ARTICLE https://doi.org/10.1038/s41467-021-22911-9 OPEN Sex differences in fear memory consolidation via Tac2 signaling in mice A. Florido 1,2, E. R. Velasco 1, C. M. Soto-Faguás1,3,4, A. Gomez-Gomez5, L. Perez-Caballero 1,2, P. Molina1,6, R. Nadal 1,2,7,8, O. J. Pozo 5, C. A. Saura 1,3,4 & R. Andero 1,2,7,8,9 ✉ 1234567890():,; Memory formation is key for brain functioning. Uncovering the memory mechanisms is helping us to better understand neural processes in health and disease. Moreover, more specific treatments for fear-related disorders such as posttraumatic stress disorder and phobias may help to decrease their negative impact on mental health. In this line, the Tachykinin 2 (Tac2) pathway in the central amygdala (CeA) has been shown to be sufficient and necessary for the modulation of fear memory consolidation. CeA-Tac2 antagonism and its pharmacogenetic temporal inhibition impair fear memory in male mice. Surprisingly, we demonstrate here the opposite effect of Tac2 blockade on enhancing fear memory con- solidation in females. Furthermore, we show that CeA-testosterone in males, CeA-estradiol in females and Akt/GSK3β/β-Catenin signaling both mediate the opposite-sex differential Tac2 pathway regulation of fear memory. 1 Institut de Neurociències, Universitat Autònoma de Barcelona, Cerdanyola del Vallès, Barcelona, Spain. 2 Departament de Psicobiologia i de Metodologia de les Ciències de la Salut, Universitat Autònoma de Barcelona, Cerdanyola del Vallès, Barcelona, Spain. 3 Department de Bioquímica i Biologia Molecular, Facultat de Medicina, Universitat Autònoma de Barcelona, Cerdanyola del Vallès, Barcelona, Spain. 4 Centro de Investigación Biomédica en Red Enfermedades Neurodegenerativas (CIBERNED), Instituto de Salud Carlos III, Madrid, Spain. 5 Integrative Pharmacology and Systems Neuroscience Research Group, Neurosciences Research Program, IMIM (Hospital del Mar Medical Research Institute), Barcelona, Spain. 6 Unitat de Fisiologia Animal, Departament de Biologia Cel·lular, Fisiologia i Immunologia. Facultat de Biociències, Universitat Autònoma de Barcelona, Cerdanyola del Vallès, Barcelona, Spain. 7 Centro de Investigación Biomédica En Red en Salud Mental (CIBERSAM), Instituto de Salud Carlos III, Madrid, Spain. 8 Unitat de Neurociència Traslacional, Parc Taulí Hospital Universitari, Institut d’Investigació i Innovació Parc Taulí (I3PT), Universitat Autònoma de Barcelona, Cerdanyola del Vallès, Barcelona, Spain. 9 ICREA, Pg. Lluís Companys 23, Barcelona, Spain. ✉email: Raul.Andero@uab.cat NATURE COMMUNICATIONS | (2021)12:2496 | https://doi.org/10.1038/s41467-021-22911-9 | www.nature.com/naturecommunications 1 ARTICLE NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-021-22911-9 M emory is the process by which knowledge of the world posttraumatic stress disorder (PTSD) or specific phobias. Not- is encoded, stored, and later retrieved by neural withstanding, the studies of the Tac2 pathway have been mostly circuits1. As a widely accepted valid model for memory, performed only with male subjects. Here, we have discovered the process of lasting memory formation is known to involve two opposite-sex effects mediated by the Tac2 pathway in behavioral different stages: short-term memory (which lasts min to hours) and molecular mechanisms of long-term memory in mice. and long-term memory (which can last days, weeks, or even In this study, we explored the role of Tac2 signaling pathway years). A major difference between them is that long-term on fear memory consolidation in both male and female young memory requires a consolidation process in which gene regula- and adult mice. Importantly, we showed that both the pharma- tion and protein synthesis are necessary. Fear memory is related cological blockade of NK3R and chemogenetic silencing of Tac2 to aversive events and allows an organism to identify threatening neurons in the CeM of male mice leads to subsequently reduced cues previously associated with a negative experience2. These cues fear expression, while in females it increases the expression of fear evoke fear responses that are oriented to preserve the organism’s when manipulations are made during proestrus. We also integrity in the face of danger, allowing the animals to reduce the described the Akt/GSK3β/β-Catenin as the molecular pathway probability of harm when the dangerous associated event appears. involved in the above-mentioned effects through its interaction In the laboratory, we use Pavlovian fear conditioning to assess with sex hormones in both, males and females. fear memory, which consists of pairing a neutral cue as a tone with an aversive event as a footshock, resulting in fear responses that may last for days when the conditioned cue is presented. Results Several data indicate notable differences in fear memory proces- Nk3R antagonism decreases memory consolidation in adult sing between the female and male brain, in both rodents and males and increases it in adult female mice. Systemic admin- humans, at molecular, systemic, and behavioral levels of istration of the Nk3R antagonist osanetant (5 mg/Kg) has pre- analysis3,4. Some of these differences include anatomical differ- viously shown to decrease fear memory consolidation in adult ences in the amygdala of rodents, as larger and denser medial male mice when administered after fear acquisition (FA) by using amygdalae in males, but without differences in basolateral or a cued-fear conditioning (FC) paradigm (Fig. 1a, b)10. First, we central amygdala5–7. Unfortunately, the study of sex differences is hypothesized a similar regulation of fear memory consolidation in still not fully considered in the scientific community researching female mice than in males in a dose-dependent manner. the brain—including memory—although women are more likely Surprisingly, 24 h after the administration of osanetant, female to present a fear-related disorder4. In the last years, researchers mice presented higher rates of freezing than the control group, have published 5.5 studies in males per 1 in females8 pointing out thus showing increased memory consolidation in a totally the evident and growing need to change our approach to neu- opposite effect than in male mice, which showed decreased roscience by including female subjects at all levels of research9. consolidation as previously reported10 (Sex x Treatment interac- Consequently, the molecular mechanisms underlying sex differ- tion: F(1,86) = 10.087, p = 0.002; males: Fig. 1c, F(1,24) = 4.964, p ences between males and females on memory processing are still = 0.036; females: Fig. 1d, F(1,62) = 6.133, p = 0.016). Another largely unclear. study reflects the fact that female mice may present different fear We previously reported that the Tachykinin 2 (Tac2) pathway behaviors that may be of interest in the form of active fear is sufficient and necessary for the modulation of fear memories10. responses instead of passive, such as darting17. Thus, we also The Tac2 gene encodes the neuropeptide Neurokinin B (NkB) studied darting behavior in male and female mice, although we that binds to the Neurokinin 3 receptor (Nk3R) (Fig. 1a). Nk3R found no significant differences between sexes (F(1,11) = 0.672, p couples to the pertussis toxin-insensitive G protein Gq, whose = 0.430). Further, female and male mice did not exhibit activation results in the production of inositol triphosphate and conditioned darting responses (Supplementary Fig. 1a, F(1,1.997) diacylglycerol, and the subsequent activation of protein kinase C = 1.086, p = 0.079). resulting in memory modulation10,11. Tachykinins are abundant Moreover, because it has already been proven that Nk3R peptides in the central nervous system and are involved in neu- systemic antagonism in humans decreases the level of circulating rotransmission and neuromodulation12. Because of its restricted sex hormones18, and these have shown to be important for fear brain expression in brain areas related to fear, such as the Bed memory consolidation in rodents, we next tested whether the Nucleus of the Stria Terminalis (BNST) or the centromedial effect of osanetant (5 mg/Kg) was modulated by the estrous cycle amygdala (CeM) and the available tools, Tac2 manipulation in female mice or the dominance status, as a predictor for allows a high degree of precision for modulating specific fear circulating testosterone concentration in both sexes. First, we memory circuits10. The use of Tac2-Cre mice and Cre-dependent explored whether an intrinsic variable among females could be viral vectors for specific manipulations of these neurons allows modulating the effect of the drug. Based on previous works by the experimenter to manipulate a small population of neurons Milad19,20 showing that the estrous cycle modulates the without affecting surrounding populations. Moreover, Nk3R consolidation of fear extinction memories, we divided the animals antagonists, such as osanetant, are safe and well-tolerated drugs of the previous experiment according to their estrous cycle stage in humans meaning that these findings could rapidly be trans- on the day of FA and drug administration using the vaginal smear lated into humans12. Interestingly, Nk3R agonists or antagonists cytology method for the estrous cycle determination21. We found are not approved for the treatment of human disorders yet. It is that osanetant is effective for increasing fear memory consolida- known that there are differences across species in the distribution tion when FA and drug administration happen during proestrus and pharmacology of the Nk3R in humans and rats13, but both (Fig. 1e, F(1,12) = 12.590, p = 0.004)—presenting high concentra- species have a similar expression of Nk3R in the amygdala, an tions of estradiol and progesterone in comparison to the rest of important area for fear memory regulation13–15. Furthermore, the the cycle—but lack any effect during estrus (Fig. 1f, F(1,13) = binding affinity for NkB in rats and humans is equivalent15 0.492, p = 0.495), metestrus (Fig. 1g, F(1,9) = 0.376, p = 0.555) or suggesting similarities in the pharmacological properties of the diestrus (Fig. 1h, F(1,22) = 1.446, p = 0.242). Nk3R in both species. Besides, in males, to assess the interaction of sex hormones Men and women have previously demonstrated differential life with the effect of osanetant as we did with females, we used the prevalence of fear-related disorders16. Specifically, females pre- Confrontation Tube Test (CTT) to investigate whether social sent a higher lifetime prevalence of fear-related disorders as hierarchy could be influencing the effect of the drug on fear 2 NATURE COMMUNICATIONS | (2021)12:2496 | https://doi.org/10.1038/s41467-021-22911-9 | www.nature.com/naturecommunications NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-021-22911-9 ARTICLE a b c d e f g h memory consolidation as suggested9. Because differential levels of of an animal and thus, differential testosterone levels in grouped- circulating sex hormones appear to be key mediating the effect of housed mice (Supplementary Fig. 2a)9,22,23. We found that both osanetant on fear memory consolidation in female mice, we next dominant and submissive male and female mice presented the tested whether differential concentrations of circulating testoster- same amount of fear expression 24 h after the administration of one also modulate the effect of the drug in male and female mice. osanetant (males: Supplementary Fig. 2b, c, F(1,12) = 0.226, p = The CTT is a tool that may be used to infer the dominance status 0.643; proestrous females: Supplementary Fig. 2d, e, F(1,14) = NATURE COMMUNICATIONS | (2021)12:2496 | https://doi.org/10.1038/s41467-021-22911-9 | www.nature.com/naturecommunications 3 ARTICLE NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-021-22911-9 Fig. 1 Systemic injection of osanetant given after 30 min of fear acquisition impairs memory consolidation in adult males and enhances it in females, especially during proestrus. a Schematic representation of the Tac2 pathway. b Experimental assessment of fear memory consolidation procedure. c Effect in the fear memory test 24 h after administration of osanetant (5 mg/Kg, ip) or vehicle 30 min after fear acquisition (FA) in adult males (n = 13 per group) (p = 0.036). d Effect in the fear memory test 24 h after administration of osanetant (5 mg/kg, ip) or vehicle 30 min after FA in adult females (n = 32 per group) (p = 0.014). This experiment was replicated three times with similar results. Main effect treatment, main effect CS, main effect Sex and interactions were analyzed using two-way repeated measures ANOVA. e Effect in the fear memory test 24 h after administration of osanetant (5 mg/Kg, ip) or vehicle 30 min after FA in adult females divided by their estrous cycle on the FA and drug administration day in proestrus (n = 7 per group) (p = 0.004), (f) estrus (vehicle: n = 7; osanetant: n = 8), (g) metestrus (vehicle: n = 5; osanetant: n = 6) or (h) diestrus (vehicle: n = 11; osanetant: n = 13). The effect of the treatment for each estrous cycle stage was analyzed using one-way repeated measures ANOVA. Data are expressed as mean ± SEM. *p ≤ 0.05 **p ≤ 0.01 vs. vehicle. Asterisks indicate a statistically significant main effect treatment in repeated measures ANOVA. 