkley, H. J. Testosterone and sex-related behavior and morphology: relationship during maturation and in the adult Japanese quail. \u003cem\u003eHormones and Behavior\u003c/em\u003e \u003cstrong\u003e11\u003c/strong\u003e, 175-182, doi:10.1016/0018-506X(78)90046-6 (1978).\u003c/li\u003e\n\u003cli\u003eOttinger, M. A. \u0026amp; Brinkley, H. J. Testosterone and sex related physical characteristics during the maturation of the male Japanese quail (Coturnix coturnix japonica). \u003cem\u003eBiology of reproduction\u003c/em\u003e \u003cstrong\u003e20\u003c/strong\u003e, 905-909, doi:10.1095/biolreprod20.4.905 (1979).\u003c/li\u003e\n\u003cli\u003ePanzica, G. C.\u003cem\u003e et al.\u003c/em\u003e Sexual differentiation and hormonal control of the sexually dimorphic medial preoptic nucleus in the quail. \u003cem\u003eBrain Research\u003c/em\u003e \u003cstrong\u003e416\u003c/strong\u003e, 59-68, doi:10.1016/0006-8993(87)91496-X (1987).\u003c/li\u003e\n\u003cli\u003eCalandreau, L.\u003cem\u003e et al.\u003c/em\u003e Higher inherent fearfulness potentiates the effects of chronic stress in the Japanese quail. \u003cem\u003eBehavioural Brain Research\u003c/em\u003e \u003cstrong\u003e225\u003c/strong\u003e, 505-510, doi:10.1016/j.bbr.2011.08.010 (2011).\u003c/li\u003e\n\u003cli\u003eJones, R. B. The tonic immobility reaction of the domestic fowl: a review. \u003cem\u003eWorld\u0026apos;s Poultry Science Journal\u003c/em\u003e \u003cstrong\u003e42\u003c/strong\u003e, 82-96, doi:10.1079/WPS19860008 (1986).\u003c/li\u003e\n\u003cli\u003eGallup, G. G. Tonic immobility: The role of fear and predation. \u003cem\u003eThe Psychological Record\u003c/em\u003e \u003cstrong\u003e27\u003c/strong\u003e, 41-61 (1977).\u003c/li\u003e\n\u003cli\u003eJones, R. B., Mills, A. D. \u0026amp; Faure, J. M. Genetic and experiential manipulation of fear-related behavior in Japanese quail chicks (Coturnix coturnix japonica). \u003cem\u003eJournal of Comparative Psychology\u003c/em\u003e \u003cstrong\u003e105\u003c/strong\u003e, 15-24, doi:10.1037/0735-7036.105.1.15 (1991).\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"mesotocin, vasotocin, aromatase, social behavior, social environment, Japanese quail","lastPublishedDoi":"10.21203/rs.3.rs-4521069/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4521069/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"Vertebrates, including humans exhibit a wide range of social behaviors that are crucial for the adaptation and survival of most species. The brain organization and function are shaped by genetic and environmental factors, although their precise contributions have been poorly explored in the context of artificial selection. We used divergent lines of quail selected on their high versus low level of motivation to approach a group of conspecifics (S+ and S-, respectively) to investigate the influence of genetic selection and early social environment on sociability. We observed distinct sex- and brain-region-specific expression patterns of three neuronal markers: mesotocin, and vasotocin, the avian homologues of mammalian oxytocin and vasopressin, as well as aromatase, the enzyme that converts androgens into estrogens. These markers displayed pronounced and neuroanatomically specific differences between S+ and S- quail. Additionally, we assessed the influence of early social environment on social skills in adolescent birds. Mixing S+ and S- resulted in more S- males approaching the group without affecting the sociability of S+ or other behaviors, suggesting that the early social environment may influence the results of genetic selection. In conclusion, the divergent quail lines offer a valuable model for unraveling the neuronal and behavioral mechanisms underlying social behaviors.","manuscriptTitle":"Exploring neuronal markers and early social environment influence in divergent quail lines selected for social motivation","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-06-19 06:01:38","doi":"10.21203/rs.3.rs-4521069/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2024-08-08T05:36:56+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-08-07T23:11:48+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-07-12T19:27:24+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"1544305877343635397386174710520532572","date":"2024-06-25T11:41:33+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"230829025676101073287709505067229393720","date":"2024-06-25T01:07:02+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2024-06-24T05:19:34+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-06-22T12:59:21+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2024-06-10T04:10:30+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2024-06-05T09:12:34+00:00","index":"","fulltext":""},{"type":"submitted","content":"Scientific Reports","date":"2024-06-03T10:07:47+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"1a3b69af-748c-447c-a31d-a13df7f6dd94","owner":[],"postedDate":"June 19th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":33307780,"name":"Biological sciences/Neuroscience/Neural circuit"},{"id":33307781,"name":"Biological sciences/Neuroscience/Social behaviour"}],"tags":[],"updatedAt":"2024-10-14T16:08:53+00:00","versionOfRecord":{"articleIdentity":"rs-4521069","link":"https://doi.org/10.1038/s41598-024-74906-3","journal":{"identity":"scientific-reports","isVorOnly":false,"title":"Scientific Reports"},"publishedOn":"2024-10-09 15:57:57","publishedOnDateReadable":"October 9th, 2024"},"versionCreatedAt":"2024-06-19 06:01:38","video":"","vorDoi":"10.1038/s41598-024-74906-3","vorDoiUrl":"https://doi.org/10.1038/s41598-024-74906-3","workflowStages":[]},"version":"v1","identity":"rs-4521069","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4521069","identity":"rs-4521069","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}