Florido et al. Biology of Sex Differences (2022) 13:28 https://doi.org/10.1186/s13293-022-00437-z RESEARCH Open Access Nk3R blockade has sex‑divergent effects on memory in mice Antonio Florido1,2 , Estefanía Moreno3 , Enric I. Canela3,4   and Raül Andero1,2,5,6,7*    Abstract Background: Memory consolidation is a process required for the formation of long-term memories. The G-protein- coupled receptor (GPCR) neurokinin-3-receptor (Nk3R) and its interactions with sex hormones seem important for the modulation of fear memory consolidation: Nk3R antagonism in male mice impairs fear memory, but enhances it in females. However, the involvement of the Nk3R as a modulator of other memories in both sexes remains unexplored. Methods: We use the novel object recognition paradigm to test the effect of a systemic blockade of Nk3R during memory consolidation. Further, we assess the expression of estrogen receptor α, estrogen receptor β, and androgen receptor and heterodimerization with Nk3R in the medial prefrontal cortex (mPFC) and dorsal hippocampus (DH) of mice. Results: Nk3R systemic antagonism elicited decreased memory consolidation in males while it enhanced it in females during proestrus. Nk3R analysis in the different subregions of the mPFC and the DH showed a higher expres- sion in males than females. Moreover, females presented upregulation of the androgen receptor in the CA1 and the estrogen receptor beta in the cingulate cortex, CA1, and dentate gyrus. Overall, males presented an upregulation of the estrogen receptor alpha. We also explored the heterodimerization of GCPR membrane sex hormone receptors with the Nk3R. We found a higher percentage of Nk3R-membrane G-protein estrogen receptors heterodimers in the prelimbic cortex of the mPFC in females, suggesting an interaction of estradiol with Nk3R in memory consolidation. However, males presented a higher percentage of Nk3R-membrane G-protein androgen receptors heterodimers compared to females, pointing to an interaction of testosterone with Nk3R in memory consolidation. Conclusion: These data propose novel ideas on functional interactions between Nk3R, sex hormones, estrogen receptors, and androgen receptors in memory consolidation. Highlights • Nk3R antagonism reduces recognition memory consolidation in male mice and increases it in proestrus females. • Androgen receptor expression is higher in the CA1 compared to DG, CA3, and the mPFC. • Estrogen repcetor α expression is higher in males than in females in the DH and mPFC. • Estrogen receptor β expression is greater in females than in males in the DG, CA1, and CG. • Over 60% of Nk3R in the DH and mPFC is heterodimerized with membrane estrogen receptor and androgen receptor. *Correspondence: Raul.Andero@uab.cat 1 Institut de Neurociències, Universitat Autònoma de Barcelona, Cerdanyola del Vallès, 08193 Barcelona, Spain Full list of author information is available at the end of the article © The Author(s) 2022. 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The Creative Commons Public Domain Dedication waiver (http://​creat​iveco​ mmons.​org/​publi​cdoma​in/​zero/1.​0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Florido et al. Biology of Sex Differences (2022) 13:28 Page 2 of 15 • Nk3R–GPAR is more abundant in males than in proestrus females, whereas Nk3R–GPER is greater in proestrus females compared to males. Keywords: Neurokinin-3 receptor, Osanetant, Sex differences, Heterodimerization, Estradiol, Testosterone Background On the other hand, the hippocampus and the medial Recognition memory is a type of declarative memory prefrontal cortex (mPFC) play a major role in the con- that allows mammals to identify an object or situation solidation of recognition memory in mice [14, 15]. Hip- as familiar, in contrast with novel objects or situations. pocampal lesions after familiarization with two objects The organism uses this memory to compare novel situ- have been shown to disrupt object recognition in male ations with past personal experiences generating a con- rats [16]. Moreover, the dorsal hippocampus (DH) is gruent response toward the novel situation [1]. Although highly interconnected with the mPFC [17, 18]. This net- rodents do not present declarative memory itself, since work has been demonstrated to mediate whether an they do not have the ability to explicitly expose informa- object was previously encountered or not [19]. Moreo- tion, analogous procedures have been developed to test ver, lesions of the prelimbic cortex (PrL) and infralimbic this type of memory in rats and mice [2]. Declarative cortex (IL) of the mPFC produce deficits in recognition memory in humans has usually been related to spatial memory [20, 21]. These findings support the role of the and recognition memory in mice [3, 4], and in both cases, mPFC–DH connections in the formation of recognition it has been established common fundamental encod- memory. ing processes in the hippocampus and its connections The Nk3R has been shown to interact with sex hor- [5]. Memory consolidation represents a crucial point mones in both males and females in the modulation of in this process when recently labile short-term memo- fear memory consolidation through the modulation ries are transformed into long-term and stable memory of the intracellular cascade comprising Akt/GSK3β/β- traces [2]. This transformation typically requires cellular Catenin [7]. In female mice, estradiol enhances recog- and molecular adaptations, such as genetic regulation nition memory consolidation, via the activation of the of immediate-early gene expression or increased density PI3K/Akt pathway in the DH [22] in a similar manner. of dendritic spines [1]. The tachykinin 2 (Tac2) pathway In addition, testosterone in male rats has been shown is characterized by the synthesis of neurokinin B (NkB) to enhance synaptic plasticity in the dentate gyrus and from the Tac2 gene and its binding to the neurokinin 3 increase reference memory measured by the inspection receptor (Nk3R) mediating sex differences in the con- of non-baited holes in a hole board [23]. solidation of fear memories [4, 6, 7]. Moreover, Nk3R In recent years, a