Neural correlates of adversity-overcoming pup rescue behavior in female mice

Neural correlates of adversity-overcoming pup rescue behavior in female mice | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Neural correlates of adversity-overcoming pup rescue behavior in female mice Kseniia Prokofeva, Mizuki Shibamiya, Rin Kawata, Chihiro Yoshihara, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7240012/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 03 Mar, 2026 Read the published version in Scientific Reports → Version 1 posted 9 You are reading this latest preprint version Abstract Rescuing infants under threat is fundamental parental behavior in mammals. However, the behavioral expression and neural correlates of adversity-overcoming infant rescue in non-parental female individuals remain poorly understood. In this study, we first established a novel pup rescue paradigm with scalable adversity, in which mothers and virgin female mice have to cross a water pool of varying depths (0, 3, or 20 mm) to retrieve pups into the nest. We unexpectedly found that virgin females were less averse to water and retrieved pups faster than mothers. Next, we implemented an additional hurdle by trapping pups into a tube, so that female mice had to cross the pool and open the tubes to rescue pups. The rescuer virgin females in this “trapped pup” rescue task showed increased neuronal activity in the anterior cingulate cortex, lateral septum, anterior commissural nucleus, basolateral amygdala, and dorsal raphe nucleus, compared with non-rescuers. The c-Fos + cell densities in these regions showed significant negative correlations with the latencies to rescue behaviors suggesting their positive impact on rescue. Given that the virgin females do not have genetic relations to the rescuee pups, our findings provide a basis of further analyses of adversity-overcoming altruistic behavior and its neural correlates. Biological sciences/Evolution Biological sciences/Neuroscience Biological sciences/Zoology Alloparental care Pup retrieval rescue c-Fos altruism Figures Figure 1 Figure 2 Figure 3 Introduction Targeted helping-like behaviors toward conspecific adults expressed as freeing them from restraint or adverse environments or as allogrooming stressed conspecifics have been observed in several rodent species under laboratory conditions 1 – 7 . Recent studies have also reported that mice engage in resuscitation-like behaviors toward unresponsive conspecifics via methodical interactions with the tongue and head of the rescuees 8 – 10 . However, the prosocial nature of these behaviors has not been confirmed, as they can be performed out of selfish intentions, such as the avoidance of being alarmed or the risk assessment for one's own survival 11 , 12 . Moreover, the aforementioned rodent behaviors do not constitute evidence of true altruism, which has been defined as the behavior that benefits the receiver at a cost (loss) to the performer 13 , because known helping behaviors in rodents often do not impose a significant cost to the rescuers. Infant care is a paramount mammalian behavior that facilitates the survival of the young, and it requires substantial cost from the parents 14 , 15 . In addition to the energy cost of lactation, the protection and transportation of infants can be associated with substantial mortality risk for mammalian parents. In laboratory mice, pup retrieval, which is an act of carrying a displaced young back into the nest, is widely used as a readout of infant care motivation, since it can be robustly and unambiguously performed by both parental and virgin female mice 14 . Ours and other groups have previously shown that mother mice and rats, respectively, retrieve (rescue) pups placed at the end of an open elevated platform, while virgin females scarcely do so 16 , 17 . This risk-taking pup rescue required specific gene expression in the anterior commissural nucleus (ACN) and the caudocentral part (cMPOA) of the medial preoptic area (MPOA) in mice, which is indispensable for infant care across mammalian species 15 , 18 . Moreover, to overcome environmental risks during pup rescue, the caregiving mice may require not only the motivation to care but also precise risk assessment and cognitive/behavioral skills to cope with the specific risk. To elucidate the neural circuit mechanism required for such an adversity-overcoming pup rescue, it is preferable to create a behavioral paradigm in which the environmental risk is scalable. However, in our previous pup-rescue paradigm on the elevated plus maze, it was not easy to modulate the width or height of the platform. In this study, we established an adversity-overcoming pup rescue task that involves crossing a water pool of variable depths to retrieve (rescue) pups, utilizing the previous findings that mice are averse to entering water 19 – 21 . We also identified the brain areas that are activated during this behavioral task, using immunohistochemistry and c-Fos analysis. As we utilized female virgin mice that are not kin to the rescuee pups, our histological study sheds light not only on the neural mechanism of costly pup rescue, but also on that of the altruistic helping-like behavior. Results Mice perceive increasing water depth levels as scalable adversity. We have implemented the adversity-overcoming pup rescue paradigm using mothers and pup care-experienced virgin females that have been cohoused from the gestational day 9 of the mother ( Fig. 1 a). To examine if repetition of the test (for virgins) and the postpartum period (for mothers) influence mouse behavior, we conducted the habituation three times across one month (corresponding to pup age of postnatal weeks 0, 2, and 4). Before undergoing each pup rescue task described in Fig. 2 , we first exposed each subject mouse to a water pool with increasing water depths (0, 3, 20mm) for 5 min (habituation) and observed their exploration of the pool area ( Fig. 1 ) to examine the water aversion of virgin females and lactating mothers. First, we manually measured the latency to the first pool entry and the number of pool entries (Fig. 1 a; Fig. 1 b, left upper panel ). We found that, as the water depth increased, both the mother and the virgin females required a significantly longer time to enter the pool and entered the pool significantly less (Figs. 1 c, f). Notably, virgin mice entered the pool significantly faster and significantly more often than mother mice (Figs. 1 c, f), suggesting that water is recognized by mothers as stronger adversity. Interestingly, both latency and the number of pool entries did not significantly vary across time, implying that the task’s repetition or the postpartum weeks did not grossly affect the perceived adversity. Altogether, we confirmed that mice are increasingly averse to water upon the rise in water depth, while maternity status exacerbates the evaluation of adversity. To optimize the analyses of behavioral parameters, we implemented a machine learning-based animal motion tracking program DeepLabCut (DLC) 22 to measure the latency to pool crossing and the number of pool crossings, which roughly correspond to pool entry parameters (Fig. 1 b, left center and bottom panels ), utilizing the middle point of mouse body to analyze its locomotion (Fig. 1 b, right panel; back_1). We found that both the latency to pool crossing and the pool crossing number followed a similar trend as the pool entry parameters (Figs. 1 d, g). The automated measures of the latencies to pool entry and the number of pool crossings were significantly correlated with the manual measures (Figs. 1 e, h), suggesting that manual and DLC analyses are essentially interchangeable. We additionally conducted intraclass correlation (ICC) analyses between the manual and DLC methods and found that they yielded high similarity. Using DLC, we also analyzed time spent in the water pool, which aligned with other parameters and confirmed mouse aversity to water (Fig. 1 i). We therefore established DLC-mediated automatic analysis for our behavioral paradigm. Female virgins overcome adversity to rescue pups. Next, we examined the adversity-overcoming rescue behavior toward displaced pups performed on postpartum days 2–5 by mothers or during postnatal week 0 by virgin females (Fig. 2 a). When mouse pups are placed outside of the nest, female mice quickly recognize them by sniffing, and generally engage in a sequence of behaviors, starting from orally carrying the pup, placing the pup into the nest (retrieving), gathering pups within the nest (grouping), and crouching over pups (designated as full parenting = crouching over all pups continuously for over a minute), as reported before (Fig. 2 b) 14 , 18 , 23 . Therefore, we examined these behaviors toward three pups of the postnatal age 2–5 days placed behind the water pool (Fig. 2 c). Additionally, we examined the latencies and numbers of pool crossing as in Fig. 1 (Fig. 2 d-f). In Fig. 2 c, the pup-directed behaviors described in Fig. 2 b were assigned ranks based on their achievement (e.g., no pup-directed behavior – 0 (lowest rank), pup sniffing – 1, and full parenting, which is only possible if all other behaviors were performed, – 7 (highest rank)). We found that a significant proportion of female virgins and mothers were able to retrieve and nurture pups at any adversity level. However, there are two notable differences between the groups. First, with increasing adversity, virgins unexpectedly displayed better pup-retrieval