0.705, p = 0.415), showing no effect of social hierarchy in the was clearly visible when administered during the proestrous stage effects of osanetant modulating fear memory consolidation. Males of the estrous cycle, but not during other stages. Osanetant was did not present differences in regard to social hierarchy in effective in modulating fear memory consolidation at 5 mg/Kg, circulating corticosterone in relatively basal conditions (F(1,13) = but not at higher (10 mg/Kg) or lower (1 mg/Kg) doses. 0.423, p = 0.528), before habituation to CTT (U = 20, p = 0.565), Moreover, social hierarchy, as a predictor of circulating before CTT (F(1,13) = 0.011, p = 0.919), before habituation to FA testosterone, did not show to modulate the effect of osanetant chamber (F(1,13) = 0.760, p = 0.400), before FA (F(1,13) = 0.260, p = on fear memory consolidation in males or females. Importantly, 0.620) or before FE test (U = 23, p = 0.848) (Supplementary Fig. 2f). P28 male and female mice that received the drug did not present In contrast, submissive females showed increased serum corticos- an effect on fear memory consolidation, highlighting the terone in relatively basal conditions (F(1,15) = 6.372, p = 0.024), importance of sexual maturity for osanetant to modulate memory before habituation to CTT (F(1,15) = 35.715, p < 0.000), before CTT consolidation in both sexes. (F(1,15) = 22.729, p < 0.000) and before habituation to FA chamber (F(1,15) = 54.978, p < 0.000); but not before FA (F(1,15) = 0.358, p = Osanetant decreases males’ testosterone and increases females’ 0.559) or before FE test (F(1,15) = 0.353, p = 0.562) (Supplementary estradiol during the consolidation window of fear memories. Fig. 2g). Because the hierarchical status remained quite stable along Taking into account previous reports that show a decrease of time in females, we discarded the role of the estrous cycle mediating circulating sex hormones in humans due to the administration of social dominance in adult females. an Nk3R antagonist18, we hypothesized that the observed beha- After demonstrating that the 5 mg/Kg dose in proestrous vioral effects were mediated by a similar decrease in circulating females and males, independent of social hierarchy, was testosterone and estradiol, without altering stress hormones. modulating fear memory consolidation, we explored whether Besides, because adult sex hormones appeared to be necessary for other doses had similar effects. For this, we tested both 1 mg/Kg osanetant to modulate memory consolidation in males and and 10 mg/Kg finding that none of them altered fear memory females, we next tested the hormonal dynamics after FA and consolidation in males (F(1,21) = 0.876, p = 0.431), females during injection of osanetant or vehicle. To prove our hypothesis, we proestrus (F(1,20) = 0,155, p = 0.858) or females during metestrus measured sex and stress hormones at two different time points (F(1,21) = 0.01, p = 0.990) (Supplementary Fig. 1b–d). Thus, the (30 min and 330 min after the injection of osanetant) that drug describes an inverted U shape dose–response curve which is reflected different moments of the consolidation window of fear similar to other drugs modulating memory24. memories. Due to the suggested role of sex hormones in modulating the Testosterone was reduced 30 min after the injection of the drug effect of osanetant on fear memory consolidation, we wanted to (1 h after FA) in males (χ2(4) = 19.661, p = 0.001; vehicle30 vs. test whether this modulation was dependent on the maturation of baseline p = 0.043, osanetant30 vs. baseline p = 0.050), females in the organism at the moment of receiving the drug, because of the proestrus (χ2(4) = 31.645, p < 0.000; vehicle30 vs. baseline p < 0.000, differences involved in circulating sex hormones and the osanetant30 vs. baseline p < 0.000) and females in metestrus (χ2(4) maturation of the brain systems involved. To that end, we tested = 17.296, p = 0.002; vehicle30 vs. baseline p < 0.000, osanetant30 vs. 5 mg/Kg of osanetant on prepubertal postnatal day (P) P28 in baseline p = 0.001) (Fig. 3a–c). When assessed 330 min after male and female mice, which have not reached sexual maturity receiving osanetant or vehicle (6 h after FA), males that received yet25. Results showed that male and female mice at day P29 had vehicle presented a peak of testosterone above the baseline similar freezing rates irrespective of treatment (males: Fig. 2a, (vehicle330 vs. baseline p = 0.051) while the osanetant treated F(1,14) = 0.171, p = 0.686; females: Fig. 2b, (F(1,14) = 0.037, p = group presented lower levels than the vehicle group (vehicle330 vs. 0.851), thereby suggesting that osanetant does not modulate fear osanetant330 p = 0.007) (Fig. 3a), thus showing that the injection memory consolidation in the absence of adult levels of sex reduced the testosterone levels during the consolidation window of hormones in males. An alternative explanation may be that the fear memories. Similar results were obtained with amygdalae Tac2 pathway circuits are not mature yet although the micropunches; osanetant decreased amygdalar concentration of neuropeptide is expressed in the brain. The lack of effect in testosterone compared to vehicle 330 min after the administration prepubertal mice might not only be due to the lack of adult levels of osanetant (6 h after FA) (Supplementary Fig. 3a, U = 9, p = of sex hormones, but developmental factors may interfere such as 0.016). Furthermore, linear regression analyses showed that the maturation of the fear-related neurocircuitry. Further, both testosterone in serum was highly predictive of amygdalae young and adult females and males, express similar levels of testosterone (Supplementary Fig. 3c, R2 = 0.786, p < 0.001) as well Nk3R in the central amygdala (CeA) (Fig. 2c, d, χ2(3) = 2.991, p as corticosterone (Supplementary Fig. 3d, R2 = 0.344, p = 0.017). = 0.393). In contrast, neither females presented an effect of the drug in Our results showed a completely opposite-sex effect of the drug testosterone when compared to the vehicle groups during proestrus in male and female mice, with males presenting a reduction of (vehicle30 vs. osanetant30 p = 0.707, vehicle330 vs. osanetant330 p = fear memory consolidation and females an increase after 0.520) nor metestrus (vehicle30 vs. osanetant30 p = 0.686, vehicle330 receiving osanetant. Further, in females, the effect of the drug vs. osanetant330 p = 0.283) (Fig. 3b, c). Females during proestrus 4 NATURE COMMUNICATIONS | (2021)12:2496 | https://doi.org/10.1038/s41467-021-22911-9 | www.nature.com/naturecommunications NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-021-22911-9 ARTICLE a b c Nk3R DAPI d Fig. 2 Systemic injection of osanetant given 30 min after fear acquisition is ineffective in modulating fear memory consolidation in prepubertal male and female mice, which present similar expression of Nk3R-positive neurons compared to adult male and female mice across the estrous cycle. a Effect of osanetant or vehicle administration 30 min after FA in freezing behavior in P28 male (n = 8 per group) and (b) female mice (n = 8 per group). c Representative confocal image of the Nk3R in the CeA of prepubertal mice. Scale bar = 60 µm. d Quantification of Nk3R-positive cells in the CeA of adult and prepubertal males and prepubertal and adult females across the cycle (n = 4 per group). Data are expressed as mean ± SEM. Main effect treatment, main effect CS, and CSxTreatment interaction were analyzed using repeated measures ANOVA, while NK3R expression was analyzed using non- parametric Krukal–Wallis’ χ2. presented increased estradiol concentration in serum compared to time between the two time points (males: vehicle30 vs. vehicle330 p the vehicle and baseline 330 min after receiving the drug (Fig. 3p, < 0.000, osanetant30 vs. osanetant330 p < 0.000; females in proestrus: χ2(2) = 8.307, p = 0.016; osanetant330 vs. baseline p = 0.022, vehicle30 vs. vehicle330 p = 0.001, osanetant30 vs. osanetant330 p < vehicle330 vs. osanetant330 p = 0.022), showing increased estradiol 0.000; females in metestrus: vehicle30 vs. vehicle330 p = 0.001, during the consolidation window of memory, while females during osanetant30 vs. osanetant330 p = 0.001) (Fig. 3d–f). Dehydrocorti- metestrus presented no effect of the drug (Fig. 3q, χ2(2) = 2.678, p costerone and deoxycorticosterone followed similar patterns as = 0.262). corticosterone as expected (Fig. 3j–o). We also tested whether osanetant produced an effect over stress- Progesterone was increased independently of the treatment related hormones corticosterone, dehydrocorticosterone, or deox- 30 min after receiving the injection in males (χ2(4) = 464.285, p ycorticosterone, as well as in progesterone in males, proestrous and < 0.000, vehicle30 vs. baseline p < 0.000, osanetant30 vs. base- metestrous females. None of these hormones were modulated by line p < 0.000) and decreased in proestrous females (χ2(4) = the administration of osanetant in any of the two chosen time 14.490, p = 0.006, vehicle30 vs. baseline p = 0.005, osanetant30 points (30 or 330 min after the drug injection) (Fig. 3d–o). This vs. baseline p = 0.003), but no changes were observed in lack of modulation was also found in amygdala micropunches for females during metestrus (χ2(4) = 2.136, p = 0.711). Males corticosterone (Supplementary Fig. 3b, t(14) = 1.263, p = 0.227). returned to relatively basal progesterone levels at 330 min We found increased corticosterone in serum in response to the after the injection (vehicle330 vs. baseline p = 0.114, osane- stress induced by FA 30 min after the treatments in males (χ2(4) = tant330 vs. baseline p = 0.072), while in proestrous females only 193.334, p < 0.000, vehicle30 vs. baseline p < 0.000, osanetant30 vs. osanetant-treated mice recovered the baseline 330 min after the baseline p < 0.000), females during proestrus (χ2(4) = 62.980, p < injection (vehicle330 vs. basal p = 0.002, osanetant330 vs. basal 0.000, vehicle30 vs. baseline p < 0.000, osanetant30 vs. baseline p < p = 0.200) (Fig. 3g–i). 0.000) and females during metestrus (χ2(4) = 52.572, p < 0.000, Interestingly, osanetant did not alter corticosterone or related vehicle30 vs. baseline p < 0.000, osanetant30 vs. baseline p < 0.000). metabolites in any of the chosen time points in both males and This increase in serum corticosterone was found to decrease over females. Importantly for our study, osanetant-treated males NATURE COMMUNICATIONS | (2021)12:2496 | https://doi.org/10.1038/s41467-021-22911-9 | www.nature.com/naturecommunications 5 ARTICLE NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-021-22911-9 presented lower testosterone than the ones administered with the a similar direction than the behavioral effects on memory vehicle at 330 min after receiving osanetant (360 min after FA). In consolidation. osanetant-treated females, we also found an increase of estradiol in proestrus, but not during metestrus. Altogether, osanetant showed a sex-opposite regulation of sex hormones 330 min after Nk3R expression is similar in males and proestrous and its administration in male and female mice that underwent FA, in metestrous females, with increased colocalization of Nk3R with 6 NATURE COMMUNICATIONS | (2021)12:2496 | https://doi.org/10.1038/s41467-021-22911-9 | www.nature.com/naturecommunications NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-021-22911-9 ARTICLE Fig. 3 Testosterone levels in serum in males after fear acquisition are decreased by osanetant while estradiol in females’ serum is increased 330 min after its administration compared to vehicle-treated mice, with no effect of the drug on progesterone, corticosterone, deoxycorticosterone, or dehydrocorticosterone. All mice received osanetant (5 mg/Kg, ip) or vehicle 30 min after fear acquisition, and trunk blood was collected either 30 min or 330 min after the injection. Estradiol measures were only determined 330 min after the injection. Dashed lines indicate the relatively basal concentration for an independent group of animals that did not receive any treatment. a–c Testosterone concentration in males (p = 0.001, treatment330min: p = 0.007), proestrous females, and metestrous females. d–f Serum corticosterone concentration in males (p = 0.000), proestrous females (p = 0.000), and metestrous females (p < 0.000). g–i Circulating progesterone concentration in serum in males (p = 0.000), proestrous females, and metestrous females. j–l Males (p < 0.000), proestrous (p < 0.000), and metestrous (p < 0.000) females’ serum dehydrocorticosterone (DHC) concentration. m–o Concentration of circulating deoxycorticosterone (DOC) in serum in males (p = 0.000), proestrous (p < 0.000) and metestrous females (p < 0.000). p Serum estradiol concentration after FA and 330 min after receiving osanetant in proestrous females (p = 0.022). q Serum estradiol concentration after FA and 330 min after receiving osanetant in metestrous females. This experiment was replicated twice with similar results. Males vehicle30: n = 11; males osanetant30: n = 10; males vehicle330: n = 16; males osanetant330: n = 16; proestrous females vehicle30: n = 8; proestrous females osanetant30: n = 8; proestrous females vehicle330: n = 7; proestrous females osanetant330: n = 8; metestrous females vehicle30: n = 8; metestrous females osanetant30: n = 8; metestrous females vehicle330: n = 8; metestrous females osanetant330: n = 8. Proestrous females vehicle330 for estradiol determination n = 5; proestrous females osanetant330 for estradiol determination n = 7; metestrous females vehicle330 for estradiol determination n = 7, metestrous females osanetant330 for estradiol determination n = 6. Data are mean ± SEM. *p ≤ 0.05 **p ≤ 0.01 ***p ≤ 0.001 vs its vehicle. When with lines, it indicates specific comparisons. +p ≤ 0.05 ++p ≤ 0.01 +++p ≤ 0.001 vs baseline. The statistic was Wald’s χ2 with pairwise comparisons between groups for testosterone, corticosterone, progesterone, DHC, and DOC. Estradiol was analyzed using non-parametric Kruskal–Wallis’ χ2. GAD65 during proestrus. Nk3R is highly expressed under basal Our results showed a similar expression of Nk3R in the CeA in conditions in limbic and prefrontal cortical areas. Especially in adult and prepubertal male and female mice, independently of the the amygdala, it is highly localized in the CeA26. We have pre- estrous cycle. Interestingly, female mice showed increased viously shown that the modulation of Nk3R in the CeA is crucial colocalization of CaMKIIα with Nk3R compared to males, for the regulation of memory consolidation10. Thus, we hypo- suggesting a higher tendency towards CaMKIIα-mediated thesized that osanetant was blocking Nk3R-expressing neurons in synaptic plasticity in females. Interestingly, although we found the CeA that were differentially colocalized with inhibitory bio- no differences in colocalization of an excitatory biomarker, as markers in males and excitatory biomarkers in females, which vGLUT2, with Nk3R in the CeA; we found increased colocaliza- could be modulated by the estrous cycle. To test our hypothesis, tion of GAD65 with Nk3R in the CeA of proestrous females, we performed an immunohistochemistry assay in the CeA to suggesting increased susceptibility to GABA-mediated synaptic measure colocalization of Nk3R with GAD65, CaMKIIα, or plasticity in the CeA of females during proestrus. vGLUT2. Further, we compared CeA Nk3R-positive neurons between males, females in proestrus or metestrus and P28 male Chemogenetic silencing of CeA-Tac2 neurons after FA and female mice to assure the lack of effect in prepubertal mice is decreases memory consolidation in males and increases it in not due to a lack of expression of this receptor in CeA. proestrous females. Because it has been previously shown that First, we found non-significant differences in Nk3R-positive local Nk3R antagonism in the CeA decreases fear memory con- neurons in the CeA in males, females in both proestrus and solidation in male mice10, we next hypothesized that a single metestrus and prepubertal male and female mice (Fig. 2c, d, χ2(3) micro-infusion of osanetant in the CeA of females mice during = 2.991, p = 0.393). To explore for possible changes of neuronal proestrus would increase fear memory consolidation in a similar markers in Nk3R-positive neurons in the CeA between males and manner than the intraperitoneal injection. Further, due to the females across the estrous cycle, which could explain the sex- high degree of colocalization between NkB (the result of the Tac2 dependent opposite effects on memory consolidation, we next gene expression) and Nk3R in the CeM27, we hypothesized that examined the colocalization of Nk3R with markers of inhibitory the silencing of Tac2 neurons in the CeM could replicate the neurons (GAD65), glutamate synaptic plasticity (CAMKIIα) and osanetant-induced alterations on fear memory consolidation in excitatory neurons (vGLUT2) (Supplementary Fig. 4c). Remark- male and female mice. Because Nk3R is also expressed in other ably, colocalization of GAD65 with Nk3R was increased in areas of the amygdala where Tac2 is not synthesizing NkB, such females during proestrus when compared to metestrous females as more lateral portions of the CeA27, we wanted to test whether and males (Supplementary Fig. 4c, e, F(2,10) = 6.001, p = 0.022, the effect on memory consolidation was replicated by specific males vs. proestrus p = 0.015, metestrus vs. proestrus p = 0.015). Tac2-CeM blockade. Furthermore, CaMKIIα immunocolocalization with Nk3R was Bilateral local injection of osanetant (625 ng/side) in the CeA of also increased in females in comparison to males (Supplementary proestrous females (Fig. 4a, Supplementary Fig. 7) after FA Fig. 4c, e, F(2,10) = 4.856, p = 0.037, males vs. proestrus p = 0.022, increased memory consolidation, as expected, as shown by males vs. metestrus p = 0.027). Immunocolocalization of increased freezing rates during the expression test when vGLUT2 with Nk3R showed no differences among groups compared to vehicle controls (Fig. 4b, F(2,7) = 5.858, p = 0.046). (Supplementary Fig. 4c, e, F(2,10) = 2.07, p = 0.189). This finding demonstrated that a population of Nk3R-positive Given the evident role of estradiol in the effects of the drug on neurons located in the CeA is required for the modulation of memory consolidation, an immunolocalization study was per- memory consolidation in an opposite-sex manner. formed to assess colocalization of Estrogen Receptors (ERs: ERβ, Next, we used Tac2-Cre mice to assess whether chemogenetic and ERα) with Nk3R in the CeA. We found a significant sex effect silencing of these neurons in the CeA replicated the effects of in colocalization of ERβ with Nk3R, showing decreased pharmacological Nk3R blockade in fear memory consolidation in colocalization of these receptors in the CeA in females compared both sexes. Male and female mice were inoculated in the CeA to males (Supplementary Fig. 4a, d, F(1,10) = 7.251 p = 0.023). On with the Cre-dependent AAV-DIO-hMD4i-mCherry. Tac2 the other hand, we found no differences in colocalization of ERα neurons were silenced with CNO administration (1 mg/Kg, ip) with Nk3R between males and proestrous or metestrous females 30 min after FA (Fig. 4c)10. The fear expression test revealed (Supplementary Fig. 4b, d, χ2(2) = 1.423, p = 0.491). reduced freezing in male mice that expressed the reporter NATURE COMMUNICATIONS | (2021)12:2496 | https://doi.org/10.1038/s41467-021-22911-9 | www.nature.com/naturecommunications 7 ARTICLE NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-021-22911-9 mCherry in the CeA when compared to wild-type injected the FA and drug administration day. Females that expressed controls. In contrast, memory consolidation was significantly mCherry in the CeA, and received FA and CNO in proestrus increased in female mice with expression of the reporter in the presented increased memory consolidation (Fig. 4e, F(1,11) = CeA (Sex x Genotype interaction: F(1,31) = 1.972, p = 0.002; 5,897, p = 0.034), while this effect was absent during metestrus males: Fig. 4d, F(1,9) = 5.738, p = 0.036; females: F(1,20) = 5.618, (Fig. 4f, F(1,7) = 0.784, p = 0.405), all compared to wild-type p = 0.028), with different effects according to the estrous cycle on controls also injected with the AAV without the reporter. 8 NATURE COMMUNICATIONS | (2021)12:2496 | https://doi.org/10.1038/s41467-021-22911-9 | www.nature.com/naturecommunications NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-021-22911-9 ARTICLE Fig. 4 CeA-Nk3R blockade increases fear memory consolidation in proestrous females while chemogenetic silencing of the CeA-Tac2 neurons impairs fear memory consolidation in males and enhances it in females during proestrus. a Schematic diagram of intracerebral injection procedure with FC. b Effect in the fear memory test 24 h after osanetant (625 ng/side) or vehicle intracerebral injections in the Central Amygdala (CeA) after fear acquisition (FA) in proestrous females (vehicle: n = 4; osanetant: n = 5) (p = 0.046). Main effect treatment, main effect CS, and CSxTreatment interaction were analyzed using one-way repeated measures ANOVA. c Schematic diagram of a fear procedure to assess Gi DREADD mediated silencing of CeA-Tac2 neurons. d Temporal silencing of CeA-Tac2 neurons after FA effect on fear memory consolidation in male mice as measured by the Fear Expression (FE) test 24 h later (wild-type: n = 5; hMD4i: n = 6) (p = 0.036). e Temporal silencing of CeA-Tac2 neurons after FA effect on fear memory consolidation measured by FE test 24 h later in proestrous females (wild-type: n = 6; hMD4i: n = 7) (p = 0.034). f Temporal silencing of CeA-Tac2 neurons after FA effect on memory fear consolidation in metestrous females (wild-type: n = 4; hMD4i: n = 5). Main effect treatment, main effect CS, main effect sex and interactions were analyzed using two-way repeated measures ANOVA. The effect of the treatment for each estrous cycle stage was analyzed using one- way repeated measures ANOVA. g Representative confocal image showing mCherry-positive neurons in infected Tac2-Cre mice (hM4Di) but not in wild- type (control). Scale bar = 30 µm. h Males and females count of CeA-Tac2 mCherry-positive neurons per mm2 (males: n = 13; females: n = 12). Data are mean ± SEM. *p ≤ 0.05 vs. vehicle or wild-type. Asterisks above a line indicate significant main effect treatment in repeated measures ANOVA. Mann–Whitney’s U was used for the histochemistry assay. The expression of the reporter mCherry was also used to assess males or proestrous females in the levels of total CREB (males: the number of Tac2-positive neurons in the CeA (Fig. 4g). F(2,21) = 0.332, p = 0.722; proestrus: χ2(3) = 0.876, p = 0.645), Supporting our previous Nk3R-positive immunolocalization phosphorylated CREB (Ser 133) (males: χ2(3) = 4.182, p = 0.124; results, no difference was found in the number of Tac2-positive proestrus: F(2,14) = 0.014, p = 0.986), total mTOR (males: F(2,21) neurons between males and females in the CeA (Fig. 4h, U = 68, = 0.210, p = 0.813; proestrus: F(2,14) = 1.441, p = 0.272) or p = 0.586). These data indicated that the opposite-sex effect on phosphorylated mTOR (Ser 2448) (males: F(2,21) = 0.049, p = memory consolidation was not due to a different number of CeA- 0.952; proestrus: F(2,14) = 1.441, p = 0.632) (Fig. 5c, Supplemen- Tac2-positive neurons. tary Fig. 6). Notwithstanding, total GSK3β protein levels were Our results suggest that osanetant-mediated alterations on fear significantly decreased in males (Fig. 5c, Supplementary Fig. 6, memory consolidation occur because of a blockade of Tac2 χ2(3) = 10,031, p = 0.007, basal vs. osanetant p = 0.011, vehicle vs neurons in the CeM rather than because of possible peripheral osanetant p = 0.006) while in females during proestrus total Akt effects caused by the drug. Moreover, we concluded that the sex- expression was increased (F(2,14) = 3.821, p = 0.050, basal vs. opposite effect observed was not due to a different expression of osanetant p = 0.029, vehicle vs. osanetant p = 0.035), but not its Tac2 in the CeM, as shown by similar expression of the reporter phosphorylation (Ser 473 and Thr 308) (respectively: χ2(3) = mCherry between sexes. 