substantial amount of evidence has manipulations in male rodents have been shown to mod- shown that G-protein-coupled receptors (GPCRs) may ulate the formation of hippocampus-dependent declara- exist as dimeric or oligomeric complexes, exhibiting bio- tive memory [8, 9]. Interestingly, Nk3R antagonists chemical properties distinct from those of the protomers are safe and well-tolerated drugs in humans [10] which in modifying cellular responses [24]. Nk3R couples to the makes them potentially attractive candidates for treating pertussis toxin-insensitive G-protein Gq, whose activa- mental disorders with memory alterations. Osanetant, tion results in the production of inositol triphosphate a potent selective Nk3R antagonist, showed safety in a and diacylglycerol, activating intracellular signaling cas- clinical trial for the treatment of psychotic and affective cades that result in memory modulation [6, 25]. Notwith- symptoms typical of schizophrenia [11]. Although this standing, heterodimerization of Nk3R with other GPCRs study was discontinued, Nk3R antagonists are currently remains unexplored. under study as a promising solution for induced vasomo- Nk3R antagonists have shown sex-dependent effects tor symptoms (iVS) during menopause [12]. Surprisingly, on fear memory and sex hormonal fluctuations [7, 26], there is only one prior study that has included females but sex differences in recognition memory after Nk3R involving the Tac2 pathway and memory formation [7]. antagonism have not been tested yet. Here, we found The abovementioned study shows how Nk3R blockade, that systemic Nk3R antagonism modulated recogni- as well as Tac2 silencing in the amygdala, reduce fear tion memory consolidation in an opposite-sex man- memory consolidation in male mice while increasing ner, reducing consolidation in males and increasing it in it in females [7]. In contrast, the role of the amygdala in females during proestrus (high levels of estradiol). Then recognition memory is limited and appears to be circum- we measured the Nk3R, estrogen receptors α (ERα) and β scribed to the familiarization phase, but not recall [13]. (ERβ), and androgen receptors (AR) in the DH and mPFC Florido et al. Biology of Sex Differences (2022) 13:28 Page 3 of 15 uncovering sex differences. Finally, we studied protein The 10 μL of the sample were unloaded and collected 5 interactions between the Nk3R and membrane G-pro- consecutive times to collect enough cells for the assess- tein estrogen receptors (GPER) and G-protein andro- ment, and later placed on an adhesion slide (Superfrost gen receptors (GPAR) measuring heterodimerization as Plus, Thermo Fisher). All vaginal smear samples were a possible mechanism of the sex differences in memory collected between 9 and 10 am. Slides were dried using a formation upon Nk3R antagonism. hot plate (HI1220, Leica) at 37 °C for 30 min before stain- ing in cresyl violet acetate (C5042, Merck) 0.1% (v/v) for Methods 5 min, washed twice for 1 min in distilled water, and read Animals in brightfield microscopy with a 10× or 20× objective in All experiments used male and naturally cycling female an Eclipse 80i microscope (Zeiss, Spain). C57BL/6J adult mice (Charles River, Barcelona, Spain), Three different cell types may appear in the prepara- 8 weeks old at the beginning of the experiments and tion: cornified epithelial cells, round nucleated epithelial housed in groups of 4 in a room with a 12:12 h light/dark cells, or leukocytes. The different stages of the estrous cycle (lights on from 8 am to 8 pm). All animals were cycle were assessed depending on the proportion of the housed with a controlled temperature of 22 ± 1 °C and abovementioned cells. Proestrus is characterized by a humidity (~ 40%). Behavioral procedures and pharma- high proportion (> 80%) of nucleated epithelial cells that cological manipulations began early in the light phase of might present very small amounts of cornified epithe- the cycle. Male and female mice were housed separately lial cells or leukocytes. Estrus is typically presented with in the same room. Experiments in mice were performed cornified epithelial cells with a lower grade of staining under the approved ethics protocol with reference than leukocytes and nucleated epithelial cells. Metestrus CEEAH 3603. All procedures were approved by the Eth- presents a mixture of cornified epithelial cells and a con- ics Committee of the Universitat Autònoma de Barcelona siderable proportion of leukocytes. Diestrus is charac- and the Generalitat de Catalunya. Experiments were also terized by > 90% of leukocytes that might present a very carried out following the European Communities Coun- small proportion of round nucleated epithelial cells [27]. cil Directive (2010-63-UE) and Spanish legislation (RD 53/2013). Novel object recognition NOR is widely accepted as a test to assess prefrontal and Drugs hippocampal-dependent learning and memory processes Osanetant was purchased from Merck (SML0798) and [28, 29]. Before starting the NOR test, a different cohort prepared for 5 and 10 mg/kg. The vehicle used for this of animals was used to test that animals did not present preparation was 0.1% (v/v) Tween 20 in 0.9% (m/v) NaCl differences in the time exploring each object of the NOR solution. Osanetant and its vehicle were administered test. at 10 mL/kg 30 min after the familiarization phase of All animals were habituated to the testing arena for the novel object recognition (NOR), to isolate the effect 5 min for 2 consecutive days before the familiarization of the drug on the consolidation window of familiariza- phase of the test. On the 3rd day, animals were placed tion consolidation. Given previous data that support an in the arena where they freely explored two identical attenuated effect of osanetant in memory during estrus, objects (either a yellow plastic pin or a purple crystal metestrus, and diestrus [7]; only females during the jar) for 15 min. Male and female mice received 5 mg/kg, proestrus stage of the estrous cycle during familiarization 10 mg/kg osanetant, or vehicle 30 min after the familiar- were used in this experiment. ization phase of the NOR test