behaviors than mothers (Fig. 2 c), probably due to the higher water aversion of mothers. Second, some of the virgins displayed no pup retrieval only in the 0mm condition (the very first task), plausibly due to their inexperience (see below), causing the non-linear correlation of the water depth and the behavioral performance. Therefore, we concluded that female virgins were more likely to overcome adversity and display nurturing behaviors than mothers. We next examined the level of water aversion in the context of pup rescue using latency to and the number of pool crossings, as well as time spent in the pool, measured using DLC. The GLMM analysis revealed a negative correlation between the latency to the pool crossing and the water level increase, however, mothers took significantly longer time to cross the pool with water depth increase, compared with virgins (Fig. 2 d). The seemingly contradictory prior result can be explained by the virgins' delay of almost all behaviors due to inexperience in the very first trial (0mm), causing the long latency to enter the pool area even though there was no water in it. The virgins entered the pool and engaged in pup retrieval much faster in the 3mm trial than in the previous trial ( Figs. 2 d, g-l). This suggests that virgins require an acclimatization period to perform the pup retrieval task even though they have cohabitated with pups, while mothers are efficient from the first trial. For water aversion, virgins appeared more resistant than mothers as in Fig. 1 . The number of pool crossings decreased with the water depth increase in both mouse groups, similarly to the habituation period (Fig. 1 g), suggesting that water is still perceived as an adversity (Fig. 2 e). Time spent in the water pool decreased with higher water depth more in mothers, confirming their high aversity (Fig. 2 f). We next identified the latencies to each pup-directed behavior to examine motivation more precisely (Figs. 2 g-m). We found that virgins sniffed pups upon crossing the water pool significantly faster than mothers (Fig. 2 g), while the latencies to both sniffing and first pup carrying were increased in both mouse groups with an increase in water depth (Fig. 2 g, 2 h). Additionally, similarly to our earlier observations, latencies to pup sniffing, carrying, and retrieving the first two pups lengthened with the increase of adversity in mothers more (Figs. 2 g-j). Similarly to previous findings (Fig. 2 c), the latencies to retrieve the second and third pup were stabilized upon the addition of water to the pool in all mice (Figs. 2 j, k), however, this effect was not as present in mothers as in virgins (Fig. 2 k). We did not observe any differences in grouping behavior and did not conduct statistics on full parenting due to its rarity (Figs. 2 l, 2 m). Therefore, we concluded that virgins are more resilient to water adversity at any depth in pup presence and rescue pups more efficiently, than mothers. Costly pup rescue increases activity in empathy-, infant care-, and adversity-associated brain areas. To identify brain regions implicated in adversity-overcoming pup rescue, we examined the expression of the cellular activity marker c-Fos across the brain during this behavior. For this purpose, we made two minor changes to the experimental protocol. First, we restricted the spontaneous locomotion of the stimulus pups by containing them in a plastic tube (two pups per tube) with a paper lid. Second, before the final pup exposure test, we trained the subject females for a period of up to four days, so that the subjects could successfully rescue pups even under adverse conditions and when pups were contained in a plastic tube (Fig. 3 a). In the first phase of training, the subjects were directly exposed to three pups (i.e., without being contained in a tube as in Fig. 2 ) placed on the opposite side of the pool with increasing water depth levels, and the test was repeated at each water depth level until the subjects succeeded or until the maximum length of the training period had been reached. In the second phase of training (tube opening), we placed two 50-ml tubes each containing two pups behind the pool and repeated the test in a similar manner, while we counted rescue success as tube opening. After the training, the subjects were isolated for one day and underwent the final experiment (“trapped pup” rescue) similarly to the last phase of training, but only at 3mm water depth. Mice that opened both tubes within the first 30 minutes were designated as “rescuers”. Out of four “rescuer” animals, most of them (3/4, 75%) opened the first tube and retrieved at least one pup before opening the second tube, while one (25%) first opened both tubes and then retrieved the pups. However, none of the animals opened the tubes without rescuing the pups. These results suggest that mice exhibited behaviors in an organized manner with the aim of pup rescue, rather than out of interest in tubes. Mice that did not open any of the tubes during the task displayed a range of behaviors, from complete lack of interest in the pool and pup-containing tubes to active pool crossing and tube sniffing without biting. Thus, these mice were designated as “non-rescuers” (control group), while mice with intermediate performance were excluded from the c-Fos analysis. Following this behavioral experiment, mice were subjected to brain sampling (Fig. 3 b). We next compared c-Fos expression across the brain between the rescuers and non-rescuers (Figs. 3 c, d). We selected the target brain regions involved in pup nurturing, emotion processing, and empathic response 5 , 8 , 10 , 23 – 29 . The “rescuer” group exhibited a significantly higher density of c-Fos-positive cells in the anterior part of the anterior commissural nucleus of the preoptic area (aACN), which has already been shown to be activated most after parental nurturing behaviors 24 . Three limbic regions, that are the area 24 of the anterior cingulate cortex (A24), lateral septum (LS), and basolateral amygdala (BLA), were also activated in rescuers. Finally, several hindbrain regions, such as the caudal part of the dorsal raphe nucleus (DRC) and the lateral parabrachial nucleus (LPB), displayed higher c-Fos cell density in rescuers compared with non-rescuers. The shell of the nucleus accumbens (AcbSh) and medial parabrachial nucleus (MPB) showed tendencies (0.05 < p < 0.1) toward an increase of the density of c-Fos-positive cells in the rescuer mice. The locus coeruleus (LC) and paraventricular nucleus (PVN), which are known to be activated by stress, exhibited relatively high c-Fos densities in both groups. No brain region in the Fig. 3 c showed higher activity in the non-rescuer group. Identification of neural correlates of adversity-overcoming pup rescue. To better describe the link between the behavior and rescue-biased brain regions, we next analyzed correlations between the latencies to the major components of the rescue behavior, such as crossing the pool, biting each tube, and retrieving each pup, and the within-region standardized densities of c-Fos-positive cells in the analyzed brain regions (Fig. 3 e). The behaviors and activation patterns of the brain regions were each sorted using unsupervised hierarchical clustering where the clusters were formed based on similarity between items. The behavior clustering aligned with the chronological sequence of the pup-rescue behaviors: investigation of the pups, opening the first tube and retrieving the first pup from it (tube 1 rescue), and opening the second tube and retrieving the second pup from the first tube (tube 2 rescue). The clustering of brain areas was based on c-Fos density variability between individual mice, highlighting the consistency of each area's response to the examined behaviors. The correlation coefficients between the brain region activities and the latencies to each behavior are color-coded in Fig. 3 e. Negative correlation (blue) means that the given brain region was more active when mice performed the given behavior with a short latency. The activities of the aACN and cMPOA were negatively correlated with the behaviors in a graded manner toward the bottom, particularly with the latency of tube 2 rescue, which reflects high caring motivation. The four limbic areas, A24, LS, BLA, and AcbSh, that showed a (trend of) higher activation in the rescuer group, were clustered together and exhibited negative correlations evenly with the behavioral latencies, suggesting their involvement through negative emotions and affective states processing generally associated with this task. The DRC and LPB also showed graded patterns yet were not classified together with the aACN and limbic regions due to higher inter-mouse variability of c-Fos density. The precise roles of these and other brain regions demonstrating significant correlations should be determined in future studies. Discussion This study has explored the adversity-overcoming pup rescue behavior in postpartum and virgin female mice, utilizing the mice's known aversion to water. Numerous studies in rodents showed that mothers care for pups better than female virgins, especially in difficult tasks or under stressful conditions 16 , 30 , 31 . Thus, we designed the present experimental paradigm to identify the brain mechanism of maternal heightened motivation for pup care and expected that mothers would outperform virgin females in this task. Surprisingly, however, in our water pool-crossing pup rescue paradigm, virgins clearly