3.640, p = 0.162; χ2(3) = 0.051, p = 0.975). In agreement with elevated Akt, phosphorylated GSK3β (Ser 9)/GSK3β ratio was Osanetant oppositely regulates the Akt/GSK3β/β-Catenin increased (F(2,14) = 4.664, p = 0.030, basal vs. osanetant p = 0.017, pathway in the amygdala during memory consolidation. Nk3R vehicle vs. osanetant p = 0.022). Inactive phosphorylated GSK3β is a G Protein-Coupled Receptor (GPCR) upstream of different may lead to decreased phosphorylation of its downstream target signaling pathways regulating the synthesis of inositol β-Catenin (Ser33/37, Thr41) (χ2(3) = 6887, p = 0.032; basal vs. triphosphate11. To detect the downstream signaling pathways vehicle p = 0.052, basal vs. osanetant p = 0.017, osanetant vs. affected by the administration of osanetant 30 min after FA, we vehicle p = 0.841) (Fig. 5c, Supplementary Fig. 6). These results performed an exploratory experiment. Next, we confirmed our are in agreement with the observed increase of Akt1 transcript results using Western Blot and further identified a specific route levels in the gene expression analyses and indicate that osanetant- altered in CeA cells in response to osanetant during the con- mediated Nk3R blockade results in upregulation of Akt leading to solidation window of fear memories. inactivation of GSK3β and upregulation of β-Catenin in To that end, we dissected both amygdalae 30 min after proestrous females. receiving osanetant after fear acquisition in males and females In summary, males and proestrous females presented a sex- during proestrus and metestrus (Fig. 5a). A qPCR mRNA array of opposite regulation of the PI3K/Akt pathway in the CeA after 84 GPCR-related genes was performed, revealing downregulation receiving osanetant as shown by bioinformatic analyses of the of Agt, Agtrap, Bcl2, Calcrl, Ccnd1, Ccne1, Cdkn1a, Cdkn1b, Cflar, GPCR-related genes expression patterns. Further, Western Blots Elk4, Galr2, Gnas, Bcl2ll in males that received osanetant confirmed these results and revealed a downregulation of the Akt/ compared to vehicle controls; while Akt1, Gcgr, Fgf2, Lhcgr, GSK3β/β-Catenin in male mice while this downstream pathway Mmp9, Ptgdr, Rho, S1pr3, Vcam1 gene expression was upregu- was upregulated in female mice during proestrus, thus, suggesting lated in females that received osanetant only during proestrus to be mediating the observed effects on fear memory consolida- (Supplementary Fig. 5). In females during metestrus, only Adrb2 tion in both sexes. and Lpar1 were downregulated and Kcnh8 upregulated after the treatment (Supplementary Table 1). Ingenuity Pathway Analysis (IPA) of these genes revealed the Akt/GSK3β/β-Catenin signaling Blockade of osanetant effect on memory consolidation by pathway as the main molecular route affected by the drug, and androgen receptor (AR) agonism or Akt activation in males specifically, Akt signaling was upregulated in proestrus (Fig. 5b, and ERs antagonist or Akt inactivation in females. Altogether, Supplementary Fig. 6). osanetant reduced testosterone in males and increased estradiol in Since PI3K/Akt signaling targets different pathways, we next proestrous females during memory consolidation. Further, we analyzed the activation and protein levels of its three main also found that osanetant decreased the Akt/GSK3β/β-Catenin downstream pathways—CREB, mTOR, and GSK3β—following a pathway in males while it increased it in proestrous females. similar approach (Fig. 5a). 10 min after the injection of the drug, Therefore, we hypothesized that local activation in the CeA of AR amygdalae were dissected and levels of total and phosphorylated in males and blockade of ERs in proestrous females would revert levels of CREB, mTOR, GSK3β, and Akt were analyzed by the consolidation effects caused by osanetant. Moreover, our Western blotting. Our results showed no effect of the drug in hypothesis also contemplated that local activation of Akt in NATURE COMMUNICATIONS | (2021)12:2496 | https://doi.org/10.1038/s41467-021-22911-9 | www.nature.com/naturecommunications 9 ARTICLE NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-021-22911-9 a b Down-regulated in males Upregulated in proestrus c males, and inhibition in females would also revert the mentioned higher freezing rates in the expression test 24 h later (Fig. 6a, b, effects of osanetant. CS x Treatment interaction F(1,8) = 4.107, p = 0.018; CS1-5: As previously hypothesized, males that received systemic vehicle vs. Akt activator p = 0.003, vehicle vs. AR agonist p = osanetant together with intra-CeA AR agonist (CI-4AS-1) or an 0.018; CS6-10: vehicle vs Akt activator p = 0.137, vehicle vs. AR Akt activator (SC 79) presented increased memory consolidation agonist p = 0.010; CS11-15: vehicle vs Akt activator p = 0.031, in comparison to those that only received osanetant, as shown by vehicle vs AR agonist p = 0.021; Supplementary Fig. 8a). In 10 NATURE COMMUNICATIONS | (2021)12:2496 | https://doi.org/10.1038/s41467-021-22911-9 | www.nature.com/naturecommunications NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-021-22911-9 ARTICLE Fig. 5 Osanetant administered 30 min after FA reduces Akt/GSK3β/β-Catenin pathway activation in males and it enhances it in proestrous females in the amygdala. a Fear procedure to obtain amygdala tissue for biochemical studies. b Schematic representation of the involved PI3K/Akt pathway revealed by the G-Coupled Protein Receptor (GCPR) qPCR array results after bioinformatics analysis of the Ingenuity Pathway Analysis (IPA) software. Red indicates downregulation and green upregulation (n = 4 per group). c Western blot analyses of the Akt/GSK3β/β-Catenin signaling pathway in amygdalar lysates of males in males (basal: n = 7; vehicle: n = 8; osanetant: n = 8) revealed downregulated total GSK3β expression in osanetant treated mice compared to vehicle (p = 0.006) and basal (p = 0.011) groups; while proestrous females (basal: n = 6; vehicle: n = 5; osanetant: n = 5) showed increased total Akt expression in osanetant treated mice compared to vehicle (p = 0.035) and basal groups (p = 0.029), with a decrease in p-β-Catenin in osanetant treated females compared to basal animals (p = 0.017). Original blots and scans are shown in Supplementary Fig. 6. Protein levels were normalized to GADPH (left graphs) and phosphorylated proteins were normalized to total protein levels (right graphs). Data are mean ± SEM. *p ≤ 0.05 vs. the other groups. For qPCR analysis, two-tailed t-test or Mann–Whitney’s U test were used for statistical analyses. Western blots were analyzed with one-way ANOVA or Kruskal–Wallis’ χ2 test. Pairwise comparisons are indicated when appropriate. contrast, proestrous females that received intra-CeA ERs electrophysiological correlate of synaptic efficacy required for long- antagonist (ICI 182,780) or an Akt inhibitor (Akti-1/2) together term memory consolidation31. Adult male and female rats show with systemic osanetant presented lower memory consolidation equivalent LTP, although the proven mechanisms in females involve when compared to those proestrous females that only received cAMP-activated protein kinase (PKA) for the initiation of poten- osanetant, as expressed by lower freezing rates in the expression tiation induced by estradiol whereas PKA is not necessary for the test 24 h after receiving the treatment (Fig. 6a, c, F(1,15) = 21.299, estradiol-induced potentiation in males32. Our current study shows p < 0.000, η2p = 0.740, vehicle vs. antiAkt p < 0.000, vehicle vs. a sex hormone-dependent modulation of memory consolidation, ERs antagonist p = 0.003, antiAkt vs. ERs antagonist p = 0.021; since testosterone results necessary for osanetant to reduce fear Supplementary Fig. 8b). memory consolidation while the increase in estradiol levels in In line with our hypothesis, local AR or Akt activation females is necessary for the enhancement of memory consolidation, normalized the decreased freezing rates caused by the blockade of as shown by the blockade of effects when we pharmacologically Nk3R by osanetant in male mice. In contrast, local blockade of reverted the hormonal fluctuations caused by osanetant. Intraper- ERs or Akt in female mice during proestrus reverted the effect of itoneal injection of a dopamine receptor 1 agonist in adult female osanetant increasing fear memory consolidation in female mice. rats during low estradiol phases enhances fear memory recall whereas females trained during high estradiol estrous phases present Discussion impaired FE recall33. However, our data provide evidence that there Taken together, our data show that the temporal inactivation of are opposite-sex effects in behavioral and molecular mechanisms the CeA-Tac2 pathway results in impaired fear memory con- (Akt/GSK3β/β-Catenin signaling) of fear memory consolidation solidation in males but increases fear memory in females. This after a drug administration in the amygdala of adult mice. memory effect is dependent on sex hormones and their specific receptors in the CeA; testosterone in males and estradiol in Dominance/submission, estrous cycle, hormones, and fear females. Additionally, Tac2 pathway inactivation is sufficient for memory. Typically, the manipulations in rodent fear memory amygdala Akt/GSK3β/β-Catenin signaling downregulation in studies only involve one sex and generally do not account for males and upregulation in proestrous females, being these traits or hormonal status which are relevant for brain molecular changes necessary to reduce fear memory consolida- functioning9. In females, it is only when osanetant is given during tion in adult males and increase it in adult females during the proestrous stage that fear memory consolidation is enhanced. proestrus. See Fig. 7 for a graphical summary of the results. These results suggest a key role of circulating sex hormones in The study of sex differences in fear memory has vastly increased females mediating the effect of osanetant on fear memory con- in the last years. Indeed, numerous previous studies have found sex solidation. In line with previous studies that demonstrate an differences in long-term memory formation and its mechanisms4. It interaction between fear memory consolidation and the estrous has recently been reported greater contextual fear conditioning and cycle, it has been already reviewed that adult female rodents in generalization in females compared to males, with context pre- proestrus present enhanced fear extinction retention20,34. More- exposure increasing conditioning in adult males and decreasing over, we have tested the effect of an Nk3R antagonist on fear generalization in females28. These results go in line with important memory consolidation depending on both dominance/submis- differences in c-fos activation between males and females during sion in males and females and the estrous cycle in females. memory retrieval and context generalization, with males showing Regarding the social hierarchy, which is highly dependent on increased c-fos activation in the dorsal hippocampus, while females androgens in male and female mice35–37, results suggest that in the basolateral amygdala28. Further, it has been described dominance status as assessed by CTT in males or females is not increased contextual fear conditioning in adult males in comparison relevant for the modulation of memory consolidation induced by to females as measured by freezing behavior, correlating with osanetant, thus suggesting a lack of effect of relatively basal tes- increased Long-Term Potentiation (LTP) in the dentate gyrus of the tosterone levels in the modulation of osanetant effects on fear hippocampus29. Quite importantly, one of the possible reasons why memory consolidation. However, an alternative explanation is adult male mice typically exhibit more freezing than females has that the stress associated with the evaluation of dominance/sub- been recently uncovered, and that is the nature of fear responses, mission in rodents interferes with subsequent memory formation with female mice exhibiting more active fear responses that directly processes. Although animals do not present increased corticos- compete with passive fear responses such as freezing in male mice17. terone concentration in serum before the FE test, we cannot Focusing on the molecular mechanisms of sex differences in fear discard an effect of stress because adrenocorticotropin releasing memory, cyclin-dependent kinase5 (CDK5) mRNA and histone hormone (ACTH) or corticotropin-releasing factor (CRF) were H3lysine 9/14 acetylation in the CA1 region of the hippocampus has not measured in our experiments. Moreover, in line with previous been shown to be upregulated in adult male mice but not in females findings38, serum corticosterone in adult female mice is increased (with the estrous cycle unmonitored) using a contextual fear con- in relatively basal conditions in submissive females compared to ditioning paradigm30. Other studies have focused on LTP an the dominants38, but not in adult males, where slightly higher NATURE COMMUNICATIONS | (2021)12:2496 | https://doi.org/10.1038/s41467-021-22911-9 | www.nature.com/naturecommunications 11 ARTICLE NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-021-22911-9 Fig. 6 Downregulation of CeA-Akt pathway and testosterone in males and upregulation of CeA-Akt signaling and estradiol in proestrous females are necessary for the systemic effect of osanetant in memory. a Schematic procedure for intra-CeA infusion of drugs after Fear Acquisition (FA). All mice received osanetant ip. Males received intra-CeA Akt activator, Androgen Receptor (AR) agonist or vehicle. Females received intra-CeA Akt inhibitor, Estrogen Receptors (ERs) antagonist or