before returning to their homecage. The percentage of time exploring each object Determination of the estrous cycle was calculated as a measure of preference for any of two Determination of the estrous stage in females was per- identical objects. formed by assessing vaginal smear cytology [27]. To On the test day, the animals returned to the arena assess the phase of the estrous cycle that females pre- where they freely explored the two objects for 15 min. sented during the familiarization phase of the NOR, all- The novel object was the yellow plastic pin for half of the female mice were monitored for 3–4 consecutive cycles animals and the purple crystal jar for the other half. Fur- (approximately 10–14 days) before familiarization to test ther, we counterbalanced the position of the novel object for regularity of the cycle. We performed a vaginal lavage (right or left) to avoid an effect of place preference. All with a 20-μL pipette that was loaded with 10 μL of stand- phases of the test were carried out under ~ 15 lux. The ard NaCl 0.9% (w/v) solution, and later the tip was softly relative time exploring the novel object, expressed in per- placed on the vaginal aperture. Before the collection centage, was calculated as a measure of recognition by an of the sample, urine was softly removed using a tissue. Florido et al. Biology of Sex Differences (2022) 13:28 Page 4 of 15 experienced observer using a stopwatch. Heatmaps were Immunoresearch). Slices were then rinsed again and obtained using Autotype for MATLAB (Fig. 1K, L). incubated for 3 min with 4′,6-diamidino-2-phenylindole (DAPI) (1:10,000, 10236276001, Merck) to stain cell nuclei before mounting and covering. Immunofluorescent determination of Nk3R, ERα, ERβ, Z-Stacks of the cingulate cortex (CG), PrL, IL, DG, and AR CA1, and CA3 (50 μm/interval) were acquired using Immunohistochemical assays were performed on naïve a Leica SP3 confocal microscope with a PL APO 40× males and proestrus females whose tissue was collected immersion objective, controlled by Zen 2010 software. early in the morning by transcardial perfusion with The integrated density of the signal was then calculated 50 mL of 4% (m/v) of buffered paraformaldehyde (PFA) using Fiji for Windows v1.52.n and used as a measure of (Casa Álvarez, Barcelona, Spain). These animals were the protein quantity of the taken pictures. naïve and had not undergone any treatment or behav- ioral procedure before tissue collection. Brains were In situ proximity ligand assays (PLA) stored in the same solution at 4 °C for postfixation over- With PLA, primary antibodies raised in different species night before being transferred to 30% (m/v) sucrose in are used to detect different proteins. Secondary antibod- Sorensen’s PB buffer for equilibration in conic Falcon ies specific to each primary antibody that include a short tubes. After 48 h of equilibration in the sucrose solution, sequence of DNA are used to detect the presence of the brains were snap-frozen in chilled isopentane and stored primary antibodies. If both primary antibodies are part at − 80 °C until sectioning. of the same protein complex, both DNA chains react and, Brains were coronally sectioned at 30 um/slice using after amplification of this DNA, fluorescent complemen- a Leica cryostat (− 20 °C for the chamber, − 18 °C for tary oligonucleotides are used to detect the heterodimers. the sample) and stored in cryoprotective solution before Naïve mice were deeply anesthetized and immediately staining. For staining, brain slices containing the mPFC perfused transcardially with buffered 4% paraformal- and DH were rinsed in KPBS 1× three times for 10 min dehyde (Casa Álvarez, Barcelona, Spain). Brains were each, before spending 1 h in incubation in blocking buffer removed and post-fixed overnight in the same solution, (5% normal donkey serum in 0.4% Triton X solution pre- cryoprotected by immersion of 30% sucrose in Sorens- pared with KPBS 1×), to avoid nonspecific binding of the en’s PB buffer for 48 h at 4 °C, and then snap-frozen in primary antibody. Then, slices were incubated overnight dry ice-cooled isopentane. Serial coronal cryostat sec- at 4 °C in 300 µL of blocking buffer containing either tions (30 μm) through the whole brain were collected in one of the following combinations: rabbit anti-Nk3R a cryoprotective solution and stored at − 20 °C until PLA (1:1500, donated by Phillipe Cioffi) and mouse mono- experiments were performed. clonal anti-ERa (1:50, sc-8002, Santa Cruz Biotechnol- Immediately before the assay, mouse brain sections ogy), rabbit anti-Nk3R and mouse monoclonal anti-ERb were mounted on glass slides, washed in PBS, permea- (1:500, ab288, Abcam) or rabbit anti-Nk3R and mouse bilized with PBS containing 0.01% Triton X-100 for monoclonal anti-AR (1:50, sc-7305, Santa Cruz Biotech- 10 min, and successively washed with PBS. Interactions nology). After primary antibody incubation, brain slices were detected using the Duolink in situ PLA detection were rinsed three times, 10 min each, with KPBS 1× and Kit (Sigma-Aldrich, St. Louis, MO, USA) following the incubated at room temperature for 2 h in blocking buffer supplier’s instructions. A mixture of the primary anti- containing donkey anti-rabbit AlexaFluor488 (1:1000, bodies rabbit anti-Nk3R antibody (1:100; donated by 115-546-072, Jackson Immunoresearch) and donkey anti- Phillipe Cioffi (INSERM)) directly linked to a plus PLA mouse Rhodamine Red (1:1000, 715-295-150, Jackson probe and rabbit anti-GPER antibody (1:100; Sigma; cat (See figure on next page.) Fig. 1 Novel object recognition (NOR) test after the administration of osanetant or vehicle. A Schematic representation of the behavioral procedure. B Males and females’ preference test between the two objects used in the test. C, E Familiarization phase of the NOR task in males before receiving osanetant (5 or 10 mg/kg) or vehicle. D NOR test after receiving 5 mg/kg or vehicle after familiarization in males. F NOR test after receiving 10 mg/kg or vehicle after familiarization in males. G, I Familiarization phase of the NOR task in females (proestrus) before receiving osanetant (5 or 10 mg/kg) or