Social experience is associated with a differential role of aromatase neurons in sexual behavior and territorial aggression in male mice

doi:10.21203/rs.3.rs-5023417/v1

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Social experience is associated with a differential role of aromatase neurons in sexual behavior and territorial aggression in male mice

https://doi.org/10.21203/rs.3.rs-5023417/v1

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Aromatase (Aro+) neurons located in the Bed Nucleus of the Stria Terminalis (BNST) are crucial for the display of both sexual behavior and territorial aggression in naive male mice. However, little is known about how Aro + neuron circuitry is influenced by social experience. Using a combination of chemogenetics, activity mapping and retrograde viral tracing we show that social experience modulates Aro + neurons during sexual behavior and territorial aggression. Chemogenetic inhibition of BNST Aro + neurons in socially experienced male mice revealed that these neurons are required for territorial aggression, but not for sexual behavior. Behavior testing in experienced animals showed a specific increase in activation in the vomeronasal organ (VNO) and the Medial Amygdala (MeA) after sexual behavior but not territorial aggression, assessed by Egr1 expression. We also observed an increase of Egr1 cells in the medial Preoptic Area (mPOA), a brain region implicated in the display of sexual behavior. Combined retrograde viral tracing and Egr1 immunodetection showed that a subset of the activated cells in the MeA are Aro + neurons projecting to the mPOA. These results highlight that social experience induces a differential neural activity in the circuitry controlling sexual behavior and aggression, which include MeA Aro + neurons projecting to the mPOA.

aromatase

aggression

sexual behavior

social experience

vomeronasal

Sexual behavior and territorial aggression are two innate social behaviors that are controlled by hard-wired circuits linking olfactory inputs to stereotyped motor outputs [1, 2]. Two brain regions are particularly important in these circuits, the principal part of the Bed Nucleus of the Stria Terminalis (BNSTpr) and the postero-dorsal Medial Amygdala (MeApd). Both regions are part of the limbic system, and receive olfactory and pheromonal information from the vomeronasal organ (VNO) via direct wiring from the accessory olfactory bulb (AOB) [3–5]. The BNSTpr and MeApd are highly interconnected and both send projections to the medial preoptic area (mPOA) [6–9], a key center for the modulation of sexual behavior, and to neurons located in the ventro-lateral ventromedial hypothalamus (VMHvl), which control male-male aggression [10]. BNSTpr and MeApd also contain the highest density of aromatase-expressing neurons (Aro+) in the brain [11]. Apart from the local production of estradiol (E2) through aromatization of testosterone, these neurons regulate various social behaviors [12, 13]. In naive male mice, inhibiting or ablating BNSTpr Aro + neurons drastically impairs sexual behavior and territorial aggression [14], while inhibiting MeApd Aro + neurons impairs different forms of aggression, but not sexual behavior [15]. However, the impact of social experience on the role played by these Aro + neurons for the display of sexual and aggressive behaviors remains largely unknown.

Sexual behavior and territorial aggression can be modified by experience [16, 17]. Sexually experienced mice or those with experience in male-male aggression show an increased motivation to initiate the corresponding behavior, as evidenced by a reduction in the latency to the first mount [18] or the first attack [19]. Sexual experience also causes motor improvements leading to a higher efficiency of the behavioral sequence with a reduced latency to the first intromission and a shorter time to reach ejaculation in sexually experienced animals compared to naive ones [20]. Experience in male-male aggression also increases overall aggression in the Resident-Intruder paradigm [19]. However, it remains unclear whether the same neurons in the BNSTpr and MeApd that control sexual and aggressive behaviors are also involved in experience-dependent behavior modulation.