vehicle. b Effect of an acute administration of an Akt activator or an AR agonist in osanetant treated adult males on fear memory consolidation (vehicle n = 3; Akt activator n = 4; androgen receptor agonist n = 4) (against vehicle: Akt activator p = 0.013; AR agonist: p = 0.006). *p ≤ 0.05 **p ≤ 0.01 ***p ≤ 0.001 indicate Akt activator vs. vehicle and +p ≤ 0.05 indicates AR agonist after decomposition of the CSxTreatment interaction. c Effect of an acute administration of an anti-Akt drug or ERs agonist after FA on fear memory consolidation in osanetant treated proestrus females (vehicle: n = 6; anti- Akt: n = 7; ERs antagonist n = 5) (against vehicle: anti-Akt p = 0.000; ERs antagonist p = 0.003). *p ≤ 0.05 **p ≤ 0.01 ***p ≤ 0.001 indicate main treatment effect in repeated measures ANOVA and pairwise comparisons. Data are mean ± SEM. Main effect treatment, main effect CS, and CSxTreatment interaction were analyzed using repeated measures ANOVA. Two-tailed pairwise comparisons are indicated for (b) and (c). values of corticosterone in dominant versus submissive mice have submissive mice. Further, and supported by existing literature38, been previously reported39. The use of different strains of mice or social hierarchies remained quite stable along the different ses- methodologies to assess hierarchies might explain this dis- sions of the CTT, and therefore we conclude there is not an effect crepancy between the reported data in males and the ones showed of the estrous cycle in the determination of the dominant/sub- here. Noteworthy, this effect in females’ relatively basal corti- missive female. However, these differences do not appear relevant costerone is abolished once the animals are habituated to a novel to modulate the enhancement of fear memory consolidation context such as the fear chamber. This exposition to a relatively induced by osanetant. mild stressor such as a novel environment might mitigate the Also, we found that our mice did not present a conditioned observed differences in corticosterone between dominant and darting response, nor a difference between males and females in 12 NATURE COMMUNICATIONS | (2021)12:2496 | https://doi.org/10.1038/s41467-021-22911-9 | www.nature.com/naturecommunications NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-021-22911-9 ARTICLE Fig. 7 Graphical summary. A graphical overview of this study. the dart count in response to the CS. These results contrast with by Dong et al.47, we hypothesize that the rapid reduction previously reported conditioned darting behavior in adult female observed in testosterone levels after acute stress might be due to a rats17, although some authors reported non-relevant levels of glucocorticoid-mediated suppression through a non-genomic darting behavior during fear conditioning40,41. Furthermore, mechanism. regarding healthy adult women, it has been previously demon- It is noteworthy in our data that osanetant prevented the peak strated that a higher concentration of circulating estradiol of testosterone in males 360 min after FA. This is not surprising enhances extinction recall, without an effect on FA or fear since it has been previously shown that Nk3R antagonism in adult extinction itself34. However, we show that osanetant given after humans reduces circulating sex hormones18. Previous studies FA at different stages of the estrous cycle can result in different show no changes on estradiol after a mild acute stressor such as effects such as enhancement of memory or no effect. Remarkably, an elevated platform even 6 h after the offset of the stressor48. In osanetant did not affect fear generalization to the context. contrast, other studies using swim stress found increased estradiol concentrations 6 h after stress inoculation in adult females49. From these results, it can be concluded that the nature and Sex hormonal but not stress hormonal response to FA is intensity of the stressor may be a key factor in the regulation of altered by systemic osanetant. Our hormonal profile showed a circulating estradiol. In our experiment, FA did not change the testosterone modulation after FA with an initial decrease after estradiol levels in the vehicle group in females, concordantly with fear acquisition and an increase hours later in those animals previous studies that show unaltered estradiol concentration after treated with vehicle. Of note, mixed results are commonly mild acute stress. Also, osanetant produced an increase in serum reported regarding testosterone regulation after exposure to mild estradiol in proestrous females 6 h after FA (330 min after the acute stressors such as the footshocks, that mice received in our administration) but not in metestrous females. Since a high experiments, as it shows to be highly dependent on the nature of variability is reported in circulating estradiol among species and the stressor, its intensity, and duration. Further, discrepancies strains50–55, we further confirmed our estradiol measurements by appear when different species or strains are tested. In male and mass spectrometry (Supplementary Fig. 3e), similar to those female adult Sprague-Dawley rats, restraint stress showed no values reported by others using the same ELISA kit56. Of note, alterations in plasma testosterone and estradiol, respectively, after progesterone was increased in males and decreased in proestrous stress42. Further, it has been reported a lack of effect on sex females 1 h after FA, and remained unaltered in metestrous hormones 24 h after restraint and footshock stress43. Some females, with no differences between vehicle and osanetant authors report increased serum concentration of testosterone treated groups in any of the cases mentioned. Thus, we can after chronic noise stress (85 dB)44 whereas others show a discard the role of progesterone in the CeA-Tac2 pathway for fear decrease after 4 days of chronic stress by paradoxical sleep memory consolidation. In line with the beforementioned results, deprivation, footshocks, or cold in adult rats45. Moreover, it has our studies with cannulated animals demonstrate that a single also been reported a reduction in plasma testosterone in restrain- intra-CeA dose of an AR agonist blocks the effect of osanetant to stressed adult male Sprague-Dawley rats after 3 h of restraint, but reduce fear memory consolidation in male mice. Furthermore, the not after 1 or 2 h46. Importantly for the interpretation of our infusion into the CeA of a dual ERs antagonist together with results, immobilization stress in male C57BL/6 mice has shown to systemic osanetant inhibits the increase in memory consolidation decrease serum testosterone during stress up to 360 min after the in proestrous female mice. Altogether, these experiments show onset of the stressor47, suggesting a rapid suppression effect of the necessary role of AR inactivation in males and ERs activation testosterone synthesis in response to stress. Following the findings NATURE COMMUNICATIONS | (2021)12:2496 | https://doi.org/10.1038/s41467-021-22911-9 | www.nature.com/naturecommunications 13 ARTICLE NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-021-22911-9 in females for osanetant to modulate memory consolidation in flexibility70. Of note, hippocampal memory has shown to require males and females. estradiol for PI3K activation71. PI3K/Akt is upstream of impor- Regarding stress hormones, it is known that circulating tant memory targets like mTOR, CREB, and GSK3β72. The PI3K/ corticosterone is increased after mild stressors57. Further, it has Akt/GSK3β pathway has been evidenced for its role in cued-fear been previously shown that Tac2 levels are increased after chronic memory consolidation through stabilization of β-Catenin73. stress exposure in adult male mice58. However, we rule out the Further, there is increasing evidence of inhibitory interactions role of the stress response in our FA experiments by showing no between estradiol and GSK3β towards the β-Catenin stabilization differences in corticosterone and related hormones after osane- through ERα promoting memory in the hippocampus74. tant injection during the consolidation window of fear memories. In our study, we show an opposite-sex regulation of the Akt/ Because ACTH and CRF levels have not been taken into account, GSK3β/β-Catenin pathway in males and proestrous females we cannot discard their effects modulating the response to treated with osanetant when high serum estradiol is present osanetant. during the consolidation window of fear memory. Our results show an increase of GPCR-related genes in females during proestrus after FA and treatment with osanetant, and a decrease GAD65 and CaMKIIα expression in CeA-Nk3R-positive neu- of GPCR-related genes in males after the same conditions, in line rons. Here, we demonstrate that there are no differences in the with the behavioral outcome. Also, bioinformatics analyses of number of Nk3R-positive neurons in the CeA between males and GPCR altered genes pointed at the Akt pathway as the central/key females neither in proestrus nor metestrus. Thus, we discard the modulator of the effects of the drug. To further understand the possibility for a distinct number of CeA-Nk3R-positive neurons involvement of Akt in the behavioral effects mentioned, we used mediating the effects of osanetant in males and females in fear biochemical approaches to analyze the different downstream memory consolidation. Notwithstanding, there are important targets of Akt. Although we found no significant alterations of physiological differences in these neurons in terms of colocali- CREB and mTOR, two well-known factors involved in synaptic zation with enzymes involved in synapse function/plasticity such plasticity and memory, we cannot discard them as contributors to as GAD65 and CaMKIIα. the osanetant-induced effect in memory consolidation because of GAD65 is one of the main enzymes that synthesize γ- different methodologies or time points may show different Aminobutyric Acid (GABA) in the brain, together with results. Notwithstanding, we show in CeA a decrease in total GAD67. There have been previously reported sex differences in GSK3β levels in males while, in females, total Akt and GAD65 synthesis, with increased synthesis in adult female rats phosphorylated GSK3β (Ser9; inactive GSK3β) are increased in dorsomedial hypothalamus and medial amygdala, while GAD67 response to the drug after FA, providing a potential target of appeared to be higher in males59. GABAergic transmission is osanetant in the modulation of fear memory consolidation in abundant in the CeA, and key in GABA-dependent synaptic male and female mice. In line with GSK3β inactivation, and plasticity60–62. Previous studies have highlighted that estradiol because β-Catenin is a direct downstream target of GSK3β, increases GAD65 mRNA while decreases GAD67 mRNA as phosphorylated β-Catenin at residues Ser33/37/Thr41 targeted by measured by in situ hybridization in adult rat brain areas like the GSK3β is reduced towards a clear tendency to elevated β-Catenin magnocellular preoptic area or the dorsomedial hypothalamus63, in female mice. This result agrees with previous findings but not in the amygdala. Further, it has been proven that estradiol indicating a role of β-Catenin in cued-fear memory is necessary for GAD enzymes to produce GABA in adult consolidation73, suggesting that activation of Akt/GSK3β/β- females64. Interestingly, we are not aware of reports studying sex Catenin mediates long-term stabilization of the recently acquired differences in GABAergic transmission in the CeA nor its role in memory in adult male and female mice. Besides, we show in fear memory. One of the tested hypotheses was that Nk3R could males that the pharmacological activation of Akt in the CeA, be expressed differentially in excitatory and inhibitory neurons, together with systemic osanetant, blocks the effects of the Nk3R and thus, it could influence in totally opposite ways the antagonist to decrease fear memory consolidation. In contrast, in consolidation of fear memory in males and females. Herein, we females during proestrus, the local administration in the CeA of found enhanced colocalization of central amygdala Nk3R in an anti-Akt blocks the effects of osanetant of increasing memory GAD65-positive neurons in females exclusively during the consolidation. Altogether, these results evidence the opposing role proestrous stage of the estrous cycle in comparison to metestrous between sexes on Akt/GSK3β/β-Catenin signaling in the regula- females and males, that present similar levels of colocalization. tion of amygdala-dependent memory consolidation. These data suggest the possibility of Nk3R to regulate As previously reported10, there is a reduction of fear memory GABAergic-dependent synaptic plasticity in proestrous