vehicle. H NOR test after receiving 5 mg/kg or vehicle after familiarization in females (proestrus). J NOR test after receiving 10 mg/kg or vehicle after familiarization in females (proestrus). The recognition index is calculated by dividing the time exploring the novel object by the total time of exploration and expressed as a percentage. The dashed line in each graph represents 50% of the relative exploration time. K Representative heatmaps of the NOR test showing males that received vehicle or osanetant (10 mg/kg). L Representative heatmaps of the NOR test showing females (proestrus) that received vehicle or osanetant (10 mg/kg). Data were analyzed using Three-way ANOVA for the familiarization phase (Sex × Object × Treatment) or Two-way ANOVA (Sex × Treatment). *p < 0.05. Obj1 = Object 1; Obj2 = Object 2 Florido et al. Biology of Sex Differences (2022) 13:28 Page 5 of 15 Fig. 1 (See legend on previous page.) Florido et al. Biology of Sex Differences (2022) 13:28 Page 6 of 15 HPA027052) directly linked to a minus PLA probe were tested for normality and homoscedasticity using the used to detect Nk3R–GPER interaction, or rabbit anti- Shapiro–Wilk test and Levene’s test, respectively. Uni- GPAR antibody (1:100; Thermo Scientific, Fremont, CA, variate ANOVAs were performed to assess the statis- USA; Cat PA5-21074) directly linked to a minus PLA tical significance of main effects and interactions, and probe were used to detect Nk3R–GPAR interaction. For significant interactions between main factors were ana- negative controls, one of the primary antibodies (anti- lyzed using Bonferroni correction. Results are presented GPER or anti-GPAR) was omitted (data not shown). as mean ± SEM, and statistical significance was set at Then, samples were processed for ligation and amplifi- p < 0.05. All graphs presented in the figures were designed cation with a Detection Reagent Red and were mounted using Prism 7 (CA, USA). using a DAPI-containing mounting medium. Samples were analyzed in a Leica SP2 confocal micro- Results scope (Leica Microsystems, Mannheim, Germany) Nk3R blockade decreases recognition memory equipped with an apochromatic 63× oil-immersion consolidation in males and increases it in females objective (1.4 numerical aperture), and 405 nm and during proestrus in a dose‑dependent manner 561 nm laser lines. For each field of view, a stack of two After assessing that animals presented no prefer- channels (one per staining) and 9 to 13 Z-stacks with a ence for any of the objects used in the test (Sex: step size of 1 µm was acquired. Images were opened and [F(1,24) = 0.057, p = 0.813]; Object: [F(1,24) = 0.072, processed with Image J software (National Institutes of p = 0.791]; Sex × Object: [F(1,24) = 0.072, p = 0.791] Health, Bethesda, MD). Quantification of cells containing (Fig. 1B), a different cohort of male and female mice one or more red dots versus total cells (blue nuclei) was underwent the familiarization phase of the object determined by using the Fiji package (http://​pacif​i c.​mpi-​ recognition. As expected, familiarization testing cbg.​de/), considering a total of 600–800 cells from 5 to 10 showed no differences in the time exploring each different fields (63×) within each region from three dif- object either before receiving osanetant 5 mg/kg (Sex: ferent animals. Nuclei and red dots were counted on the [F(1,56) = 0.000, p = 1.000]; Object: [F(1,56) = 0.031, maximum projections of each image stack. After getting p = 0.860]; Treatment: [F(1,56) = 0.000, p = 1.000], the projection, each channel was processed individually. Sex × Object: [F(1,56) = 0.359, p = 0.552], Sex × Treat- The blue nuclei and red dots were segmented by filtering ment: [F(1,56) = 0.000, p = 0.000], Object × Treatment: with a median filter, subtracting the background, enhanc- [F(1,56) = 1.128, p = 0.293]; Sex × Object × Treatment: ing the contrast with the Contrast Limited Adaptive [F(1,56) = 0.006, p = 0.939]) or 10 mg/kg (Sex: Histogram Equalization (CLAHE) plug-in, and finally [F(1,50) = 0.000, p = 1.000]; Object: [F(1,50) = 0.000, applying a threshold to obtain the binary image and the p = 1.000; Treatment: [F(1,50) = 0.103, p = 0.749]; regions of interest (ROIs). Red spots were counted in Sex × Object: [F(1,50) = 0.091, p = 0.764]; Sex × Treat- each of the ROIs obtained in the nuclei images. ment: [F(1,50) = 0.000, p = 1.000]; Object × Treatment: [F(1,50) = 1.638, p = 0.206]; Sex × Object × Treatment: Experimental design and statistical analyses F(1,50) = 2.651, p = 0.110]) (Fig. 1C, E). The NOR test was analyzed using a multifactorial design The recognition test, performed 24 h after receiving Sex × Treatment (2 × 2) and the percentage of explora- the drug, revealed no differences in recognition between tion of the new object as the dependent variable. The males and females (no estrous cycle monitorization) familiarization phase was analyzed using a multifactorial that received 5 mg/kg (Sex: [F(1,28) = 1.748, p = 0.197; design Sex × Object × Treatment (2 × 2 × 2). Immunohis- Treatment: [F(1,28) = 0.227, p = 0.603; Sex × Treat- tochemistry assays included main factors Sex and Area ment: [F(1,28) = 0.002, p = 0.965) (Fig. 1D, H). Given (2 × 6) to assess statistical differences or interactions of that high levels of circulating estradiol have shown to integrated density between groups on Nk3R, ERα, ERβ, be necessary for osanetant to modulate memory con- and AR expression in the mPFC and the DH. Heterodi- solidation in a cued-fear conditioning task [7], we then merization analysis included Area and Sex as main fac- tested females that were in the proestrus stage of the tors (6 × 2), and the percentage of cells including dimers estrous cycle during the familiarization phase and the among Nk3R positive cells was used as a dependent vari- administration of the drug. In a different cohort of mice, able across the mPFC and the DH. The result of all statis- males that received 10 mg/kg after the familiarization tical tests is summarized in Table 2. phase presented reduced memory consolidation com- All statistical analyses were performed using IBM pared to vehicle administered males, while proestrus