surpassed mothers in pup retrieval (Fig. 2 c), plausibly due to mothers’ heightened aversion to water compared with female virgins (Figs. 1 c, 1 d, 1 f, 1 g ). This strong water aversion in mothers might have occurred directly from the altered physiology of postpartum and lactating animals. However, time since parturition (postpartum weeks) did not significantly affect water aversion in mothers, implying that water aversion persists beyond weaning. The precise mechanism of heightened water aversion of postpartum females should be deciphered in future studies. Additionally, our study indicates that the modality of environmental stress can be a critical determinant for parental care motivation in various reproductive contexts. For more comprehensive understanding of adversity-overcoming pup care, it may be beneficial to compare the results of experiments using different modalities of risks, such as water, bright light, or height, with different risk levels, such as tolerable versus barely tolerable water depth. We observed that the “trapped pup” rescue activated the preoptic area (aACN), limbic brain regions (A24, LS, BLA), and several hindbrain regions (LPB, DRC) that also showed strong relations with some particular rescue behaviors, such as the tube opening (Figs. 3 c, e). These brain regions may have been activated in response to pup cues and associated reward 18 , 27 , 32 , 33 or as a reaction to an adverse and sensory-heavy environment 26 , 34 . The major limitation of our study is that we did not show functional importance of these regions for the pup rescue behavior. Thus, detailed behavioral manipulation studies on identified regions could yield valuable insights into the neural mechanism of the adversity-overcoming rescue. The brain activity mapping in Fig. 3 was performed with virgin females, which do not have genetic relations to the rescuee pups. Therefore, these findings are also relevant for altruistic behavior and its neural correlates. Recent studies have identified a wide network of brain areas involved in prosocial behaviors, including helping, rescue, and harm avoidance behaviors toward adult and adolescent conspecifics in rats and mice 5 , 8 – 10 , 25 , 28 , 29 , 35 , 36 . Our findings partially aligned with the results of these studies, as we showed that A24, LS, BLA, and to a lesser extent AcbSh were activated during pup rescue, and we specifically identified brain areas responding to cost-bearing rescue. We were unable to confirm increased activity in the medial amygdala (MeA) and PVN in our behavioral paradigm, which may have been due to the differences in stimulus animals (pup versus adult) and their consciousness level, as well as related to environmental adversity and experienced stress. Future studies examining the neural mechanism of cost-bearing rescue of adults and pups of different ages are awaited and would greatly contribute to the existing mechanistic understanding of the neural basis of altruism. Materials and methods Animals. All mouse experiments were approved by the Animal Care and Use Committee of the Institute of Science Tokyo and were thus in accordance with NIH guidelines (NIH Publications No. 8023, revised 1985), no procedures required euthanasia. This study followed the ARRIVE Guidelines 2.0. 37 Animals were maintained under a 12:12 h light/dark cycle (7:00 lights on, 19:00 lights off) with food and water ad libitum . C57BL/6N mice were originally obtained from Japan SLC or Jackson Laboratory and raised in our breeding colony. Pups were weaned at 4 weeks of age and housed with their littermates and/or mice of the same age in groups up to five. 2- to 10-months-old female mice were used for experiments. Adversity-overcoming pup rescue. A pregnant wild-type female mouse (gestational age 7-14) was housed together with two virgin females in an experimental cage (44 × 24 × 15 cm, Fig. 1a ) until delivery. The experimental cage included a living area, where a mouse nest was located, and an experimental area that was separated from the living area with a water pool and a sham fence to force mice to enter the pool from the center. Virgin females underwent a pup retrieval test in advance to confirm their nurturing behavior. Experiments, consisting of combined habituation and pup rescue tests, started from the postpartum day 2 (PPD2) using the pups of the age of postnatal day 2 (PND2) and were conducted between 9:30–13:00. Experiments were conducted on the postpartum week 0 (PPD2-5), postpartum week 2 (PPD10-17), and postpartum week 4 (PPD28-32). However, the results of pup rescue tests on the postpartum weeks 2 and 4 will be reported elsewhere due to substantial differences in experimental design. We created three levels of adversity: 1) 0 mm, 2) 3 mm, and 3) 20 mm water depth in the pool, based on the results of a previous study 21 . We used room temperature water (temperature range between +21.0°C and +23°C). The habituation and pup rescue experiments were structured in the following way. One subject (a mother or a virgin) was left in the experimental cage, while other animals, including pups, were temporarily transferred to a clean husbandry cage. The subject was habituated to the empty cage with no water in the pool for 5 min. After that, 3 healthy pups were placed behind the pool in the experimental area and the subject’s behavior was observed for 30 min. Next, the subject was habituated to the empty cage with 3mm-deep water in the pool 21 and then 3 healthy pups were placed behind the pool in a similar way to observe subject’s behavior for 30 min. Finally, the same experiments were conducted with the 20mm-deep water in the pool. Upon the end of these experiments, all animals were reunited in the experimental cage. Next day, another subject underwent the experiments. All experiments were recorded on videos and analyzed manually and automatically using DeepLabCut 22 . Manual analysis was performed by several raters with exceptional inter-rater reliability (the absolute‑agreement, two‑way random‑effects Intraclass Correlation Coefficient (ICC) for k = 4 rater ICC(A,4) = 0.996 (95% CI 0.995–0.997; F(85, 257) = 262, p < 1 × 10⁻²¹⁰)). Adversity-overcoming “trapped pup” rescue for c-Fos sampling. To examine c-Fos activity during the pup rescue test, only virgin female mice were used. First, we trained subjects to retrieve pups as described above at each water depth for one day (max three tests per day). If the subject failed to retrieve pups at a particular water depth, we started from this water depth the next day and repeated the tasks for one more day. Next, we conducted similar experiments but placed pups in two 50 ml falcon tubes (2 pups/tube) closed with paper lids (“trapped pup”) to make pup rescue comparable with the one of adult conspecifics or older pups 1,2,28,29 and repeated these tests for up to two days. After that, each subject was isolated in the husbandry cage overnight and then underwent the “trapped pup” rescue test at the 3mm water depth in the pool. Mice were labeled as “rescuers” (= opened both tubes within 30 min) and “non-rescuers” (= did not open the tubes within 30 min), while subjects with intermediate performance were excluded from future studies. Two hours from the start of the test, mice were perfused, and brains were sampled. Brain tissue processing. Mice that underwent “trapped pup” rescue experiment were deeply anesthetized with 8% (v/v) Midazolam (Sandoz Group AG, Switzerland), 10% (v/v) Vetorphale (Meiji Animal Health Co., Ltd., Japan), 7.5% (v/v) Domitor (Nippon Zenyaku Kogyo Co., Ltd., Japan) in saline, then perfused transcardially with 4% (w/v) paraformaldehyde (PFA) in 1x phosphate buffered saline (PBS, pH 7.4). The brains were removed, immersed in the same fixative at 4°C overnight, followed by cryoprotection in the series of 20% and 30% (w/v) sucrose in PBS for two days, embedded in O.C.T. Compound (Sakura Finetek Japan, Tokyo, Japan), and stored at -80°C until cryosectioning. Brains were cryosectioned coronally at a thickness of 40 mm according to the mouse brain atlas 38 . Every third section from the serial sections was used for immunohistochemistry. Immunohistochemistry (IHC). Immunohistochemical detection of c-Fos on free-floating sections was performed as described previously 24 . The sections were washed with PBS containing 0.2% Triton-100 (PBST), incubated with 0.3% H 2 O 2 in methanol for 5 minutes, washed with PBST, blocked with 0.8% Block Ace (Dainihon-Seiyaku, Osaka, Japan) in PBST, and incubated at 4°C overnight with rabbit primary antibody against c-Fos (1:5000, Cat# sc-52, RRID: AB_2106783, Santa Cruz Biotechnology, Inc., Dallas, TX, USA). The following morning, the sections were washed and incubated with biotin-conjugated horse anti-rabbit secondary antibody (1:2000, Cat# BA-1100, RRID: AB_2336201, Vector Laboratories, Inc., Burlingame, CA, USA) for 2 hours and then in ABC peroxidase reagent (Cat# PK-6100; Vectastain ABC Elite kit; Vector Laboratories) for 1 hour according to the manufacturer’s instructions. The labeling was visualized by incubation in 3,30’–diaminobenzidine (DAB) solution with nickel intensification (DAB peroxidase substrate kit Cat#SK-4100, Vector Laboratories) for 5 minutes. Finally, Nissl staining using cresyl violet was performed. Histological analysis. Obtaining brightfield photomicrophotographs, image processing, and calculation of c-Fos + cell density were performed as described elsewhere 16,24,39 . Brain regions abbreviations are based on Paxinos G. and Franklin