Experience-dependent improvement of sexual behavior is associated with a synaptic and molecular plasticity in the mPOA evidenced by an increase in mature dendritic spine density [18] and increased androgen receptor expression [21]. Similarly, a testosterone-dependent synaptic plasticity has been described in Esr1-positive neurons of the VMHvl after receiving inputs from the posterior amygdala [22]. This mechanism promotes increased aggression in experienced animals, while its depression diminishes the behavioral effect of aggression training. Experience has also been shown to induce plastic changes at the sensory processing level. For example, Arc expressing interneurons in the AOB undergo synaptic plasticity following aggression training, and chemogenetic inhibition of these neurons during training prevents an increase in aggression [23]. Yet, little is known about the effects of social experience on neurons downstream to the AOB, especially on aromatase neurons.

Here, we used a chemogenetic approach to inhibit BNSTpr Aro + neurons in socially-experienced male mice (trained for both aggression and sexual behavior) to determine differential contributions of this cell population in the display of sexual behavior and territorial aggression. We next used Egr1 as a reporter of neural activity to investigate the differential neural plasticity in the form of increased local activity driven by social experience in the context of sexual behavior and male-male aggression. Our results revealed that social experience triggers a behavior-specific plasticity in the VNO and MeApd. Thus, our data expose an experience-dependent link between sensory inputs in the accessory olfactory pathway with limbic regions containing Aro + neurons.

Mice

We employed B6.129S(SJL)-Cyp19a1^{tm2.1(cre)Shah}/J Stock#: 027038; RRID: IMSR_JAX:027038 from the Jackson Laboratory (denoted as Aro^cre mice) that were crossed to 129sv wild type mice (Janvier Laboratories, France) to produce a mixed C57Bl/6 x 129sv background. To generate Aro^Cre Ribo^Tag mice, we crossed homozygotes Aro^Cre mice with RiboTag^loxP/loxP mice (B6N.129(Cg)-Rpl22^tm1.1Psam/J from the Jackson laboratory Stock#: 011029; RRID: IMSR_JAX: 011029 [24]. Adult C57Bl/6 males and ovariectomized receptive females (see supplementary material) were used as stimulus animals (Janvier). Mice were housed under standard 12h light/dark cycles with regular chow diet and water ad libitum.

Social Experience Protocol

Sexual Behavior. Sexually naive male mice (> 8 weeks) were individually housed from 4 days before training. The training began when an unfamiliar ovariectomized receptive female was introduced in their home cage overnight (bedding not changed for at least 4 days). Ejaculation was confirmed by video recordings and latency to ejaculation was scored. From this time point, mice remained individually housed until euthanasia. A second session was performed 14 days later with the introduction of a new unfamiliar receptive female in their home cage overnight. Only males that were successful in the two sessions, with a significant reduction of the latency to ejaculation (Figure S1, left) were kept for the following experiments.

Territorial Aggression. Sexually experienced males (as described above) were trained for territorial aggression by 3 consecutive Resident-Intruder tests (1 per day) [19, 25]. Briefly, an unfamiliar and gonadally intact naive male was introduced into the resident home cage (bedding not changed for at least 4 days) for 10 minutes and latency to the first attack was scored. Aggressive behavior was defined as lunging, biting, chasing, tail rattling, wrestling and kicking. If aggression was too intense, the test was interrupted. Residents were not exposed twice to the same intruder.

Stereotaxic injections. BNSTpr and mPOA coordinates were experimentally determined considering a ten-degree angle (distance from bregma: BNSTpr - AP: -0.18; ML: ±1.28; DV: 5.07 and 4.07 / mPOA – AP: 0; ML: ±1.3; DV: -5.4 and − 5,1). All injections were performed at a rate of 100 nL/min with a 5µL Hamilton Neurosyringe (33 gauge, not beveled). To target the whole BNSTpr in the dorso-ventral axis, two consecutive injections of 250 nL of virus placed 1mm apart were used. Mice were thus injected bilaterally with 500 nL of virus. In the mPOA, 150nL were injected more ventrally and 300µm above, representing a total of 300nL per hemisphere. After injection, the needle was kept in place for two minutes and slowly removed. For details about the whole surgery, see supplementary material.