SPSS v23.0. Outliers were detected using Grubb’s test females that received 10 mg/kg showed increased per- and removed when it was appropriate. Before proceed- formance in the recognition test compared to the vehi- ing with the null hypothesis testing, all datasets were cle control group (Sex: [F(1,25) = 10.595, p = 0.003]; Florido et al. Biology of Sex Differences (2022) 13:28 Page 7 of 15 Treatment: [F(1,25) = 0.022, p = 0.884]; Sex × Treat- Heterodimerization of Nk3R with the membrane ment: [F(1,25) = 17.128, p = 0.000; treatment in males: estrogen receptor is enhanced in females, while males [F(1,25) = 9.496, p = 0.005], and in proestrus females: present higher rates of heterodimerization of Nk3R [F(1,25) = 7.714, p = 0.010]) (Fig. 1F, J). with the membrane androgen receptor This study extends our knowledge on Nk3R antago- We hypothesized a possible interaction between the nism showing that it does not only exert an opposite-sex Nk3R and the membrane GPER in females and with the regulation of memory consolidation in fear memories as membrane GPAR in male mice. Data showed significant it has previously been demonstrated, but also in object interactions between Nk3R-ERα, Nk3-ERβ, and Nk3R- recognition. AR in proestrus females and males using in situ proxim- ity ligand assay (PLA) in the DH and the mPFC. Given Nk3 and AR expression are higher in males compared that estradiol and testosterone appear to be necessary to females (proestrus), while ERα and ERβ expression for modulation of fear memories upon antagonism of depend on sex and area the Nk3R in female and male mice, respectively [7], we Our previous research suggests the necessity of adult aimed to test whether an activational effect of sex hor- concentration of sex hormones for osanetant to modu- mones could mediate the efficacy of Nk3R in the modula- late fear memory consolidation [7]. Following up on this tion of memory consolidation. To test that hypothesis, we data, we measured the expression of sex hormone recep- assessed the presence of Nk3R–GPER and Nk3R–GPAR tors (androgen receptor -AR-, estrogen receptor alpha heterodimers in male and proestrus females through -ERα-, and estrogen receptor beta -ERβ-) in areas related PLA in the DH and the mPFC. Results suggest significant to NOR memory consolidation where the Nk3R might be interactions of area by sex [F(5,120) = 2.461, p = 0.037] exerting a regulating function. and dimer by sex [F(1,120) = 159.754, p = 0.000] in the To understand a possible role for Nk3R and sex hor- number of positive cells, while the interactions area by mones in memory consolidation, we performed immu- dimer ([F(5,120) = 0.383, p = 0.860]) and area by sex by nolocalization assay to assess the expression of Nk3R, dimer ([F(5,120) = 1.250, p = 0.290]) did not reach statis- ERα, ERβ and AR in the DH (CA1, CA3 and DG) and the tical significance (Fig. 3A, B, Tables 1, 2). mPFC (CG, PrL and IL), areas that are typically related Interaction between main effects area by sex and area to the consolidation of recognition memory. This analy- by dimer describes an increased proportion of heter- sis revealed an overall increased expression of Nk3R in odimerized Nk3R with GPER in proestrus females com- males compared to females in the DH and the mPFC pared to males in all areas examined ([F(1,120) = 213.664, (Sex: [F(1,43) = 7.770, p = 0.008]; Area: [F(5,44) = 1.631, p = 0.000]), with an increased proportion of heterodimer- p = 0.257]; Sex × Area: [F(5,44) = 0.768, p = 0.758]) ized Nk3R with GPAR in all assessed areas in male mice (Fig. 2A, E). The expression of ERα was also enhanced in compared to proestrus female mice ([F(1,120) = 10.616, male mice compared to proestrus female mice in the DH p = 0.001]). Differences in the number of heterodimers and the mPFC (Sex: [F(1, 47) = 7.001, p = 0.011]; Area describe increased number of heterodimers in proes- [F(5,47) = 1.789, p = 0.133]; Sex × Area: [F(5,47) = 1.326, trus females compared to males in the DH in CA1 p = 0.270]) (Fig. 2D, E). Contrarily, the expression of ([F(1,120) = 15.100, p = 0.000]), CA3 ([F(1,120) = 28.687, ERβ was greater in proestrus females compared to p = 0.000]) and the PrL ([F(1,120) = 8.884, p = 0.003]) males in CA1, DG and CG (Sex: [F(1,41) = 115.275, and IL ([F(1,120) = 17.899, p = 0.000]), but not in the CG p = 0.000]; Area [F(5,41) = 28.398, p = 0.000]; Sex × Area ([F(1,120) = 0.779, p = 0.379]) (Fig. 3A, B, Tables 1, 2), [F(5,41) = 26.475, p = 0.000], CA1 [F(1,41) = 21.410, probably because of the higher rate of heterodimeriza- p = 0.000], CA3 [F(1,41) = 0.001, p = 0.973], DG tion with GPER in all areas examined. [F(1,41) = 98.405, p = 0.000], CG [F(1,41) = 105.908, p = 0.000], PrL [F(1,41) = 0.029, p = 0.866], IL Discussion [F(1,41) = 0.000, p = 0.985]) (Fig. 2C, E). Interestingly, This study shows for the first time that the systemic block- no differences between areas or sexes were found in the ade of the Nk3R reduces novel object recognition memory analyses AR expression in the DH and mPFC of male consolidation in male mice and enhances this same process and proestrus females, but a sex-independent increase in female mice that present high levels of circulating sex of AR expression (Sex: [F(1,42) = 1.388, p = 0.245]; Area hormones. The Nk3R antagonist osanetant is a good candi- [F(5,42) = 9.144, p = 0.000]; Sex × Area [F(5,41) = 0.475, date to study memory consolidation since it presents high p = 0.793]; CG vs rest of the areas p = 0.000) (Fig. 2B, E). blood–brain barrier penetration, a half-life of ~ 2 h [30], and a high clearance [31]. Notwithstanding, since the Nk3R is a GPCR, the effect of its blockade remains in the brain until phosphatases, kinases, and other metabolite levels Florido et al. Biology of Sex Differences (2022) 13:28 Page 8 of 15 Fig. 2 Immunolocalization of neurokinin 3 receptor (Nk3R) with androgen receptor (AR), estrogen receptor α (ERα), and estrogen receptor β (ERβ). A Immunolocalization of Nk3R in the medial prefrontal cortex (mPFC) or the dorsal hippocampus (DH). B Immunolocalization of AR in the mPFC or the DH. C Immunolocalization of ERβ in the mPFC or the DH. D Immunolocalization of ERα