K. B. J. mouse brain atlas 38 with additional specifications of anterior (a) and posterior (p) parts. Due to ~60% success rate of the “trapped pup” rescue in virgin females, we counted c-Fos in both left and right hemispheres of “rescuer” and “non-rescuer” group brains (technical replicates) to increase the number of samples and reliability of results. Statistical analysis. All statistical analyses were conducted using R v. 4.4.0 (R Development Core Team, 2024; https://www.r-project.org/). No statistical methods were used to predetermine sample sizes. For analyses of the latencies to the first pool entry and pool crossing ( Figs. 1c-d ), the numbers of pool entries and pool crossings ( Figs. 1f-g ), and the time spent in the pool (Fig. i ) during the 5-min habituation test, as well as during the pup rescue test ( Figs. 2d-f ); for the analysis of multiple pup nurturing behaviors ( Fig. 2c ); for the analysis of latency to each pup nurturing behavior ( Figs. 2g-l ), the generalized linear mixed model (GLMM) from glmmTMB package (v1.1.10) was used 40 . For analysis of correlation between latencies to pool entry and pool crossing, as well as between numbers of pool entry and pool crossing, Pearson correlation and Intraclass Correlation Coefficient (ICC) from the irr package (v0.84.1; https://cran.r-project.org/web/packages/irr/index.html) were used. For the analysis of density c-Fos-positive neurons ( Fig. 3c ), Welch`s t-test was used. Normality was assessed using the Kolmogorov–Smirnov test. For the analysis of correlation between the latencies to rescue behaviors and density of c-Fos-positive neurons ( Fig. 3e ), tydiverse (v2.0.0) 41 , ggdendro (v0.2.0; https://cran.r-project.org/web/packages/ggdendro/index.html), and corr (v0.4.4; https://cran.r-project.org/web/packages/corrr/index.html) packages were used. Detailed information about statistical tests is provided in corresponding figure legend. Error bars represent mean ± standard error of mean (S.E.M.). Data from censored observations were replaced by the maximum observation time (1800 s (=30 min) for all mice). The sample size is the same as the number of animals (biological replicate) in all cases, except for the c-Fos + cell density data where technical replicates (left and right hemispheres) were utilized together with biological replicates. Declarations Author contributions statement K.P. and K.O.K. designed research with help from M.S. and R.K; K.P., M.S., and R.K. performed the experiments with help from C.Y. and K.O.K.; K.P., M.S., R.K., and K.O.K analyzed data; K.P., M.S., and R.K. wrote the first draft of the paper; all authors reviewed and edited the paper; K.O.K. conceived the research; C.Y. and K.O.K. contributed unpublished reagents/analytic tools. Additional Information Authors declare no competing interests. Acknowledgements We thank Eri Miyazawa, Gulimire Yilihan, and Haruto Ijiri for technical assistance, Dr. Hiroshi Ueno for fruitful discussion, the Center for Integrative Biosciences of Institute of Science Tokyo for animal husbandry. Funding Declaration This work was supported by Japan Society for the Promotion of Science KAKENHI Grant-in-Aid for Scientific Research (B) (JP23K23927), the Bioscience Research Grant of Takeda Science Foundation to K.O.K, Japan Society for the Promotion of Science KAKENHI Grant-in-Aid for Early-Career Scientists (JP25K18581) to K. P., Japan Society for the Promotion of Science KAKENHI Grant-in-Aid for Scientific Research (C) (JP22K12236) to C.Y., Mitsubishi Electric Software Scholarship to M. S. Data Availability The datasets used and/or analyzed during the current study are available from the corresponding authors on reasonable request. 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Chapter 25 Assessing Postpartum Maternal Care, Alloparental Behavior, and Infanticide in Mice: With Notes on Chemosensory Infl uences. Methods in Molecular Biology 1068 , 331–47 (2013). Numan, M. The Parental Brain: Mechanisms, Development, and Evolution. The Parental Brain 499 (2020). Yoshihara, C. et al. Calcitonin receptor signaling in the medial preoptic area enables risk-taking maternal care. Cell Rep 35 , 109204 (2021). Yang, Y. et al. Behavioral and pharmacological investigation of anxiety and maternal responsiveness of postpartum female rats in a pup elevated plus maze. Behavioural Brain Research 292 , 414–427 (2015). Kuroda, K. O. et al. Parental brain through time: The origin and development of the neural circuit of mammalian parenting. Annals of the New York Academy of Sciences vol. 1534 24–44 Preprint at https://doi.org/10.1111/nyas.15111 (2024). Ellenbroek, B. & Youn, J. Rodent models in neuroscience research: Is it a rat race? DMM Disease Models and Mechanisms 9 , 1079–1087 (2016). Wahlsten, D. Mouse Behavioral Testing: How to Use Mice in Behavioral Neuroscience . (Academic Press, 2010). Ueno, H. et al. Mice can recognise water depths and will avoid entering deep water. Transl Neurosci 13 , 1–10 (2022). Mathis, A. et al. DeepLabCut: markerless pose estimation of user-defined body parts with deep learning. Nature Neuroscience 2018 21:9 21 , 1281–1289 (2018). Kuroda, K. O., Tachikawa, K., Yoshida, S., Tsuneoka, Y. & Numan, M. Neuromolecular basis of parental behavior in laboratory mice and rats: with special emphasis on technical issues of using mouse genetics. Prog Neuropsychopharmacol Biol Psychiatry 35 , 1205–1231 (2011). Tsuneoka, Y. et al. Functional, anatomical, and neurochemical differentiation of medial preoptic area subregions in relation to maternal behavior in the mouse. J Comp Neurol 521 , 1633–1663 (2013). De Waal, F. B. M. & Preston, S. D. Mammalian empathy: behavioural manifestations and neural basis. Nat Rev Neurosci 18 , 498–509 (2017). Malezieux, M., Klein, A. S. & Gogolla, N. Neural Circuits for Emotion. Annu Rev Neurosci 46 , 211–231 (2023). Corona, A., Choe, J., Muñoz-Castañeda, R., Osten, P. & Shea, S. D. A circuit from the locus coeruleus to the anterior cingulate cortex modulates offspring interactions in mice. Cell Rep 42 , 112771 (2023). Bartal, I. B. A. et al. Neural correlates of ingroup bias for prosociality in rats. Elife 10 , (2021). Breton, J. M. et al. Neural activation associated with outgroup helping in adolescent rats. iScience 25 , (2022). Stolzenberg, D. S. & Rissman, E. F. Oestrogen-Independent, Experience-Induced Maternal Behaviour in Female Mice. J Neuroendocrinol 23 , 345–354 (2011). Erskine, M. S., Barfield, R. J. & Goldman, B. D. Postpartum aggression in rats: II. Dependence on maternal sensitivity to young and effects of experience with pregnancy and parturition. 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Reporting animal research: Explanation and elaboration for the ARRIVE guidelines 2.0. PLoS Biol 18 , e3000411 (2020). Franklin, K. B. J. & Paxinos, G. The Mouse Brain in Stereotaxic Coordinates . (Academic Press, 2019). Tsuneoka, Y. et al. Distinct preoptic‐ BST nuclei dissociate paternal and infanticidal behavior in mice . EMBO J 34 , 2652–2670 (2015). Brooks, M. E. et al. glmmTMB balances speed and flexibility among packages for zero-inflated generalized linear mixed modeling. R Journal 9 , 378–400 (2017). Wickham, H. et al. Welcome to the Tidyverse. J Open Source Softw 4 , 1686 (2019). Additional Declarations No competing interests reported. Supplementary Files SupplementaryTables12.xlsx Cite Share Download PDF Status: Published Journal Publication published 03 Mar, 2026 Read the published version in Scientific Reports → Version 1 posted Editorial decision: Revision requested 06 Oct, 2025 Reviews received at journal 28 Sep, 2025 Reviews received at journal 24 Sep, 2025 Reviewers agreed at journal 17 Sep, 2025 Reviewers agreed at journal 14 Sep, 2025 Reviewers invited by journal 06 Sep, 2025 Editor assigned by journal 01 Sep, 2025 Submission checks completed at journal 13 Aug, 2025 First submitted to journal 13 Aug, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-7240012","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":511259326,"identity":"78c05fab-3fdb-4705-ac61-565adef9a366","order_by":0,"name":"Kseniia Prokofeva","email":"data:image/png;base64,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","orcid":"","institution":"Institute of Science Tokyo","correspondingAuthor":true,"prefix":"","firstName":"Kseniia","middleName":"","lastName":"Prokofeva","suffix":""},{"id":511259327,"identity":"29b6cae2-3b53-489d-a202-c96cc3ed9535","order_by":1,"name":"Mizuki Shibamiya","email":"","orcid":"","institution":"Institute of Science Tokyo","correspondingAuthor":false,"prefix":"","firstName":"Mizuki","middleName":"","lastName":"Shibamiya","suffix":""},{"id":511259328,"identity":"586c8a3d-704d-4b8d-b140-1085ba9fa3e0","order_by":2,"name":"Rin Kawata","email":"","orcid":"","institution":"Institute of Science Tokyo","correspondingAuthor":false,"prefix":"","firstName":"Rin","middleName":"","lastName":"Kawata","suffix":""},{"id":511259329,"identity":"4e30ccf8-6db5-40ae-8218-ed7628ddfd86","order_by":3,"name":"Chihiro Yoshihara","email":"","orcid":"","institution":"Institute of Science Tokyo","correspondingAuthor":false,"prefix":"","firstName":"Chihiro","middleName":"","lastName":"Yoshihara","suffix":""},{"id":511259330,"identity":"c339e480-12b2-47a4-ad5e-e2a93ed2e711","order_by":4,"name":"Kumi O. Kuroda","email":"","orcid":"","institution":"Institute of Science Tokyo","correspondingAuthor":false,"prefix":"","firstName":"Kumi","middleName":"O.","lastName":"Kuroda","suffix":""}],"badges":[],"createdAt":"2025-07-29 07:08:17","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-7240012/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-7240012/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1038/s41598-026-35639-7","type":"published","date":"2026-03-03T15:57:32+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":91195698,"identity":"a4ed6d69-8c5e-4830-9a88-87386eb5d6ed","added_by":"auto","created_at":"2025-09-12 14:58:52","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":319155,"visible":true,"origin":"","legend":"\u003cp\u003eExamination of aversity to water in female virgins and mothers and establishment of automatic video analysis.