females. consolidation by Nk3R inactivation in males, in line with what is CaMKIIα is a postsynaptic effector critical for memory because published and expected. Notwithstanding, blocking Nk3R in of its involvement in long-term potentiation through phosphor- females increases Akt/GSK3β/β-Catenin signaling which suggests ylation of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic that this pathway mediates the increase in consolidation observed acid (AMPA) receptors65,66. In the CeA, CaMKIIα is expressed in behavior. In line with the immunohistochemistry assays in cell bodies and neuropil of glutamatergic neurons67. To the described above, in which Nk3R/CaMKIIα colocalization is best of our knowledge, no studies have addressed differences in upregulated in females in comparison to males, it is possible CaMKIIα expression between males and females in the amygdala. that the increase observed in memory consolidation could be Here, proestrous and metestrous females showed increased partially dependent on CaMKIIα, which is part of a different colocalization of CaMKIIα with Nk3R in the CeA in comparison molecular pathway related to PKA and CREB. to males. This result may indicate a tendency to increased These data thereby provide evidence that there exist opposite- CaMKIIα-dependent synaptic plasticity related to fear memories sex effects after a drug administration in behavioral and in females than in males. molecular mechanisms in memory formation through the involvement of the Akt/GSK3-β/β-Catenin signaling pathway in The CeA Akt/GSK3β/β-Catenin pathway is oppositely regu- Nk3R-positive neurons of the CeA in interaction with sex lated between sexes in memory consolidation. The Akt/GSK3β/ hormones. Thus, our discovery proposes a diverse perspective on β-Catenin pathway is widely known for its role in amygdalar- and why and how memory processes differ between females hippocampal-dependent learning and memory68,69 and cognitive and males. 14 NATURE COMMUNICATIONS | (2021)12:2496 | https://doi.org/10.1038/s41467-021-22911-9 | www.nature.com/naturecommunications NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-021-22911-9 ARTICLE Methods assess for differences in the effect of the drug between dominant and Ethics and biosecurity protocols. Ethics protocols approved for the experiments submissive mice. in mice ref. CEEAH 3603 and biosecurity protocols 345-16 and 407-17. All pro- We extracted 50 μl of blood from each animal by tail-nick two days before the cedures were approved by the Committee of Ethics of the Universitat Autònoma de first habituation to the tube (under relatively basal conditions), 2 h before the first Barcelona and the Generalitat de Catalunya. They were also carried out in habituation session, 2 h before the first test and 2 h before the first habituation to accordance with the European Communities Council Directive (2010-63-UE) and the fear conditioning chamber, thus, with every two days since the first extraction. Spanish legislation (RD 53/2013). Blood was centrifuged (8000 g, 15 min, 4 °C) and serum was stored at −80 °C. In female mice, we also monitored the estrous cycle daily from the first habituation to the tail-nick extraction until the FE test. Mice. All experiments used male and naturally cycling female adult mice (8 weeks old has been used for all the experiments except, P28 for prepubertal mice and 2–6 months of age for inbred Tac2-Cre mice) housed in groups of 4 (except when Vaginal smear cytologies. Determination of the estrous stage in females was mentioned) in a room with a 12:12 h light/dark cycle (lights on from 8 am to 8 pm). performed by assessing vaginal smear cytologies76,77. To assess the phase of the All animals were housed with a controlled temperature of 22 ± 1 °C and humidity estrous cycle that female mice presented during FA, all female mice were mon- (~40%). Behavioral procedures and pharmacological manipulations began early in itored for 3–4 consecutive cycles (approximately 10-14 days) before learning to test the light phase of the cycle. B6.129-Tac2 /J (Tac2-cre) (stock# for regularity of the cycle. We performed a vaginal lavage with a 20 μl pipette that 018938 Jackson Labs) were bred within our animal facility. Wild-type C57BL/6J was loaded with 10 μl of standard NaCl 0.9% (w/v) solution, and later the tip was were purchased from Charles River (Barcelona, Spain). Male and female mice were softly placed on the vaginal aperture. In case of urination when grabbing the housed separately in the same room. animal, urine was cleaned using a regular tissue. The 10 μl of saline were unloaded and collected for 5 consecutive times to collect enough amount cells for the assessment, and later placed on an adhesion slide (Superfrost Plus, Thermo Fisher, Cued-Fear Conditioning. For Fear Acquisition (FA) and Fear Expression (FE) Barcelona, Spain). All vaginal smear samples were collected between 9:30 and 11:30 test, a computerized Startle system was employed (Panlab-Harvard, Barcelona, am. Slides were dried using a hot plate (HI1220, Leica, Madrid, Spain) at 37 °C for Spain)75. Delivery of tones and shocks was simultaneously controlled by Freezing 30 min and later stained in Cresyl Violet Acetate (C5042, Sigma-Aldrich, Spain) v1.3.04 software (Panlab-Harvard, Barcelona, Spain). The fear chamber consisted 0.1% (v/v), washed twice for 1 min in distilled water and read in brightfield of a black methacrylate box with a transparent front door (25 × 25 × 25 cm3) microscopy with a 10× or 20× objective in an Eclipse 80i microscope (Zeiss, Spain). inside a sound-attenuating chamber (67 × 53 × 55 cm3). The same boxes were used Three different cell types may appear in the preparation: cornified epithelial cells, for FA and FE. round nucleated epithelial cells, or leukocytes. The different stages of the estrous Animals were habituated to the chambers for 5 min/day for two consecutive cycle were assessed depending on the proportion of the abovementioned cells. days before FA. For cue-dependent fear conditioning, all animals remained 5 min Proestrus is characterized by a high proportion (>80%) of nucleated epithelial cells, in the fear chamber before the onset of the first tone. During FA, all groups that might present very small amounts of cornified epithelial cells or leukocytes. received 5 trials consisting of a tone as the Conditioned Stimulus (CS) (30 s, 6 kHz, Estrus is typically presented with cornified epithelial cells with a lower grade of 75 dB) that coterminated with a footshock which served as the Unconditioned staining than leukocytes and nucleated epithelial cells. Metestrus presents a mixture Stimulus (US) (1 s, 0.3 mA). The intertrial interval (ITI) was 3 min, and 3 of cornified epithelial cells and a considerable proportion of leukocytes. Diestrus is additional min followed the last trial. All animals received the treatments 30 min characterized by >90% of leukocytes that might present a very small proportion of after FA to manipulate the consolidation of memory and therefore avoid any effect round nucleated epithelial cells. After assessment of regular cycling, females were during acquisition. The FE test was performed 24 h after FA. Mice remained 5 min distributed in groups according to the stage of the estrous cycle they presented in the chamber before trials and afterward were exposed to 15 trials of the 30 s CS before FA, which is also the day they receive the different treatments after FA. tone alone (cued-fear) with a 0.5 min of ITI interval. An additional 0.5 min interval followed the last trial of FE. Freezing behavior was used as an index of fear. Freezing behavior understood as a lack of any movement of the animal but for Drugs. Intraperitoneal osanetant (Sigma-Aldrich, Spain) dose was 5 mg/Kg 10, 1 those related to breathing, was recorded using the StartFear system, which allows mg/Kg or 10 mg/Kg, and the intracerebral dose was 30 nmol per side as it has recording and analysis of the signal generated by the animal movement through a shown to modulate fear memory consolidation in male mice when administered high sensitivity weight transducer system. To that end, a movement threshold was intra-Central amygdala (CeA)10 using 0.1% Tween 20 in saline as the vehicle. The established for low mobility (13/100) and a freezing episode was considered when dose of intra-CeA Androgen Receptor (AR) agonist CI-4AS-1 (Tocris, UK) was the animal spent at least 500 ms under this threshold. This system was validated by 100 nM based on previous research showing decreased depolarization-induced 5 experimented researchers through stopwatch analyses of video recordings. suppression of excitation in POMC neurons78, dissolved in 1% DMSO (v/v). We Darting behavior, as known as rapid peaks of locomotor behavior much above the used the AR agonist instead of testosterone to avoid its possible conversion to 17-β- normal locomotor activity of the mouse was counted by an experimented estradiol by aromatase. The Akt activator SC79 (Tocris, UK) was used at a dose of observer17. These recordings were performed throughout the session and separated 0.1 µM in 1% DMSO (v/v) because it has shown to have a protective effect on in bouts of 5 min of habituation and then, independently, during each CS and ITI. dopaminergic neurons against oxidative stress79. Anti-Akt1/2 (Tocris, UK) was To assure that freezing behavior was exclusive of the previous tone-shock used at 5 µM in 1% DMSO (v/v) due to its ability to block Akt phosphorylation80. conditioning, different contexts were utilized for FA and FE. FA context consisted Estrogen Receptors (ERs) antagonist ICI 182,780 (Tocris, UK) was used at a 10 µM of a yellow light source (~10 lux), a grid floor of 25 bars (3 mm Ø and 10 mm in 1% DMSO (v/v) as it has shown previously to be effective in decreasing EtOH between bars) that dispensed the footshocks, background noise of 60 dB produced excitation of DA neurons in the VTA81. CNO in 0.5% DMSO (v/v) systemically by a ventilation fan and a solution of ethanol (EtOH) 70% (v/v) odor was used for was dosed at 1 mg/Kg10. cleaning between sessions. FE context consisted of a red light source (~10 lux), a gray floor covering the bars, no background noise, and CR36—bronopol 0.26 % (v/ v), benzalkonium chloride 0.08% (v/v), and isopropyl alcohol 41% (v/v)—(José Surgery. All surgeries were performed using isoflurane 5% (v/v) for induction, and Collado, Barcelona, Spain) for cleaning, with changes in the length and turns of the 2–3% (v/v) for maintenance, in oxygen, at a constant rate of 1.5 l/min. After skin transportation route from the vivarium to the testing room between FA and FE. shave and skin disinfection with EtOH 95% (v/v) and iodine povidone 10% (v/v), ovariectomies were performed making a bilateral incision on the back of the ani- mal, 1 cm lateral to the midline and right over the back limbs line. Adipose tissue Confrontation tube test (CTT). Female and male mice were pair-housed for was extracted, and the ovary was localized and isolated making a knot with sterile 5 weeks before testing to establish stable social hierarchies between them. The absorbable suture thread (Centauro, Spain) around the oviduct. The ovary was confrontation tube consisted of a methacrylate tube (30 cm length, 3.6 cm inner Ø) extirpated and the adipose tissue, containing the rest of the oviduct, was returned with two lids at 13 cm from each end. All animals were habituated to the tube for 2 to the abdominal cavity. The muscle was sewed with sterile absorbable thread and consecutive days before the test. The habituation consisted of 3 crossings from one skin was sewed using sterile silk suture (Centauro, Spain). Mice remained resting end to the other of the tube and the tube was always cleaned with EtOH 70% (v/v) for 6 weeks until trunk blood was collected to avoid any effect of previous estradiol. between animals. Mice were tested for 6 consecutive days, with 8 trials per day with For stereotaxic surgeries, after induction of anesthesia, mice were placed in the ~20 min between trials. For each trial, each mouse from each couple entered the stereotaxic frame (Kopf Model 962, Harvard-Panlab, Barcelona, Spain). After tube simultaneously by the extremes. When both animals reached the lids, these alignment of the Antero-Posterior (AP) and Latero-Medial (LM) axis with the were lifted for animals to face each other and that’s when the test started. The first frame, injections of AAV8-hSyn-DIO-hM4D(Gi)-mCherry were performed at a animal that put its four limbs outside the tube was considered the loser while the rate of 1 μl per 15 min to the CeA using the following coordinates: AP −1.3, LM ± other one won the trial. When both animals remained 2 min inside the tube 2.5, Dorso-Ventral (DV) –4.4 mm from Bregma10. For cannulation, the same AP without confronting each