in the mPFC or the DH. D Representative confocal images of AR, ERβ, and ERα with Nk3R in the CG and CA1. CG cingulate cortex, IL infralimbic cortex, PrL prelimbic cortex, CA1 field CA1 of hippocampus, CA3 field CA3 of hippocampus, DG dentate gyrus. Data are mean ± SEM. Yellow pixels represent colocalization between red and green. White pixels represent the colocalization of red, green, and blue. Datasets were analyzed using two-way ANOVA. **p < 0.01, ***p < 0.001. Asterisks above a line in A and D indicate a significant main effect of sex independently of the area. Asterisks in B and C indicate significant main effect sex in discrete areas after finding a significant Sex × Area interaction. E Yellow pixels indicate colocalization between Nk3R and AR, ERβ, or ERα. White pixels indicate colocalization between DAPI, Nk3R, and AR, ERβ, or ERα. Scale bar = 25 µm Florido et al. Biology of Sex Differences (2022) 13:28 Page 9 of 15 return to the baseline. We measured the expression of the [35]. Further, activation of the Akt/GSK3β pathway in the Nk3R and sex hormone receptors in the mPFC–hippocam- presence of estradiol has been shown to increase object pal circuit that underlie the formation of novel object rec- recognition [22]. Taking all these data into account, we ognition memories. We also uncovered potential new hypothesize a possible confluence in the mechanisms of protein interaction mechanisms between the Nk3R and memory consolidation after Nk3R antagonism, with the membrane receptors for sex hormones (GPER and GPAR), GSK3β downstream target β-Catenin modulating the for- in male and female mice, that may lead to opposite-sex mation of long-lasting memories. effects of Nk3R targeted drugs on memory consolidation In previous works [6, 7], we demonstrated that a sin- (Fig. 4). gle dose of 5 mg/kg reduces fear memory consolidation in males and increases it in females. In contrast, this dose Sex differences in object recognition was not effective in modulating recognition memory Sex differences in recognition memory performance have consolidation, but a 10 mg/kg dose reproduced this effect been previously studied, although it is still controversial. in the NOR task. As commented before, the neural net- While some authors report better performance for males works supporting consolidation for fear memories and in the NOR test [32], others report an outperformance of recognition memories remain similar between humans female mice [33]. This better performance of females has and rodents and differ consistently. Consolidation of been hypothetically related to better object discrimina- cued-fear memories typically requires molecular and sys- tion compared to male mice, as shown in other work [34]. temic changes in the amygdala and extra-amygdalar com- In contrast, male mice have been shown to present a bet- plexes [36, 37]. In contrast, consolidation of recognition ter discrimination index when the novel object resembles memory has been widely associated with the hippocam- the object that they have been previously familiarized pus and its connections with the mPFC [2, 22, 38]. Each with [34]. Our study does not show a sex-dependent per- of these brain regions presents a unique organization and formance on the NOR test, nor in the preference test per- information flow, which may vary its sensitivity to osan- formed before the recognition test, suggesting that both etant modulation of synaptic and systemic consolidation objects used as novel and old were sufficiently different to processes. produce a similar discrimination rate between sexes. Previous work has demonstrated that senktide, a potent Nk3R agonist, increases memory consolidation in male Nk3R opposite‑sex regulation of memory consolidation rats [9]. Also, senktide reverted the scopolamine-induced The Nk3R has been previously linked to the performance memory impairment, suggesting that the muscarinic of male subjects in the object recognition test. The admin- cholinergic receptors mediate the memory enhance- istration of the Nk3R agonist Senktide enhances per- ment of senktide [39]. Senktide has also been shown to formance in the NOR test in male Wistar rats [9]. At a increase acetylcholine release in the mPFC and the DH molecular level, our previous studies in fear memory show [40, 41]. A tentative explanation of the observed behav- that the Tac2 pathway modulations in the central amygdala ioral outcome is a decrease of ACh release in males and (CeA) reduce the activity of the Akt/GSK3β/β-Catenin in an increase in females, after osanetant administration. males and increases the activity of this molecular route Notwithstanding, NkB also interacts with the dopamin- in females leading to opposite-sex effects in memory [6, ergic system regulating dopamine release in the striatum 7]. Our present study suggests that Nk3R antagonism in healthy and pathological individuals [42]. We suggest may modulate in a similar manner recognition memory that further research should evaluate ACh release in the in both sexes with potential similarities in the mecha- mentioned areas as well as the behavioral outcome of nisms that mediate both fear and recognition memory. site-directed manipulations of Nk3R receptors. Increased activity of GSK3β prevents the phosphorylation of β-Catenin and, therefore, its role in the consolidation of Sex hormones interaction with Nk3R on recognition memories. In the same line, increased activity of GSK3β memory consolidation has been shown to impair novel object recognition, as well Estradiol has been shown in female mice to increase rec- as mood regulation and hippocampal cell proliferation ognition memory consolidation by enhancing the activity (See figure on next page.) Fig. 3 Heterodimerization of neurokinin 3 receptor with membrane sex hormones receptors. A Quantification of heterodimerization Nk3R with G-protein androgen receptor (GPAR) and G-protein estrogen receptor (GPER) in male and female mice during proestrus. CG cingulate cortex, IL infralimbic cortex, PrL prelimbic cortex, CA1 field CA1 of hippocampus, CA3 field CA3 of hippocampus, DG dentate gyrus. Data are mean ± SEM. Datasets were analyzed using