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ea\u003c/strong\u003e, Scheme of the habituation test and water depth levels.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eb\u003c/strong\u003e, Visualization of the pool entry and pool crossing parameters (left panels) and DeepLabCut (DLC) model (right panel).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ec\u003c/strong\u003e, Latency to the first pool entry in seconds during 5 min habituation test (generalized linear mixed model (GLMM), latency ~ mouse type + water depth + week, gamma distribution). Water depth and pup age independent variables were set as ordinal in this and following GLMM models.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ed\u003c/strong\u003e, Latency to the first pool crossing in seconds during 5 min habituation test (GLMM, latency ~ mouse type + water depth + week, gamma distribution).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ee\u003c/strong\u003e, Linear regression plot between latencies to the first pool entry and the first pool crossing (Pearson correlation, r = 0.955, p \u0026lt;0.001; ICC(A,2) = 0.955 (95% CI 0.939 – 0.967; F(170,171) = 22.3, p \u0026lt; 0.001)).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ef\u003c/strong\u003e, Number of pool entries during habituation test (GLMM, latency ~ mouse type + water depth + week, negative binomial distribution). Zero enrichment was added to the models with number count data due to excess of zeroes.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eg\u003c/strong\u003e, Number of pool crossings during habituation test (GLMM, latency ~ mouse type + water depth + week, negative binomial distribution). The number of pool crossings in virgin females demonstrated a non-linear relationship with water depth (GLMM, water depth (quadratic) = 0.4046, p = 0.027), suggesting that the rate of pool crossing decrease slows down after 3mm depth.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eh\u003c/strong\u003e, Linear regression plot between numbers of pool entries and pool crossings (Pearson correlation, r = 0.79, p \u0026lt;0.001; ICC(A,2) = 0.758 (95% CI 0.653– 0.828; F (170,92.9)= 4.41, p \u0026lt; 0.001)).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ei\u003c/strong\u003e, Time spent in the water pool during habituation test (GLMM, latency ~ mouse type + water depth + week, tweedie distribution). Data are shown as mean ± S.E.M. Virgin mice, n = 10 (week 0) or n = 9 (weeks 2 and 4). Mother mice, n = 10 (week 0) or n = 9 (weeks 2 and 4).\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-7240012/v1/b798cce81764db84ff9bb65d.png"},{"id":91195696,"identity":"a3887a00-102b-493f-b602-59de3818013c","added_by":"auto","created_at":"2025-09-12 14:58:52","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":221452,"visible":true,"origin":"","legend":"\u003cp\u003eAnalysis of pup care behaviors in adversity-overcoming rescue paradigm in female virgins and mothers.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ea\u003c/strong\u003e, Scheme of the pup rescue test.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eb\u003c/strong\u003e, Visual representation of measured pup-directed behaviors: sniffing, carrying, retrieving of pups 1-3, pup grouping, and full parenting.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ec\u003c/strong\u003e, Stacked bar graph displaying ratio of mice that performed or did not perform pup-directed behaviors depending on mouse type (virgins, mother) and water depth (0, 3, or 20 mm). For analysis of pup-directed behaviors, first, each analyzed behavior was changed to numbers according to its rank (e.g., 7 – full parenting, 0 – no behavior). Next, for each individual mouse, the highest achieved behavior was identified, and it was changed to the corresponding number. These numbers were then compared. The distribution was set to “binomial” (due to presence of ceiling value of 7), and water depth independent variable were set as ordinal. Adjustment for multiple comparisons was done using Holm method.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ed\u003c/strong\u003e, Latency to the first pool crossing in seconds during 30 min (1 800 s) pup rescue test (GLMM, latency ~ mouse type * water depth, gamma distribution, followed by Holm multiple comparisons).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ee\u003c/strong\u003e, Number of pool crossings in seconds during pup rescue test (GLMM, latency ~ mouse type * water depth, negative binomial distribution, followed by Holm multiple comparisons).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ef\u003c/strong\u003e, Time spent in the water pool during pup rescue test (GLMM, latency ~ mouse type * water depth, tweedie distribution, followed by Holm multiple comparisons).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eg-m\u003c/strong\u003e, Latencies to the pup sniffing (\u003cstrong\u003eg\u003c/strong\u003e), pup carrying (\u003cstrong\u003eh\u003c/strong\u003e), first pup retrieval (\u003cstrong\u003ei\u003c/strong\u003e), second pup retrieval (\u003cstrong\u003ej\u003c/strong\u003e), third pup retrieval (\u003cstrong\u003ek\u003c/strong\u003e), pup grouping (\u003cstrong\u003el; \u003c/strong\u003eGLMM, latency ~ mouse type * water depth, gamma distribution, followed by Holm multiple comparisons), and full parenting (\u003cstrong\u003em\u003c/strong\u003e) in seconds during pup rescue test. For multiple comparisons between mouse groups at the same water depth levels, Holm adjustment method was used. Due to rarity of the full parenting behavior in \u003cstrong\u003eFig 2m\u003c/strong\u003e, statistical analysis was not applied to this behavior. Data are shown as mean ± S.E.M. Virgin mice, n = 10. Mother mice, n = 10.\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-7240012/v1/baf0f87a993773a3038f66ff.png"},{"id":91196827,"identity":"0a09919d-3fd4-429c-b993-fb92b8a00c52","added_by":"auto","created_at":"2025-09-12 15:06:52","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":1171954,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ee\u003c/strong\u003e, A heatmap demonstrating Spearman correlation results between c-Fos\u003csup\u003e+\u003c/sup\u003e cell densities in various brain regions and latencies to measured behaviors in the “trapped pup” rescue paradigm. Each region’s individual value was divided by the region’s average value for standardization. The pairwise Euclidean distances were calculated between latencies to each behavior and between standardized c-Fos\u003csup\u003e+\u003c/sup\u003e cell densities in each brain region. The computed distance matrix was then used in the hierarchical clustering function (hclust) using Ward.D2 method for latencies and densities separately. In each step of clustering, the two clusters that lead to the smallest increase in the overall variance were merged. The resulting hierarchical clustering objects were then converted into dendrograms (as.dendrogram). Finally, the latency and density data were combined as a heatmap based on the results of Spearman correlation between each behavior latency and c-Fos+ cell density of each region. Behaviors that were displayed after the second tube opening were cut off due to their rarity and growing unreliability of the correlation results. Panels below the heatmap mark brain regions that have significant correlation coefficients with different behavioral clusters where the blue color corresponds to negative correlation and red corresponds to positive correlation. Spearman correlation coefficients, unadjusted and adjusted p-values are reported in Supplementary Table 2.\u003c/p\u003e\n\u003cp\u003eBrain regions activated during adversity-overcoming “trapped pup” rescue in female virgins.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ea, \u003c/strong\u003eScheme of the test followed by brain sampling for c-Fos examination\u003cstrong\u003e.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eb, \u003c/strong\u003eBehavior achievement during training and “trapped pup” rescue on each day by each mouse. Tm2, opening both tubes; Tm1, opening the first tube only.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ec\u003c/strong\u003e, Density of c-Fos-positive cells in rescuers and non-rescuers in each examined brain region (independent Welch’s t-test for each brain region). Adjusted p-values are reported in the Supplementary Table 1. Data are shown as mean ± S.E.M. Rescuer, n = 5-8 (including technical replicates of the left and right brain hemispheres), non-rescuer, n = 4-6. We obtained four brain samples from the first group and three samples from the second group.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ed\u003c/strong\u003e, Representative images of brain regions with significantly higher c-Fos density in rescuers compared with non-rescuers. Scale bar: 500 μm.