other, the trial was considered null. The animal that and ML coordinates were utilized, although cannulae were implanted at DV −3.4 presented more won trials throughout each session was considered the winner of mm due to the need to remain the CeA intact and because of the extra mm the session. At the end of the six sessions, the animal that presented more won projection of the internal cannula. Cannulae were secured to the skull with anchor sessions was considered dominant over its cage mate. The day after the 6th session screws for mice (Plastics One, Germany) and dental cement (Fortex Fájula, of the tube test, animals underwent the FC protocol as abovementioned. All mice Cibertec, Spain) For further details on the virus and microinfusions, please see received osanetant 30 min after FA and were tested for memory recall 24 h later to Adeno associate virus (AAV) infection and microinfusions section below. NATURE COMMUNICATIONS | (2021)12:2496 | https://doi.org/10.1038/s41467-021-22911-9 | www.nature.com/naturecommunications 15 ARTICLE NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-021-22911-9 Adeno-associate virus (AAV) infection and microinfusions. We used AAV and the extracts were reconstituted in 100 µl water:methanol (50:50). Ten obtained from the pAAV-hSyn-DIO-hM4D(Gi)-mCherry (hM4di-mCherry microliters were injected into the LC-MS/MS system consisting of an Acquity UPLC DREADDs). The plasmid was obtained from Addgene and the AAV from the viral system coupled to a triple quadrupole (TQS Micro) mass spectrometer. vector production unit at Universitat Autònoma de Barcelona. Serotype 8 of the Chromatographic separation was performed in an Acquity BEH C18 column (100 AAV was bilaterally injected (0.5 μl/side) using a microinjection pump into the mm × 2.1 mm i.d., 1.7 μm) (Waters Associates) at 55 °C and at a flow rate of 300 μl/ CeA (coordinates and infusion rate abovementioned). Cannulae were placed using min. Mobile phases consisted in water with ammonium formate (1 mM) and formic coordinates mentioned in the surgery section. For microinfusions of drugs in acid (0.01 % v/v) and methanol with ammonium formate (1 mM) and formic acid awake animals, internal cannulae projected 1 mm from the tip of the internal guide, (0.01 % v/v). The selected gradient program linearly changed the organic solvent reaching DV coordinate −4.4 mm from Bregma for injections, corresponding to percentage as follows: 0 min, 65%; 1 min, 65%; 3.7 min, 68%; 3.8 min, 99%; 4.8 min, the CeA. Bilateral injections were performed simultaneously and manually using 99%; 4.9 min, 65%; 6.0 min, 65%. Quantification of estradiol was performed in the two knurled hub, type 3 tip, 1 μl Hamilton syringe (external Ø 0.52 mm and SRM mode by monitoring the transitions of 431 > 96, 431 > 161 and 431 > 367 for internal Ø 0.26 mm) (Cibertec-Harvard, Madrid, Spain) coupled on one end to a estradiol and 434 > 96, 434 > 161 and 434 > 370 for estradiol-d3. polyethylene (PE) 50 tube (~20 cm) (Plastics One, Germany) with the internal cannula coupled to the other end of the tube. The PE-50 tube was filled with distilled water, leaving a 7.5 mm gap of air between the internal cannula and the Immunohistochemistry. For immunohistochemistry assays, mice were transcar- water to avoid dilution of the drugs with water. Microinfusions were performed at a dially perfused with 50 ml of 4% (v/v) paraformaldehyde (PFA) (Casa Álvarez, rate of 0.5 μl over 2 min and internal cannulae remained one extra min in place to Spain) for 5–6 min, then decapitated, and brains were extracted and stored in 4% prevent the backflow of the drug. (v/v) PFA for 24 h. After this time, brains were rinsed (3 times, 10 min each) with 1× Sorenson’s PB consisting of 10.9 g/l Sodium phosphate dibasic (Sigma-Aldrich, Spain), 3.2 g/L Sodium phosphate monobasic (Sigma-Aldrich, Spain) and trans- ferred into 30% Sucrose (Sigma-Aldrich, Spain) in 1× Sorenson’s PB in conic Steroids determination. For steroids determination in Fig. 3, trunk blood was Falcon tubes (Thermo Fisher, Spain) until the brain reached the bottom of the tube collected after sacrificing the animals. We used animals under relatively basal (~48–72 h). Right after, brains were snap-frozen in a metal cube containing Iso- conditions that did not undergo any procedure whose blood was collected within 3 pentane (Sigma-Aldrich, Spain) cooled with dry ice and stored at −80 °C until min of touching the homecage, animals with FA and an injection of osanetant 30 sectioning. min after that were euthanized 30 min after receiving the injection, and animals For Neurokinin 3 Receptor (Nk3R), Glutamic Acid Decarboxylase 65 (GAD65), with FA and osanetant 30 min after that were euthanized 330 min after the Calmodulin Kinase II α (CaMKIIα), and Vesicular Glutamate Transporter 2 injection for the three cohorts of study: males, proestrous females, and metestrous (vGLUT2) fluorescent immunostaining brain sections (30 μm/section) were rinsed females. Corticosterone determination was performed 30 min after receiving osa- three consecutive times with 1× KPBS. All incubations were performed on top of a netant or vehicle since these hormones have shown to be altered after fear con- shaking platform. Right after washing the slices, these were incubated for 60 min in ditioning procedures57. While we chose 330 min after treatments for estradiol blocking buffer (5% Donkey Serum and 0.4% Triton-X in 1× KPBS) at 4 °C. After assessment since previous studies have shown a long-term (4–6 h) regulation of incubation in blocking buffer, sections were incubated overnight with the following this hormone after stress48,49. For Fig. 2, males and females’ blood was collected by primary antibodies: rabbit anti-Nk3R (Donated by Philip Cioffi, INSERM, 1:1500), tail-nick as described in the CTT section. Blood was centrifuged (8000 g, 15 min, 4 ° chicken anti-GAD65 (139958, Abcam, 1:500), goat anti-CaMKIIα (87597, Abcam, C). Serum was collected using a 200 µl pipette, transferred into a 2.5 ml Eppendorf 1:500) and mouse monoclonal anti-vGlut2 (ab79157, Abcam, 1:300). Primary tube, and stored at −80 °C for later analysis. Brains from animals euthanized 330 antibodies were diluted in 0.4% Triton-X in 1× KPBS. After incubation in primary min after treatment (6 h after FA) were snap-frozen using Isopentane cooled with antibodies solution, brain slices were rinsed 3 times with KPBS 1× and then dry ice, and later stored at −80 °C until sectioning. Fresh frozen brains were then incubated in a secondary antibodies solution for 2 h at room temperature. The coronally sectioned until reaching a coronal plane 0.58 mm behind Bregma. 1 mm secondary antibodies solution was prepared in Triton X (×100, Sigma-Aldrich, Ø micropunches from both Amygdalae were extracted reaching coronal plane 1.94 Spain) 0.4% (v/v) in 1× KPBS, and included the following secondary antibodies mm behind Bregma, transferred into a 2.5 ml Eppendorf tube, and stored until conjugated to fluorophores: donkey anti-rabbit AlexaFluor488 (115-546-072, processing. 400 μl of Formic Acid (Sigma-Aldrich, Spain) 0.1% (v/v) were added to Jackson Immunoresearch, 1:1000), donkey anti-chicken Cyanine3 (703-166-155, each tube and sonicated for five consecutive cycles. The sonication probe was then Jackson Immunoresearch, 1:1000), donkey anti-goat AlexaFluor594 (705-586-147, rinsed with 800 μl of Acetonitrile (Sigma-Aldrich, Spain) that were added to the Jackson Immunoresearch, 1:1000) and donkey anti-mouse AlexaFluor647 (715- tubes, and these were vortexed for 2 s. Micropunches extracts were stored at 606-150, Jackson Immunoresearch, 1:1000). 4′,6-diamidino-2-phenylindole −80 °C until analysis. Both serum and amygdalar testosterone, progesterone, (DAPI) (10236276001, Sigma-Aldrich, 1:10000) was used to stain cell nuclei. dehydrocorticosterone, and deoxycorticosterone were evaluated based on pre- Z-Stacks of the central amygdala (0.50 μm/interval) were acquired using a Leica viously reported papers82,83. Briefly, 20 µl of plasma was mixed with 20 µl of a SP5 confocal microscope (Leica, Spain) with a PL APO 40x/1.25-0.75 immersion labeled internal standard solution. After protein precipitation with 100 µl of acet- objective controlled by LAS X v2.7.3.9729 software (Leica, Spain). Nk3R-positive onitrile, samples were centrifuged (3000g, 5 min) and the supernatant transferred neurons were determined using the cell count plugin from Fiji for Windows v1.52. to a clean tube. In the case of micropunches 20 µl of the labeled internal standard n. Colocalization analyses were performed using Fiji for Windows v1.52.n. Z- solution was added to the 1 ml of extract from micropunches. The mixture was Projections of Nk3R signal was used to create a mask. This mask was then applied vortexed and transferred to a clean tube. Both plasma and micropunches under- to Z-Projections (0.50 μm/interval) of GAD65, CaMKIIα, and vGLUT2 stacks went a liquid-liquid extraction by the addition of 1 ml of NaCl (saturated solution) using LungJ plugin. Mander’s colocalization coefficient between Nk3R and GAD65, and 4 ml of ethyl acetate. Extracts were centrifuged (3000 g, 5 min) and the organic CaMKIIα, and vGLUT2 was calculated using Just Another Colocalization Plugin layer was transferred into a clean tube and dried under a nitrogen stream. Dried (JACoP). Average colocalization index between right and left CeA was used as a extracts were reconstituted with 100 µl of methanol and 10 µl were injected into the measure of colocalization for each animal. LC-MS/MS system consisting of an Acquity UPLC system coupled to a triple For Estrogen Receptor β (ERβ) and Estrogen Receptor α (ERα) fluorescent quadrupole (TQS Micro) mass spectrometer. Steroids detection was performed by immunostaining, the staining protocol was similar to the one mentioned above. selected reaction monitoring (SRM) including two transitions for each analyte. The Primary antibody solution contained rabbit anti-Nk3R (Donated by Philip Cioffi, most specific one was selected for the quantification. Quantification was performed 1:1500) and mouse monoclonal anti- ERα (sc-8002, Santa Cruz, 1:50) or mouse by an external calibration approach using the TargetLynx module of the MassLynx monoclonal anti- ERβ (288, Abcam, 1:500). Secondary antibodies solution included v4.1 software (Water Associates, USA). Estradiol was measured with the ELISA kit donkey anti-rabbit AlexaFluor488 (115-546-072, Jackson Immunoresearch, 1:1000) ES180S-100 (Calbiotech, California, USA). Ovariectomized (OVX) mice were used and donkey anti-mouse Rhodamine Red-X (715-295-150, Jackson Immunoresearch, for the ELISA analyses of estradiol with the ELISA kit. For standard curve pre- 1:1000). DAPI (Sigma-Aldrich #10236276001) was used to stain cell nuclei. Z-Stacks paration, serum from OVX mice was used adding known concentrations of of the central amygdala (0.50 μm/interval) were acquired using a Zeiss LSM 700 estradiol (Sigma-Aldrich, Spain)—0, 3, 10, 30, 100, and 300 ρg/ml—utilizing confocal microscope (Zeiss, Spain) with a PL APO 40x/1.25-0.75 immersion denaturalized EtOH (Casa Álvarez, Spain) as a vector for estradiol. Kit instructions objective controlled by ZEN v2010 software (Zeiss, Spain). Cell counter plugin for were followed as stated, samples were loaded in duplicates and absorbance was read Fiji v1.52.n was employed to measure the number of Nk3R-positive cells in the at 450 nm with the microplate reader Varioskan Lux (Thermo Fisher, Spain) CeM. Mander’s colocalization coefficients for ERα and ERβ were calculated as controlled with Skanit for microplates v6.0 software (Thermo Fisher, Spain). The abovementioned for Nk3R, GAD65, CaMKIIα and vGLUT2 immunohistochemistry average of duplicates was used as estradiol determinations for each sample. using JACoP plugin in Fiji v1.52.n for Windows. Estradiol results were confirmed by the LC-MS/MS method based on chemical derivatization and detection in the SRM mode. Briefly, plasma samples (c.a. 100 µl) were mixed with 20 µl of internal standard (estradiol-d3 at 2 ng/ml) and 1 ml of Cannula placement verification. For cannula placement verification, amygdala water and were subjected to two consecutive liquid-liquid extractions with tert-butyl brain sections were stained with a standard Nissl staining protocol. Brains were methyl ether. The organic layers were mixed and evaporated under nitrogen stream sectioned (30 μm/section) using a Leica Cryostat (−20 °C for the chamber, −18 °C for (<40 °C, < 15 psi). Derivatization with 1,2-Dimethylimidazole-5-sulfonyl chloride the sample), direct to mount and stored at −20 °C until staining. Slides were dried on was performed at 60 °C for 15 min. Derivatized extracts were evaporated under a hot plate at 37 °C overnight before staining. First, slides were dehydrated in con- nitrogen stream (<40 °C, < 15 psi) and reconstituted in 1 ml of water. Two secutive decreasing concentrations of denaturalized EtOH (Casa Álvarez, Spain): 3 additional liquid-liquid extractions with 4 ml of hexane were performed. The min in EtOH 100% (v/v), 3 min in EtOH 95% (v/v) and 3 min in 70% (v/v). Right organic layers were mixed and evaporated under nitrogen stream (<40 °C, < 15 psi) after that, slides were rinsed in distilled water three times for 2 min each to extract any 16 NATURE COMMUNICATIONS | (2021)12:2496 | https://doi.org/10.1038/s41467-021-22911-9 | www.nature.com/naturecommunications NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-021-22911-9 ARTICLE trace of sucrose. Samples were then stained for 8 min in a Cresyl Violet Acetate Data analyses. Statistics analyses were performed using IBM SPSS Statistics 23.0. Solution consisting of 1 mg/ml Cresyl Violet Acetate in Walpole Solution, prepared Detection of outliers was using Grubb’s test and removed when it was appropriate. with Glacial Acetic Acid (Sigma-Aldrich, Spain) 60% (v/v) and Sodium Acetate 0.2 M For experiments involving independent samples, when these presented normal (Sigma-Aldrich, Spain) 40% (v/v). After staining, slides are rinsed twice in distilled distributions and equality of variances, one-way ANOVA (GLM) was employed; water for 2 min each and then dehydrated using increasing concentrations of EtOH: 3 otherwise, non-parametric analyses were utilized for one-factor analyses. Wald’s χ2 dips in EtOH 50% (v/v), 3 dips in EtOH 70% (v/v), 3 dips in EtOH 96% (v/v) and 3 with pairwise comparisons was used in Generalized Linear Model for multifactorial min in EtOH 100% (v/v). After this last dehydration, slides are incubated in Xylene analyses that did not accomplish normality or homoscedasticity. Additional indi- (Sigma-Aldrich, Spain) 3 times for 3 min each and then covered using DPX mountant vidual comparisons were performed when appropriated. for histology (Sigma-Aldrich, Spain). Slides remain untouched for 24 h in the dark For related sample analyses, two-way repeated measures ANOVA was used to until stored in a regular box for slides at room temperature. assess Sex x Treatment interaction. To assess the effect of the treatments across the Cannulae placement verification was performed using brightfield photographs estrous cycle, one-way repeated measures ANOVA was performed as one factor obtained using a 10x objective in an Eclipse 80i microscope (Leica, Spain). Animals may “overshadow” the effect of other when using multiple factors with several that did not present a bilateral cannula lesion in the central amygdala were levels each. For repeated measures ANOVA, we used trials for FA or mean of discarded from experiments. groups of 5 trials for FE test as within-subject variable. Equality of variances and sphericity were tested. When sphericity could not be assumed, Greenhouse-Geisser statistics were used for assessing the significance and the corrected degrees of mRNA qPCR array. 30 min after receiving osanetant (1 h after FA), animals were freedom were provided. ANOVA was used for the evaluation of simple main effects decapitated, and brains were immediately fresh frozen in isopentane cooled with and when an interaction between main effects was significant. Linear regressions dry ice and stored at −80 °C. Amygdala tissue from both hemispheres was were calculated to understand the correlational effects between serum and extracted by 1 mm micropunch as previously described and each structure from amygdala hormones. The results are presented as means ± or + SEM, and each mouse was individually stored. Total RNA was isolated and purified from the statistical significance was set at P ≤ 0.05. Specific sample sizes are reported in tissue with the RNeasy Mini Kit (74106, Qiagen) following the manufacturer’s figure legends for each dataset. instructions. Total RNA was isolated with Maxwell RSC simplyRNA Tissue Kit Normality and homoscedasticity tests, as well as statistics with main effects, (AS1390, Promega). Quantus Fluorometer (E6150 Promega) was used to ensure interactions, pairwise analyses, and effect size, when appropriate, are represented in the quality of RNA before the qPCR array (PAMM-071ZC-24, Qiagen). Gusb was Supplementary Data 1. In Supplementary Data 2, 95% confidence intervals are the housekeeping gene used for the normalization of qPCR results. qPCR array was represented. performed following the instructions of the kit as stated, using the thermocycler All graphs presented in the figures were designed using Prism 7 (CA, USA). 7500FAST, controlled by 7500FAST v2.0.6 software both from Applied Biosystems (Thermo Fisher, Spain). Reporting summary. Further information on research design is available in the Nature Research Reporting Summary linked to this article. Ingenuity pathway analysis—bioinformatics. The gene list obtained on the mRNA qPCR array was analyzed with Bioinformatics. We used Ingenuity Pathway Analysis (IPA) software version #44691306. The male and female lists of genes Data availability All datasets obtained and analyzed in the reported study are available from the selected according to their significant p-value were uploaded separately. All genes corresponding author upon reasonable request. Source data are provided with this article were introduced with their respective expression fold change value. The female’s and deposited in the Digital Repository of Documents from the Autonomous University gene list contained the genes Akt1, Gcgr, Fgf2, Lhcgr, Mmp9, Ptgdr, Rho, S1pr3, of Barcelona https://doi.org/10.5565/ddd.uab.cat/237256. Source data are provided with Vcam1. The males’ gene list contained the genes Agt, Agtrap, Bcl2, Calcrl, Ccnd1, Ccne1, Cdkn1a, Cdkn1b, Cflar, Elk4, Galr2, Gnas, Bcl2l1. We performed a Core this paper. Analysis/Expression Analysis using the fold change as a measurement to base the analysis on. These analyses were performed separately for males and females by Received: 26 June 2020; Accepted: 25 March 2021; using most of the predetermined criteria by IPA. The general settings of this analysis included both direct and indirect relationships based on the reference set “Ingenuity Knowledge Base (Genes Only)”. For the network analysis, we selected 35 molecules per network and 25 networks per analysis. We also choose all Node Type and all Data Sources. For Confidence, we selected only “Experimentally observed”. For Species we selected all. The results we obtained with this IPA analysis were focused on two categories: Canonical Pathways (Fig. 5b) and References Upstream analysis (Supplementary Fig. 5b). 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Distribution of CaMKIIα estradiol determination. Procedural images were created with BioRender.com. RA was expression in the brain in vivo, studied by CaMKIIα-GFP mice. Brain Res. supported by a NARSAD Young Investigator Grant #22434, Ramón y Cajal program 1518, 9–25 (2013). RYC2014-15784, RETOS-MICINN SAF2016-76565-R FEDER funds, and ERANET- 68. Lai, Z., Zhang, L., Su, J., Cai, D. & Xu, Q. Sevoflurane postconditioning Neuron JTC 2019 ISCIII AC19/00077. CAS is supported by Ministerio de Ciencia e improves long-term learning and memory of neonatal hypoxia-ischemia brain Inovación (MICINN) grant SAF2016-80027-R, PID2019-106615RB-100, and ISCIII damage rats via the PI3K/Akt-mPTP pathway. Brain Res 1630, 25–37 (2016). (CIBERNED CB06/05/0042). RN was supported by MICINNgrant SAF2017-83430 and 69. Jung, S. Y. & Kim, D. Y. Treadmill exercise improves motor and memory was a recipient of an ICREA Academia Award (Generalitat de Catalunya, 2015-2019). 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Neurosci. 30, 4390–4400 Author contributions This study was planned and conceptualized by R.A. and A.F., A.F. carried all the (2010). behavioral experiments, stereotaxic surgeries, vaginal cytologies, ELISA, qPCR, immu- 72. Hemmings, B. A. & Restuccia, D. F. PI3K-PKB/Akt pathway. Cold Spring nohistochemistry, histology, DREADDs, and helped with Western blot experiments. E.V. Harb. Perspect. Biol. 4, a011189 (2012). contributed with help for the vaginal cytology readings. L.P.C. and A.F. performed the 73. Maguschak, K. A. & Ressler, K. J. β-catenin is required for memory CTT in females. P.M. performed IHC. in prepubertal mice. A.G. and O.P. contributed to consolidation. Nat. Neurosci. 11, 1319–1326 (2008). the mass spectrometry data. R.N. contributed with statistical analyses of datasets, pro- 74. Cardona-Gomez, P., Perez, M., Avila, J., Garcia-Segura, L. & Wandosell, F. vided infrastructures for behavioral assays as well as the set-up for video and freezing Estradiol inhibits GSK3 and regulates interaction of estrogen receptors, GSK3, recordings. C.M.S. and C.A.S. contributed to the biochemical experiments. The paper and beta-catenin in the hippocampus. Mol. Cell. Neurosci. 25, 363–373 (2004). was written by R.A. and A.F. and commented on and discussed by all authors. 75. Daviu, N., Andero, R., Armario, A. & Nadal, R. Sex differences in the behavioural and hypothalamic-pituitary-adrenal response to contextual fear conditioning in rats. Horm. Behav. 66, 713–723 (2014). Competing interests 76. Byers, S. L., Wiles, M. V., Dunn, S. L. & Taft, R. A. Mouse estrous cycle R.A. declares a potential conflict of interest with the patent PCT/US2015/037629. The identification tool and images. PLoS ONE 7, e35538 (2012). other authors declare no competing interests. 77. Caligioni, C. S. Assessing reproductive status/stages in mice. Curr. Protoc. Neurosci. Appendix 4. https://doi.org/10.1002/0471142301.nsa04is48 (2009). 78. Conde, K. et al. Testosterone rapidly augments retrograde endocannabinoid Additional information signaling In proopiomelanocortin neurons to suppress glutamatergic input Supplementary information The online version contains supplementary material from steroidogenic factor 1 neurons via upregulation of diacylglycerol lipase- available at https://doi.org/10.1038/s41467-021-22911-9. α. Neuroendocrinology 105, 341–356 (2017). 79. Xu, Y., Gao, Y.-W. & Yang, Y. SC79 protects dopaminergic neurons from Correspondence and requests for materials should be addressed to R.A. oxidative stress. Oncotarget 9, 12639–12648 (2018). Peer review information Nature Communications thanks Liisa Galea and the other, 80. Tsuchiya, A., Kanno, T. & Nishizaki, T. PI3 kinase directly phosphorylates anonymous, reviewer(s) for their contribution to the peer review of this work. Peer Akt1/2 at Ser473/474 in the insulin signal transduction pathway. J. Endocrinol. reviewer reports are available. 220, 49–59 (2014). 81. Vandegrift, B. J., You, C., Satta, R., Brodie, M. S. & Lasek, A. W. Estradiol Reprints and permission information is available at http://www.nature.com/reprints increases the sensitivity of ventral tegmental area dopamine neurons to dopamine and ethanol. PLoS ONE 12, e0187698 (2017). 82. Gomez-Gomez, A. et al. Determination of the steroid profile in alternative Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in matrices by liquid chromatography tandem mass spectrometry. J. Steroid published maps and institutional affiliations. Biochem. Mol. Biol. 197, 105520 (2020). 83. Marcos, J. et al. Investigation of endogenous corticosteroids profiles in human urine based on liquid chromatography tandem mass spectrometry. Anal. Open Access This article is licensed under a Creative Commons Chim. Acta 812, 92–104 (2014). Attribution 4.0 International License, which permits use, sharing, 84. Van Der Jeugd, A. et al. Reversal of memory and neuropsychiatric symptoms adaptation, distribution and reproduction in any medium or format, as long as you give and reduced tau pathology by selenium in 3xTg-AD mice. Sci. Rep. 8, 1–12 appropriate credit to the original author(s) and the source, provide a link to the Creative (2018). Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the Acknowledgements article’s Creative Commons license and your intended use is not permitted by statutory The authors thank Mar Castillo, Dr. Javier Carrasco, and Núria Barba, technicians at the regulation or exceeds the permitted use, you will need to obtain permission directly from Institute of Neuroscience, UAB. We especially thank Dr. Antonio Armario for their the copyright holder. To view a copy of this license, visit http://creativecommons.org/ valuable contribution to commenting on the manuscript as a member of the excellence licenses/by/4.0/. 2017 SGR-00457 group (Generalitat de Catalunya). We thank Nicole Gouws for her help with the graphic design. We thank Gleb Shumyatsky for help with IPA and Cristina Márquez for help with CTT. We would also like to thank Noemí Haro for help with © The Author(s) 2021 NATURE COMMUNICATIONS | (2021)12:2496 | https://doi.org/10.1038/s41467-021-22911-9 | www.nature.com/naturecommunications 19