Viruses. For the BNSTpr chemogenetic experiments, an AAV1/2-hSyn-flx-hM4Di-mCherry-flx (5.9*10^12 viral genomes/mL, reference # V-84-1) or an AAV1/2-hSyn-flx-mCherry-flx (5.4*10^12 viral genomes/mL, reference # V-116-1) were purchased from the Viral Vector Facility of the University of Zurich (Switzerland). Undiluted stock solutions were bilaterally injected in the BNSTpr as described above. For mPOA retrograde tracing experiments, we used a canine adenovirus 2 (CAV2) hSyn1-flx-mCherry-flx (10*10^12 pp/mL) purchased from EJ Kremer in the Plateforme de Vectorologie de Montpellier, France (PVM) [26]. On the day of the injection, a viral aliquot was thawed on ice and diluted just before injection to a final concentration of 1*10^9/150nL in sterile saline.

Chemogenetics. Individually housed and trained Aro^Cre males were tested in their home cage for a 10 min Resident-Intruder aggression test on day 1, and for a 30 min sexual behavior test on day 4. All animals were habituated to the whole behavioral protocol (handling, injections, red light) during 3 days. We randomly assigned animals to receive IP injections of either CNO (3.5mg/kg) or sterile saline 30 minutes before the test. The timing and dose of CNO injection were chosen to ensure CNO concentrations in the brain higher than its EC₅₀ for hM4Di [27]. CNO (Selleck) was dissolved in sterile saline at 12.5mg/mL, aliquoted and stored at -20°C. On each test day, a CNO aliquot was thawed and diluted to 0.35 mg/mL in sterile saline. On a given week, the assigned treatment was the same on day 1 and day 4 (CNO or saline). The experiment was repeated the following week and the treatment was switched (animals that previously received CNO received then sterile saline on day 1 and 4 and vice versa). This whole 2-week experiment was repeated twice. Bedding was changed 1 week before the start of the whole experiment. To keep the animals in a standardized bedding state during the whole experiment (1 month), 1/3 of the soiled bedding was replaced by fresh one following each territorial aggression test (once a week). Behaviors were video recorded and scored manually by a blind annotator using Boris software [28].

Histology

Fixed and frozen coronal brain slices were obtained as described in supplementary material. For chemogenetic experiments, only mice densely expressing mCherry bilaterally along the dorso-ventral axis of the BNSTpr were analyzed. For retrograde tracing experiments, mice were analyzed when the injection site was in the mPOA and mCherry expression was observed in the target regions (BNSTpr and MeApd). Immunolabeling and in situ hybridization were performed as described in supplementary material. 2–3 brain sections per animal were imaged using a Zeiss LSM780 confocal microscope and for each region of interest, 3–4 z-series optical sections were collected. All confocal images were z-normalized using a custom Python script (G-Node DOI: 10.12751/g-node.cs3tdm) and cells in each region of interest (ROI) was classified as described in supplementary material.

Statistical Analysis and Software

Statistical analyses were performed using the statistical software R version 4.2.3. (RRID:SCR_001905) Since most of our data did not meet normality and homoscedasticity requirements, we chose to perform permutation-based statistical tests for the whole data analysis [29–31]. The significance threshold (alpha) was set to 0.05. Sample size (N), p-values, and the specific statistical test performed for each experiment are indicated in the main text and figure legends, and recapitulated in the Supplementary Table 1.

Training. Latency to ejaculation and latency to the first attack were evaluated before and after the training to confirm an improvement in the performance of the animals (Figure S1). Behavioral data were compared using a unilateral Wilcoxon test with permutations.