\u003c/p\u003e","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-7240012/v1/ca558d7d106e5fa1f4b63165.png"},{"id":104250614,"identity":"b1b33bf9-d00f-48d9-a1e7-68e0d75c5466","added_by":"auto","created_at":"2026-03-09 16:02:00","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2527586,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7240012/v1/a54cbcfa-8e11-4fdf-b5a2-0eb4380eb909.pdf"},{"id":91198372,"identity":"f40bbd98-03c7-4a02-a5e3-34f1c3f137ed","added_by":"auto","created_at":"2025-09-12 15:14:52","extension":"xlsx","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":31641,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryTables12.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-7240012/v1/6f24fd8b611d2ff9ba9c723d.xlsx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Neural correlates of adversity-overcoming pup rescue behavior in female mice","fulltext":[{"header":"Introduction","content":"\u003cp\u003eTargeted helping-like behaviors toward conspecific adults expressed as freeing them from restraint or adverse environments or as allogrooming stressed conspecifics have been observed in several rodent species under laboratory conditions \u003csup\u003e\u003cspan additionalcitationids=\"CR2 CR3 CR4 CR5 CR6\" citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e. Recent studies have also reported that mice engage in resuscitation-like behaviors toward unresponsive conspecifics via methodical interactions with the tongue and head of the rescuees \u003csup\u003e\u003cspan additionalcitationids=\"CR9\" citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e. However, the prosocial nature of these behaviors has not been confirmed, as they can be performed out of selfish intentions, such as the avoidance of being alarmed or the risk assessment for one's own survival \u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e,\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e. Moreover, the aforementioned rodent behaviors do not constitute evidence of true altruism, which has been defined as the behavior that benefits the receiver at a cost (loss) to the performer \u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e, because known helping behaviors in rodents often do not impose a significant cost to the rescuers.\u003c/p\u003e\u003cp\u003eInfant care is a paramount mammalian behavior that facilitates the survival of the young, and it requires substantial cost from the parents \u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e,\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e. In addition to the energy cost of lactation, the protection and transportation of infants can be associated with substantial mortality risk for mammalian parents. In laboratory mice, pup retrieval, which is an act of carrying a displaced young back into the nest, is widely used as a readout of infant care motivation, since it can be robustly and unambiguously performed by both parental and virgin female mice \u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e. Ours and other groups have previously shown that mother mice and rats, respectively, retrieve (rescue) pups placed at the end of an open elevated platform, while virgin females scarcely do so \u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e,\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e. This risk-taking pup rescue required specific gene expression in the anterior commissural nucleus (ACN) and the caudocentral part (cMPOA) of the medial preoptic area (MPOA) in mice, which is indispensable for infant care across mammalian species \u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e,\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e. Moreover, to overcome environmental risks during pup rescue, the caregiving mice may require not only the motivation to care but also precise risk assessment and cognitive/behavioral skills to cope with the specific risk. To elucidate the neural circuit mechanism required for such an adversity-overcoming pup rescue, it is preferable to create a behavioral paradigm in which the environmental risk is scalable. However, in our previous pup-rescue paradigm on the elevated plus maze, it was not easy to modulate the width or height of the platform.\u003c/p\u003e\u003cp\u003eIn this study, we established an adversity-overcoming pup rescue task that involves crossing a water pool of variable depths to retrieve (rescue) pups, utilizing the previous findings that mice are averse to entering water \u003csup\u003e\u003cspan additionalcitationids=\"CR20\" citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e. We also identified the brain areas that are activated during this behavioral task, using immunohistochemistry and c-Fos analysis. As we utilized female virgin mice that are not kin to the rescuee pups, our histological study sheds light not only on the neural mechanism of costly pup rescue, but also on that of the altruistic helping-like behavior.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cb\u003eMice perceive increasing water depth levels as scalable adversity.\u003c/b\u003e\u003c/p\u003e\u003cp\u003eWe have implemented the adversity-overcoming pup rescue paradigm using mothers and pup care-experienced virgin females that have been cohoused from the gestational day 9 of the mother \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea). To examine if repetition of the test (for virgins) and the postpartum period (for mothers) influence mouse behavior, we conducted the habituation three times across one month (corresponding to pup age of postnatal weeks 0, 2, and 4). Before undergoing each pup rescue task described in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e, we first exposed each subject mouse to a water pool with increasing water depths (0, 3, 20mm) for 5 min (habituation) and observed their exploration of the pool area \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e) to examine the water aversion of virgin females and lactating mothers.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eFirst, we manually measured the latency to the first pool entry and the number of pool entries (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea; Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eb, \u003cb\u003eleft upper panel\u003c/b\u003e). We found that, as the water depth increased, both the mother and the virgin females required a significantly longer time to enter the pool and entered the pool significantly less (Figs.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ec, f). Notably, virgin mice entered the pool significantly faster and significantly more often than mother mice (Figs.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ec, f), suggesting that water is recognized by mothers as stronger adversity. Interestingly, both latency and the number of pool entries did not significantly vary across time, implying that the task\u0026rsquo;s repetition or the postpartum weeks did not grossly affect the perceived adversity. Altogether, we confirmed that mice are increasingly averse to water upon the rise in water depth, while maternity status exacerbates the evaluation of adversity.\u003c/p\u003e\u003cp\u003eTo optimize the analyses of behavioral parameters, we implemented a machine learning-based animal motion tracking program DeepLabCut (DLC)\u003csup\u003e\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e to measure the latency to pool crossing and the number of pool crossings, which roughly correspond to pool entry parameters (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eb, \u003cb\u003eleft center and bottom panels\u003c/b\u003e), utilizing the middle point of mouse body to analyze its locomotion (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eb, \u003cb\u003eright panel;\u003c/b\u003e back_1). We found that both the latency to pool crossing and the pool crossing number followed a similar trend as the pool entry parameters (Figs.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ed, g).\u003c/p\u003e\u003cp\u003eThe automated measures of the latencies to pool entry and the number of pool crossings were significantly correlated with the manual measures (Figs.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ee, h), suggesting that manual and DLC analyses are essentially interchangeable. We additionally conducted intraclass correlation (ICC) analyses between the manual and DLC methods and found that they yielded high similarity. Using DLC, we also analyzed time spent in the water pool, which aligned with other parameters and confirmed mouse aversity to water (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ei). We therefore established DLC-mediated automatic analysis for our behavioral paradigm.\u003c/p\u003e\u003cp\u003e\u003cb\u003eFemale virgins overcome adversity to rescue pups.\u003c/b\u003e\u003c/p\u003e\u003cp\u003eNext, we examined the adversity-overcoming rescue behavior toward displaced pups performed on postpartum days 2\u0026ndash;5 by mothers or during postnatal week 0 by virgin females (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea). When mouse pups are placed outside of the nest, female mice quickly recognize them by sniffing, and generally engage in a sequence of behaviors, starting from orally carrying the pup, placing the pup into the nest (retrieving), gathering pups within the nest (grouping), and crouching over pups (designated as full parenting\u0026thinsp;=\u0026thinsp;crouching over all pups continuously for over a minute), as reported before (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb) \u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e,\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e,\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e. Therefore, we examined these behaviors toward three pups of the postnatal age 2\u0026ndash;5 days placed behind the water pool (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ec). Additionally, we examined the latencies and numbers of pool crossing as in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ed-f).