two-way ANOVA. **p < 0.01, ***p < 0.001. B Representative confocal images of the previously reported quantification. Scale bar = 20 µm Florido et al. Biology of Sex Differences (2022) 13:28 Page 10 of 15 Fig. 3 (See legend on previous page.) Florido et al. Biology of Sex Differences (2022) 13:28 Page 11 of 15 Fig. 4 Schematic representation of Nk3R–GPER and Nk3R–GPAR in the dorsal hippocampus and the medial prefrontal cortex. Males are represented in blue on the left and females in green on the right. The percentage of positive cells is represented with the sign +. ++ indicates a medium expression (25–75%) and +++ indicates a high proportion (< 75%) of the Akt/GSK3β pathway in the DH [22]. Interestingly, Table 1 Mean values and standard deviation (mean (SD)) for wild-type mice, but not ERβ-KO, increased their perfor- Nk3R–GPAR and Nk3R–GPER heterodimers in the mPFC and the mance on the object recognition task when treated with DH estradiol [43], suggesting a role for ERβ in the modula- Area GPAR GPER tion of the enhancement effect of estradiol in object rec- Males Females Males Females ognition memory consolidation. Our study shows that ERβ is more expressed in the CA1 and DG of the DH and CA1 63.43 (8.92) 63.65 (11.09) 59.36 (5.56) 82.00 (4.01) the CG of the mPFC in females compared to males, sug- CA3 59.47 (5.80) 57.72 (5.79) 49.47 (7.04) 82.73 (6.38) gesting possible target areas of estradiol in the enhance- DG 65.73 (6.02) 59.26 (7.09) 60.48 (5.66) 80.70 (5.88) ment of recognition memory consolidation, especially CG 68.42 (9.60) 58.77 (7.08) 63.13 (3.21) 77.98 (4.78) in those that represent the direct output of the DH. Not- PrL 67.78 (7.72) 58.75 (6.14) 57.37 (9.17) 83.94 (6.31) withstanding, ERα is also present in the DH and mPFC IL 66.57 (6.85) 60.07 (12.02) 50.69 (7.67) 82.07 (6.25) of female mice although its expression is lower expressed The mean value was calculated based on the quantification of cells containing than in males. Data in female Swiss mice have also high- one or more heteromers versus total cells lighted the role of hippocampal ERα in enhancing mem- GPAR G-protein androgen receptor, GPER G-protein estrogen receptor, CG cingulate cortex, IL infralimbic cortex, PrL prelimbic cortex, CA1 field CA1 of ory consolidation [44]. hippocampus, CA3 field CA3 of hippocampus, DG dentate gyrus Our study demonstrates a high degree of heterodimeriza- tion of the Nk3R with GPER in the DH and the mPFC, find- ing that around 80% of Nk3R found in these areas present activating function over the GPER that allows the inhibi- heterodimerization with GPER in female mice (Fig. 4A, B). tion of the Nk3R to increase intracellular cascades respon- With these data, we hypothesize that estradiol exerts an sible for an increase in memory consolidation. Although Florido et al. Biology of Sex Differences (2022) 13:28 Page 12 of 15 Table 2 Statistical tests for independent sample analyses Table 2 (continued) Between-subjects variables Snedecor’s F p-value Between-subjects variables Snedecor’s F p-value df Value df Value Preference test AR expression Sex 1,24 0.057 0.813 Sex 1,42 1.388 0.245 Object 1,24 0.072 0.791 Area 5,42 9.144 0.000 Sex × Object 1,24 0.072 0.791 Sex × Area 5,42 0.475 0.793 Familiarization (5 mg/kg) CG vs rest of areas Bonferroni correction 0.000 Sex 1,56 0.000 1.000 Nk3R–GPAR positive cells Object 1,56 0.031 0.860 Sex 1,60 8.402 0.005 Treatment 1,56 0.000 1.000 Area 5,60 0.688 0.635 Sex × Object 1,56 0.359 0.552 Sex × Area 5,60 0.717 0.613 Sex × Treatment 1,56 0.000 1.000 Nk3R–GPER positive cells Object × Treatment 1,56 1.128 0.293 Sex 1,60 290.103 0.000 Sex × Object × Treatment 1,56 0.006 0.939 Area 5,60 1.609 0.171 NOR test (5 mg/kg) Sex × Area 5,60 3.810 0.005 Sex 1,28 1.748 0.197 Sex in CA1 1,10 0.001 0.972 Treatment 1,28 0.227 0.603 Sex in CA3 1,10 0.274 0.612 Sex × Treatment 1,28 0.002 0.965 Sex in DG 1,10 2.906 0.119 Familiarization (10 mg/kg) Sex in CG 1,10 3.929 0.076 Sex 1,5 0.000 1.000 Sex in IL 1,10 3.929 0.076 Object 1,5 0.000 1.000 Sex in PrL 1,10 5.033 0.049 Treatment 1,5 0.103 0.749 Two or three-way ANOVAs were performed to assess statistical significance for Sex × Object 1,5 0.091 0.764 independent sample analyses. Significant main effects and interactions between main effects are indicated in italic. Bonferroni post hoc analysis was used to Sex × Treatment 1,5 0.000 1.000 assess significance between categories of a significant multiple-category main Object × Treatment 1,5 1.638 0.206 effect Sex × Object × Treatment 1,5 2.651 0.110 NOR test (10 mg/kg) Sex 1,25 10.595 0.003 not at a similar level, approximately 60% of Nk3R in the Treatment 1,25 0.022 0.884 DH and the mPFC presents heterodimerization with mem- Sex × Treatment 1,25 17.128 0.000 brane GPAR (Fig. 4C, D). Previous experiments have dem- Treatment in males 1,25 9.496 0.005 onstrated that the memory consolidation effects observed Treatment in females 1,25 7.714 0.010 after the administration of osanetant are dependent on the Nk3R expression sexual maturity of the mouse [7], either male or female. Sex 1,43 7.770 0.008 Interestingly, given the involvement of sex hormones in Area 5,44 1.631 0.257 the regulation of Nk3R activity on memory formation [7], Sex × Area 5,44 0.768 0.758 estrogen GPCR has recently been shown to modulate hip- ERα expression pocampal-dependent memory and long-term potentiation Sex 1,47 7.001 0.011 [45]. Although the Nk3R presents a significant number of Area 5,47 1.789 0.133 heterodimers with AR in males and proestrus females, the Sex × Area 5,47 1.326 0.270 Nk3R–GPER heterodimer appears quite more abundant ERβ expression than the rest, pointing to a possible explanation for the Sex 1,41 115.275 0.000 increased efficacy of Nk3R during high estradiol levels. A Area 5,41 28.389 0.000 tentative hypothesis for this phenomenon is that the high Sex × Area 5,41 26.475 0.000 degree of heterodimerization of Nk3R with sex hormones Sex in CA1 1,41 21.410 0.000 receptors, with new and unique pharmacological and func- Sex in CA3 1,41 0.001 0.973 tional properties different from those of its components Sex in DG 1,41 98.405 0.000 [24, 46, 47], would produce a sex-specific activation