Chemogenetic. Behavioral data was scored with Boris (RRID:SCR_021434) and analyzed using a custom R script. For latency values, animals not displaying the behavior were given a value of the total duration of the test (i.e., aggression = 600s and sexual behavior = 1800s). Individual values represent the mean of all behavioral parameters (except for proportions) over two trials for each individual mouse. A linear mixed model with permutations was used to test a two-factor model including the group (hM4Di vs. mCherry) and the treatment (CNO vs. Saline). When the interaction was significant, we performed a planned comparison testing the effect of CNO in the hM4Di group with a unilateral paired Wilcoxon test with permutations. Since we tested 4 behavioral parameters, p-values were corrected for 4 comparisons using the Bonferroni method. For proportions, only the second session is presented in Fig. 2 and results from hM4Di animals receiving CNO vs. Saline were compared using a unilateral exact McNemar test. Given that 3 variables were studied for sexual behavior (mount, intromission, ejaculation), p-values were corrected for 3 comparisons using the Bonferroni method. Since only one parameter was analyzed for aggression (attack), the p-value was not corrected.

Confocal Images. Proportions of the different cell types were computed for each slice. Plots represent the mean proportion in each animal. Outliers were identified using the IQR method and removed for further analyses. We performed unilateral two-sample Fisher-Pitman permutation tests for the comparison of the total Egr1 cell proportions between naive and experienced animals, for each behavior. Given that we tested 9 brain regions, all p-values were adjusted for multiple comparisons using the Bonferroni method. For the retrograde tracing analysis, we used the same statistical test, but p-values were corrected for 4 comparisons (2 regions x 2 cell classes). The VNO was analyzed separately using a unilateral two-sample Fisher-Pitman permutation test. The non-parametric Spearman correlation was computed for all correlation analysis.

BNSTpr Aro + cells are necessary for the display of male-male aggression, but not for sexual behavior, in socially experienced male mice.

To evaluate whether Aro + neurons are required in socially-experienced animals, we first crossed Aro^Cre males with a mouse line expressing the Cre-dependent reporter Ribotag [24] and visualized Cre + cells in the BNSTpr. We observed a dense population of Tag + cells present in the BNSTpr after immunodetection (Fig. 1a, left). Aromatase (Cyp19a1) gene mRNA labeling via in situ hybridization (Fig. 1a, right) showed a similar cell distribution in the BNSTpr, indicating that Cre recombinase is largely expressed in Aro + neurons in the Aro^Cre mouse. We then performed a 3-week training of naive Aro^Cre males for sexual behavior and territorial aggression (see methods) (Figs. 1b and S1). Next, we injected the experienced males bilaterally in the BNSTpr with a Cre-dependent adeno associated virus (AAV) encoding for hM4Di-mCherry (Fig. 1c). Then, we used a chemogenetic approach [32] to inhibit Aro + cells by injecting clozapine n-oxide (CNO) to socially experienced Aro^Cre animals prior to behavior testing. As controls, we used saline-injected males, as well as CNO-treated Aro^Cre animals expressing mCherry reporter alone (Fig. 1b-c). Behavior scoring showed that chemogenetic inhibition of BNSTpr Aro + cells had a limited impact on sexual behavior when compared to saline-injected or mCherry-expressing male mice (Fig. 2a-b). The proportion of CNO-hM4Di males mounting and intromitting was slightly lower compared to saline controls (61% vs 84% and 61% vs 76%, respectively), although this difference was not statistically significant (p.adj = 0.37, 0.93, respectively). 38% of CNO-treated animals ejaculated, compared to 76% of controls, but this difference was not significant (p.adj = 0.18) (Fig. 2b). These results show that a high proportion of treated animals still displayed full sexual behavior. We further analyzed other behavioral parameters during the motivational phase such as the time spent in anogenital investigation (Z = 1.15, p.adj = 0.49) and the latency to the first mount (Z = -1.54, p.adj = 0.24), but did not find significant differences between saline and CNO groups (Fig. 2a). Similarly, these animals displayed a comparable mounting time during the copulatory phase (Z = -0.34, p.adj = 1) (Fig. 2a), although we observed a significant increase in the latency to ejaculation in treated vs. control animals (Z = -3.32, p.adj = 0.0017) (Fig. 2a), indicating an incomplete inhibition of sexual behavior.