\u003c/p\u003e\u003cp\u003eIn Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ec, the pup-directed behaviors described in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb were assigned ranks based on their achievement (e.g., no pup-directed behavior \u0026ndash; 0 (lowest rank), pup sniffing \u0026ndash; 1, and full parenting, which is only possible if all other behaviors were performed, \u0026ndash; 7 (highest rank)). We found that a significant proportion of female virgins and mothers were able to retrieve and nurture pups at any adversity level. However, there are two notable differences between the groups. First, with increasing adversity, virgins unexpectedly displayed better pup-retrieval behaviors than mothers (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ec), probably due to the higher water aversion of mothers. Second, some of the virgins displayed no pup retrieval only in the 0mm condition (the very first task), plausibly due to their inexperience (see below), causing the non-linear correlation of the water depth and the behavioral performance. Therefore, we concluded that female virgins were more likely to overcome adversity and display nurturing behaviors than mothers.\u003c/p\u003e\u003cp\u003eWe next examined the level of water aversion in the context of pup rescue using latency to and the number of pool crossings, as well as time spent in the pool, measured using DLC. The GLMM analysis revealed a negative correlation between the latency to the pool crossing and the water level increase, however, mothers took significantly longer time to cross the pool with water depth increase, compared with virgins (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ed). The seemingly contradictory prior result can be explained by the virgins' delay of almost all behaviors due to inexperience in the very first trial (0mm), causing the long latency to enter the pool area even though there was no water in it. The virgins entered the pool and engaged in pup retrieval much faster in the 3mm trial than in the previous trial \u003cb\u003e(\u003c/b\u003eFigs.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ed, g-l). This suggests that virgins require an acclimatization period to perform the pup retrieval task even though they have cohabitated with pups, while mothers are efficient from the first trial. For water aversion, virgins appeared more resistant than mothers as in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. The number of pool crossings decreased with the water depth increase in both mouse groups, similarly to the habituation period (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eg), suggesting that water is still perceived as an adversity (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ee). Time spent in the water pool decreased with higher water depth more in mothers, confirming their high aversity (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ef).\u003c/p\u003e\u003cp\u003eWe next identified the latencies to each pup-directed behavior to examine motivation more precisely (Figs.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eg-m). We found that virgins sniffed pups upon crossing the water pool significantly faster than mothers (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eg), while the latencies to both sniffing and first pup carrying were increased in both mouse groups with an increase in water depth (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eg, \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eh). Additionally, similarly to our earlier observations, latencies to pup sniffing, carrying, and retrieving the first two pups lengthened with the increase of adversity in mothers more (Figs.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eg-j). Similarly to previous findings (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ec), the latencies to retrieve the second and third pup were stabilized upon the addition of water to the pool in all mice (Figs.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ej, k), however, this effect was not as present in mothers as in virgins (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ek). We did not observe any differences in grouping behavior and did not conduct statistics on full parenting due to its rarity (Figs.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003el, \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003em). Therefore, we concluded that virgins are more resilient to water adversity at any depth in pup presence and rescue pups more efficiently, than mothers.\u003c/p\u003e\u003cp\u003e\u003cb\u003eCostly pup rescue increases activity in empathy-, infant care-, and adversity-associated brain areas.\u003c/b\u003e\u003c/p\u003e\u003cp\u003eTo identify brain regions implicated in adversity-overcoming pup rescue, we examined the expression of the cellular activity marker c-Fos across the brain during this behavior. For this purpose, we made two minor changes to the experimental protocol. First, we restricted the spontaneous locomotion of the stimulus pups by containing them in a plastic tube (two pups per tube) with a paper lid. Second, before the final pup exposure test, we trained the subject females for a period of up to four days, so that the subjects could successfully rescue pups even under adverse conditions and when pups were contained in a plastic tube (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea). In the first phase of training, the subjects were directly exposed to three pups (i.e., without being contained in a tube as in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e) placed on the opposite side of the pool with increasing water depth levels, and the test was repeated at each water depth level until the subjects succeeded or until the maximum length of the training period had been reached. In the second phase of training (tube opening), we placed two 50-ml tubes each containing two pups behind the pool and repeated the test in a similar manner, while we counted rescue success as tube opening. After the training, the subjects were isolated for one day and underwent the final experiment (\u0026ldquo;trapped pup\u0026rdquo; rescue) similarly to the last phase of training, but only at 3mm water depth. Mice that opened both tubes within the first 30 minutes were designated as \u0026ldquo;rescuers\u0026rdquo;. Out of four \u0026ldquo;rescuer\u0026rdquo; animals, most of them (3/4, 75%) opened the first tube and retrieved at least one pup before opening the second tube, while one (25%) first opened both tubes and then retrieved the pups. However, none of the animals opened the tubes without rescuing the pups. These results suggest that mice exhibited behaviors in an organized manner with the aim of pup rescue, rather than out of interest in tubes. Mice that did not open any of the tubes during the task displayed a range of behaviors, from complete lack of interest in the pool and pup-containing tubes to active pool crossing and tube sniffing without biting. Thus, these mice were designated as \u0026ldquo;non-rescuers\u0026rdquo; (control group), while mice with intermediate performance were excluded from the c-Fos analysis. Following this behavioral experiment, mice were subjected to brain sampling (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eb).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eWe next compared c-Fos expression across the brain between the rescuers and non-rescuers (Figs.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ec, d). We selected the target brain regions involved in pup nurturing, emotion processing, and empathic response \u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e,\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e,\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e,\u003cspan additionalcitationids=\"CR24 CR25 CR26 CR27 CR28\" citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e. The \u0026ldquo;rescuer\u0026rdquo; group exhibited a significantly higher density of c-Fos-positive cells in the anterior part of the anterior commissural nucleus of the preoptic area (aACN), which has already been shown to be activated most after parental nurturing behaviors\u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e. Three limbic regions, that are the area 24 of the anterior cingulate cortex (A24), lateral septum (LS), and basolateral amygdala (BLA), were also activated in rescuers. Finally, several hindbrain regions, such as the caudal part of the dorsal raphe nucleus (DRC) and the lateral parabrachial nucleus (LPB), displayed higher c-Fos cell density in rescuers compared with non-rescuers. The shell of the nucleus accumbens (AcbSh) and medial parabrachial nucleus (MPB) showed tendencies (0.05\u0026thinsp;\u0026lt;\u0026thinsp;p\u0026thinsp;\u0026lt;\u0026thinsp;0.1) toward an increase of the density of c-Fos-positive cells in the rescuer mice. The locus coeruleus (LC) and paraventricular nucleus (PVN), which are known to be activated by stress, exhibited relatively high c-Fos densities in both groups. No brain region in the Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ec showed higher activity in the non-rescuer group.