of the Sex in CG 1,41 105.908 0.000 heterodimer in the presence of sex hormones, allowing the Sex in IL 1,41 0.000 0.985 antagonism to modulate memory consolidation in a sex- Sex in PrL 1,41 0.029 0.866 opposite way. It would be interesting for future studies to follow up our PLA analyses with co-immunoprecipitation Florido et al. Biology of Sex Differences (2022) 13:28 Page 13 of 15 to further understand protein–protein interactions of the the treatment of anterograde amnesic affection in women, Nk3R, ERα, ERβ, and AR. Additionally, an important ques- suggesting a path for future research on personalized tion that could be further addressed is whether this results medicine that tests the possibilities of Nk3R antagonists in heterodimerization of receptors that might interact with in the treatment of memory loss in women. Moreover, drug treatments, experience, or during the normal hormo- the impaired memory consolidation of osanetant in males nal cycling or life-span in male and female mice. Further, in the consolidation of both fear memory and episodic we would like to highlight that our treatments take place memory may be a tool for the treatment of aversive events 30 min after the familiarization phase of the NOR task, with an emotional and episodic memory component. therefore, avoiding a possible effect of the drug in the test Acknowledgements phase. Notwithstanding, we believe that the consolidation Procedural images were created under license with BioRender.com. effect observed is affecting the storage of memories and therefore, retrieval. Further, we would also highlight that Author contributions This study was planned and conceptualized by RA and AF. AF performed during the retrieval phase osanetant is no longer present behavioral and immunolocalization experiments, vaginal cytologies, and data- in the animal’s body, so we could isolate the effect of the sets analyses. EM and EIC performed In Situ Proximal Ligand Assays. The paper drug in the consolidation phase. Notwithstanding, it will was written by RA and AF and commented on and discussed by all authors. All authors read and approved the final manuscript. be quite interesting to manipulate Tac2/Nk3R before or during retrieval to have a more complete knowledge of the Funding phenomenon explained here. R.A. was supported by a NARSAD Young Investigator Grant #22434, Ramón y Cajal program (Agencia Estatal de Investigación) RYC2014-15784, RETOS- MICINN SAF2016-76565-R and PID2020-112705RB-I00 FEDER funds, Fundación Alicia Koplowitz, ERANET Neuron JTC 2019 (Ministerio de Ciencia, Innovación Perspectives and significance y Universidades) ISCIII AC19/00077, and CIBER—Consorcio Centro de Investi- This study demonstrates for the first time a sex-opposite gación Biomédica en Red—(CB19/09/00029); and SAF2017-87629-R to E.I.C. effect of an Nk3R antagonist on recognition memory A.F. was the recipient of Generalitat de Catalunya (Agència de Gestió d’Ajuts Universitaris i de Recerca) 2018 FI_B00030 predoctoral fellowship. consolidation, together with a high degree of heterodi- merization between Nk3R and membrane sex hormone Availability of data and materials receptors. Osanetant is a safe and well-tolerated drug All data and materials are available from the corresponding author upon reasonable request. in humans [10], an important finding that may lead to a rapid translation of these results into the clinic. Osan- Declarations etant has not been approved yet for the treatment of mental disorders in humans, but there are clinical trials Ethics approval and consent to participate currently studying this possibility. As indicated in “Methods” section, ethics protocols were approved for the experiments in mice with ref. CEEAH 3603. All procedures were approved Previous research has shown that Nk3R is expressed by the Ethics Committee of the Universitat Autònoma de Barcelona and the in Tac2 neurons in the centromedial amygdala (CeM) Generalitat de Catalunya. Experiments were also carried out following the [4]. As previously demonstrated [7], these neurons in the European Communities Council Directive (2010-63-UE) and Spanish legislation (RD 53/2013). CeM are GABAergic neurons, concordantly with the ana- tomical data available on the distribution of GABA and Consent for publication glutamate in the amygdala. Notwithstanding, few articles Not applicable. address the colocalization of Tac2 with other neuropep- Competing interests tidergic systems. One example of this colocalization is R.A. declares a potential conflict of interest with the patent PCT/ the Tac2-Kisspeptin neurons in the hypothalamus [48]. US2015/037629. The other authors declare no competing interests. Following this, Kisspeptin is not one of the representa- Author details tive neuropeptides found in the CeM, suggesting that the 1 Institut de Neurociències, Universitat Autònoma de Barcelona, Cerdanyola del colocalization with other neuropeptidergic systems may Vallès, 08193 Barcelona, Spain. 2 Departament de Psicobiologia i Metodologia de les Ciències de la Salut, Universitat Autònoma de Barcelona, Cerdanyola vary depending on the area. The specific expression of del Vallès, 08193 Barcelona, Spain. 3 Departament de Bioquímica i Biome- Tac2 and its colocalization in the medial prefrontal cor- dicina Molecular, Facultat de Biologia, Universitat de Barcelona i Institut de tex and the dorsal hippocampus has not been addressed Biomedicina de la Universitat de Barcelona (IBUB), 08028 Barcelona, Spain. 4 Instituto de Salud Carlos III, 28029 Madrid, Spain. 5 Centro de Investigación in this article and could be of interest for future research. Biomédica en Red en Salud Mental (CIBERSAM), Instituto de Salud Carlos III, Interestingly, Nk3R pharmacological manipulations 28029 Madrid, Spain. 6 Unitat de Neurociència Translacional, Parc Taulí Hospital have aroused as a promising new tool for memory defi- Universitari, Institut d’Investigació I Innovació Parc Taulí (I3PT), Universitat Autònoma de Barcelona, Cerdanyola del Vallès, 08193 Barcelona, Spain. cits through its interaction with the cholinergic system 7 ICREA, Pg. 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