By contrast, chemogenetic inhibition of BNSTpr Aro + neurons of socially experienced males showed a higher impact on territorial aggression. The number of attacks (Z = 3.30, p.adj = 0.0019), the attack duration (Z = 3.58, p.adj = 0.0006) and the time spent tail rattling (Z = 2.74, p.adj = 0.012) were 4 to 10-fold lower in treated animals, and they took longer to initiate the first attack compared to controls (Z = -2.27, p.adj = 0.046) (Fig. 2c). The proportion of males attacking at least once was reduced from 76% in saline-hM4Di to 38% in CNO-hM4Di animals (p.adj = 0.062) (Fig. 2d). We next examined the percentage of males that displayed a decrease in mounting and attack behavior after CNO injection, and found that 90% of the treated animals attacked less, while only 41% showed less mounting after CNO, a value similar to controls (Fig. 2e). Together, these results indicate that chemogenetic inhibition of BNSTpr Aro + neurons in socially-experienced male mice causes a robust reduction of territorial aggression, while only causing limited impact on sexual behavior.

Social experience triggers a behavior-specific activity increase in the MeApd

We hypothesized that the incomplete inhibition of sexual behavior in socially experienced males may result from experience-induced plasticity. This may be denoted in the form of higher BNSTpr activity, which may render inhibition less effective and/or the recruitment of an alternative/complementary olfactory pathway, bypassing Aro + BSNTpr neurons. To test whether an increase in activity in the BSNTpr occur after experience, we performed immunohistochemistry (IHC) for the immediate early gene Egr1 as a proxy of neural activity in coronal brain slices of naive versus experienced animals after sexual behavior or territorial aggression (Figs. 3a and 4). We found a 4.3-fold significant increase of Egr1 + cells in the BNSTpr of socially experienced animals after territorial aggression (Z = -3.32, p.adj = 0.004) (Fig. 4a). A similar tendency (4.4-fold Egr1 + cell increase) was found after sexual behavior, but the difference was not statistically significant (Z = -2.24, p.adj = 0.11; Fig. 4a).

We also quantified the number of Egr1 + cells in the MeApd (Fig. 4b), a region containing a high density of Aro + neurons that is directly innervated by the vomeronasal system [5]. Following territorial aggression, the proportion of Egr1 + cells in this region between naive and experienced animals was not significantly different (Z =-2.16, p.adj = 0.13; Fig. 4b). By contrast, we observed a much larger 7.5-fold increase in the mean proportion of Egr1 + cells after sexual behavior in experienced (10%) vs. naive (1.3%) animals (Z = -2.94, p.adj = 0.014; Fig. 4b). These results indicate that social experience causes a robust increase in the number of Egr1 + cells in the MeApd following sexual behavior, but not after territorial aggression.

Social experience triggers a behavior-specific activity increase in the VNO

Sexual behavior and territorial aggression in rodents are highly dependent on olfactory signals detected by VNO sensory neurons (VSNs) [33–36]. The BSNTpr and MeApd receive direct and indirect inputs from the VNO via the AOB [5, 37]. We thus explored whether the higher activity observed in the MeApd after social experience is accompanied by a higher activity in the VNO. We performed IHC staining for Egr1 on coronal VNO slices in naive vs. experienced animals after sexual behavior or territorial aggression (Fig. 5). We found that experienced animals showed a significantly higher (1.9-fold) number of Egr1 + cells in the VNO after sexual behavior (Z = -2.0028, p = 0.022; Fig. 5a-b), but not after territorial aggression (Z = -1.43, p = 0.07; Fig. 5c-d). Consistently, Egr1 activity in the VNO strongly correlates with MeApd Egr1 activity following sexual behavior (sex: r_s = 0.75, p = 0.02; aggression: r_s = 0.48, p = 0.19; Fig. 5b). These data suggest that social experience causes a behavior-specific neural plasticity in the VNO that result in higher sensory cell activation rate.

Social experience triggers a behavior-specific plasticity in MeApd Aro + neurons projecting to the mPOA.

BNSTpr and MeApd neurons innervate hypothalamic regions, such as the VMH and mPOA, that play an important role for the display of aggressive and sexual behaviors. Notably, the mPOA receives direct projections from the BNSTpr and the MeApd [8, 9], and mPOA lesions drastically inhibit sexual behavior [38]. To better understand how social experience modulates hypothalamic activity, we screened for Egr1 activity in six hypothalamic regions (mPOA, Arc, DMH, VMHvl, VMHc, VMHdm) (Figs. 6 and S2). Among all these regions, only the mPOA experienced an increase in Egr1 + cells. We observed a 3.1-4-fold increase of Egr1 + cells after experience following both sexual behavior (Z = -2.58, p.adj = 0.044) and territorial aggression (Z = -2.80, p.adj = 0.022), respectively (Fig. 6a-b). We also found a high correlation between MeApd and mPOA activity following sexual behavior (r_s = 0.88, p < 0.00001), but not after territorial aggression (r_s = 0.5, p = 0.22; Fig. 6a).

Given the high density of Aro + neurons in the MeApd, we reasoned that MeApd Aro + cells projecting to the mPOA may be specifically activated in experienced mice following sexual behavior. To test this possibility, we injected a Canine Adenovirus 2 (CAV2) coding a floxed mCherry in the mPOA of Aro^Cre males to retrogradely trace Aro + neurons projecting to the mPOA (Fig. 3). We then performed IHC staining for Egr1 in naive and experienced animals after sexual behavior or territorial aggression and quantified the proportion of cells co-expressing mCherry and Egr1 in the BNSTpr and MeApd. We observed a 3.5–4.9 -fold experience-dependent increase of Egr1+/mCherry + neurons in the BNSTpr following both sexual and aggressive behaviors (sex: Z = -2.21, p.adj = 0.053; aggression: Z = -2.24, p.adj = 0.049; Fig. 6c). A similar 3–3.6-fold increase was observed for Egr1+/mCherry- cells (sex: Z = -2.04, p.adj = 0.08; aggression: Z = -2.48, p.adj = 0.025; Fig. 6c), indicating a global increase in Egr1 activity in the BNSTpr following the two behaviors. In the MeApd, the proportions of both cell populations showed a significant 5-7-fold increase after sexual behavior (mCherry+: Z = -2.79, p.adj = 0.01; mCherry-: Z = -2.89, p.adj = 0.007; Fig. 6d). Importantly, and in contrast to the BNSTpr, territorial aggression did not induce any significant increase of Egr1 + mCherry + or - cells in experienced animals (mCherry+: Z = -0.68, p.adj = 0.98; mCherry -: Z = -1.76, p.adj = 0.15; Fig. 6d). These results indicate that a fraction of Aro + neurons in BNSTpr and MeApd that project to the mPOA, is activated during social behaviors. Experienced animals show a higher proportion of activity in these (and other) cells in the MeApd after sexual behavior, but not after territorial aggression, suggesting that the MeApd is subjected to a functional plasticity specifically after sexual experience.

Although sexual behavior and territorial aggression can be initiated without prior experience, both the motivation to engage in these behaviors and the performance increases dramatically in experimented animals [19, 39]. Here we show that chemogenetic inhibition of BNSTpr Aro + cells in socially experienced male mice drastically reduces territorial aggression. These results contrast with sexual behavior performance, which is barely affected by the inhibition. One relevant aspect of our results lies in the behavior-specificity of the observed phenomenon. Territorial aggression improves in experienced animals (Figure S1) [19], and BNSTpr neurons (including Aro+) play a crucial role in the display of aggression in socially experienced animals [40]. We observed that BNSTpr Aro + inhibition largely impairs aggression, but not sexual behavior, suggesting that these two behaviors are controlled by different Aro + neuron subsets after social experience. In this scenario, neural plasticity in one specific Aro + subpopulation may explain the mechanisms behind this differential regulation. In addition, social experience may induce the recruitment of parallel redundant circuits bypassing the BNSTpr, such as direct olfactory inputs to the MeApd. Consistent with these hypotheses, we found a sexual behavior-specific plasticity in the form of higher number of Egr1 + cells in the MeApd and VNO after experience. While we also observe an increase of Egr1 + cells in the BNSTpr following social experience, this phenomenon occurred following both behaviors and it is thus unlikely to explain the behavior-specificity of the chemogenetic inhibition. Consistent with our results, a study using in vivo calcium imaging of the MeA in mice showed a higher number of neurons recruited specifically when presented with a female congener in sexually experienced compared to naive male mice [41]. Furthermore, we show that the increase of Egr1 expression in experienced animals affects a subpopulation of MeApd Aro + cells which project to the mPOA, suggesting a crucial role of the MeApd as a substrate of neural plasticity following sexual behavior experience in male mice.

We also observed altered sensory function in the VNO, which can be modulated by internal factors including hormonal changes and social experience, leading to behavioral changes in sexual behavior and pup care [42, 43]. Sensory detection of social cues by the VNO is followed by MeA coding of the different olfactory stimuli to produce the appropriate behavioral response [44]. We show an increase in Egr1 + VSNs in experienced male mice following sexual behavior, but not after territorial aggression. This increase in Egr1 staining in the VNO correlates with the increase in Egr1 staining in the MeApd, suggesting that, together with the MeApd and mPOA, a specific sensory plasticity in the VNO may also play a role in sexual behavior learning.

Overall, our data indicate that in experienced animals, BNSTpr Aro + neurons are essential for the display of territorial aggression, but not for sexual behavior. In this context, we observe an increase in Egr1 activity in the MeApd, mPOA and VNO after sexual behavior, suggesting that social experience induces neural plasticity changes in these areas to modulate the behavior. Future experiments aimed to inhibit MeApd Aro + cells in naive animals during learning may prevent the plasticity in the MeApd resulting in a lack of increase in their performance during future encounters. This would help to elucidate whether synaptic plasticity in the MeApd is specifically required for sexual behavior learning.

Statements & Declarations

Acknowledgements

We thank all the people implicated in animal husbandry from the Unité Experimentale de Physiologie Animale de l’Orfrasière (UEPAO), especially Déborah Crespin, Aurélie Gasnier and Flora Martin (https://uepao.val-de-loire.hub.inrae.fr; DOI: 10.15454/1.5573896321728955E12) We also thank the people from the the Plateforme d’Imagerie Cellulaire (PIC; https://pic.val-de-loire.hub.inrae.fr), especially Marie-Claire Blache, Maryse Meurisse and Renaud Fleurot.

Funding

This work was supported by the Agence National de la Recherche (ANR) grant ANR-20-CE92-0003 (PC), and Region Centre Val de Loire project 201900134883 (PC). ET was supported by a grant from the INRAE and Region Centre Val de Loire.

Competing Interests

The authors have no relevant financial or non-financial interests to disclose.

Author Contributions

ET, HV, MK and PC designed the research; ET, TSN, CP and HV performed research; ET and CP analyzed data; PC provided funding acquisition; ET and PC wrote the manuscript with edits from all authors. All authors read and approved the final manuscript.

Data Availability

The datasets generated and analyzed during the current study, with detailed data analysis reports including used packages, codes and outputs are available in a G-Node repository (https://doi.gin.g-node.org/10.12751/g-node.cs3tdm/).

Ethics approval

All animal experiments have been approved by the local ethical committee (Comité d’Ethique en Expérimentation Animale Val de Loire) and complied with French and European guidelines (Apafis reference number 35299).

Consent to participate

Not applicable.

Consent to publish

Not applicable.

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Social experience is associated with a differential role of aromatase neurons in sexual behavior and territorial aggression in male mice

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Abstract

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Introduction

Materials and Methods

Mice

Social Experience Protocol

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Statistical Analysis and Software

Results

Social experience triggers a behavior-specific activity increase in the MeApd

Social experience triggers a behavior-specific activity increase in the VNO

Discussion

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References

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