\u003c/p\u003e\u003cp\u003e\u003cb\u003eIdentification of neural correlates of adversity-overcoming pup rescue.\u003c/b\u003e\u003c/p\u003e\u003cp\u003eTo better describe the link between the behavior and rescue-biased brain regions, we next analyzed correlations between the latencies to the major components of the rescue behavior, such as crossing the pool, biting each tube, and retrieving each pup, and the within-region standardized densities of c-Fos-positive cells in the analyzed brain regions (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ee). The behaviors and activation patterns of the brain regions were each sorted using unsupervised hierarchical clustering where the clusters were formed based on similarity between items.\u003c/p\u003e\u003cp\u003eThe behavior clustering aligned with the chronological sequence of the pup-rescue behaviors: investigation of the pups, opening the first tube and retrieving the first pup from it (tube 1 rescue), and opening the second tube and retrieving the second pup from the first tube (tube 2 rescue). The clustering of brain areas was based on c-Fos density variability between individual mice, highlighting the consistency of each area's response to the examined behaviors.\u003c/p\u003e\u003cp\u003eThe correlation coefficients between the brain region activities and the latencies to each behavior are color-coded in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ee. Negative correlation (blue) means that the given brain region was more active when mice performed the given behavior with a short latency. The activities of the aACN and cMPOA were negatively correlated with the behaviors in a graded manner toward the bottom, particularly with the latency of tube 2 rescue, which reflects high caring motivation. The four limbic areas, A24, LS, BLA, and AcbSh, that showed a (trend of) higher activation in the rescuer group, were clustered together and exhibited negative correlations evenly with the behavioral latencies, suggesting their involvement through negative emotions and affective states processing generally associated with this task. The DRC and LPB also showed graded patterns yet were not classified together with the aACN and limbic regions due to higher inter-mouse variability of c-Fos density. The precise roles of these and other brain regions demonstrating significant correlations should be determined in future studies.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eThis study has explored the adversity-overcoming pup rescue behavior in postpartum and virgin female mice, utilizing the mice's known aversion to water. Numerous studies in rodents showed that mothers care for pups better than female virgins, especially in difficult tasks or under stressful conditions \u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e,\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e,\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u003c/sup\u003e. Thus, we designed the present experimental paradigm to identify the brain mechanism of maternal heightened motivation for pup care and expected that mothers would outperform virgin females in this task. Surprisingly, however, in our water pool-crossing pup rescue paradigm, virgins clearly surpassed mothers in pup retrieval (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ec), plausibly due to mothers\u0026rsquo; heightened aversion to water compared with female virgins (Figs.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ec, \u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ed, \u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ef, \u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eg\u003cb\u003e).\u003c/b\u003e This strong water aversion in mothers might have occurred directly from the altered physiology of postpartum and lactating animals. However, time since parturition (postpartum weeks) did not significantly affect water aversion in mothers, implying that water aversion persists beyond weaning. The precise mechanism of heightened water aversion of postpartum females should be deciphered in future studies. Additionally, our study indicates that the modality of environmental stress can be a critical determinant for parental care motivation in various reproductive contexts. For more comprehensive understanding of adversity-overcoming pup care, it may be beneficial to compare the results of experiments using different modalities of risks, such as water, bright light, or height, with different risk levels, such as tolerable versus barely tolerable water depth.\u003c/p\u003e\u003cp\u003eWe observed that the \u0026ldquo;trapped pup\u0026rdquo; rescue activated the preoptic area (aACN), limbic brain regions (A24, LS, BLA), and several hindbrain regions (LPB, DRC) that also showed strong relations with some particular rescue behaviors, such as the tube opening (Figs.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ec, e). These brain regions may have been activated in response to pup cues and associated reward\u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e,\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e,\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e,\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u003c/sup\u003e or as a reaction to an adverse and sensory-heavy environment \u003csup\u003e\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e,\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u003c/sup\u003e. The major limitation of our study is that we did not show functional importance of these regions for the pup rescue behavior. Thus, detailed behavioral manipulation studies on identified regions could yield valuable insights into the neural mechanism of the adversity-overcoming rescue.\u003c/p\u003e\u003cp\u003eThe brain activity mapping in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e was performed with virgin females, which do not have genetic relations to the rescuee pups. Therefore, these findings are also relevant for altruistic behavior and its neural correlates. Recent studies have identified a wide network of brain areas involved in prosocial behaviors, including helping, rescue, and harm avoidance behaviors toward adult and adolescent conspecifics in rats and mice \u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e,\u003cspan additionalcitationids=\"CR9\" citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e,\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e,\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e,\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e,\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e,\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e\u003c/sup\u003e. Our findings partially aligned with the results of these studies, as we showed that A24, LS, BLA, and to a lesser extent AcbSh were activated during pup rescue, and we specifically identified brain areas responding to cost-bearing rescue. We were unable to confirm increased activity in the medial amygdala (MeA) and PVN in our behavioral paradigm, which may have been due to the differences in stimulus animals (pup versus adult) and their consciousness level, as well as related to environmental adversity and experienced stress. Future studies examining the neural mechanism of cost-bearing rescue of adults and pups of different ages are awaited and would greatly contribute to the existing mechanistic understanding of the neural basis of altruism.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cp\u003e\u003cstrong\u003eAnimals.\u0026nbsp;\u003c/strong\u003eAll mouse experiments were approved by the Animal Care and Use Committee of the Institute of Science Tokyo and were thus in accordance with NIH guidelines (NIH Publications No. 8023, revised 1985), no procedures required euthanasia. This study followed the ARRIVE Guidelines 2.0.\u003csup\u003e37\u003c/sup\u003e Animals were maintained under a 12:12 h light/dark cycle (7:00 lights on, 19:00 lights off) with food and water \u003cem\u003ead libitum\u003c/em\u003e. C57BL/6N mice were originally obtained from Japan SLC or Jackson Laboratory and raised in our breeding colony. Pups were weaned at 4 weeks of age and housed with their littermates and/or mice of the same age in groups up to five. 2- to 10-months-old female mice were used for experiments.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAdversity-overcoming pup rescue.\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003eA pregnant wild-type female mouse (gestational age 7-14) was housed together with two virgin females in an experimental cage (44 \u0026times; 24 \u0026times; 15 cm, \u003cstrong\u003eFig. 1a\u003c/strong\u003e) until delivery. The experimental cage included a living area, where a mouse nest was located, and an experimental area that was separated from the living area with a water pool and a sham fence to force mice to enter the pool from the center. Virgin females underwent a pup retrieval test in advance to confirm their nurturing behavior. Experiments, consisting of combined habituation and pup rescue tests, started from the postpartum day 2 (PPD2) using the pups of the age of postnatal day 2 (PND2) and were conducted between 9:30\u0026ndash;13:00. Experiments were conducted on the postpartum week 0 (PPD2-5), postpartum week 2 (PPD10-17), and postpartum week 4 (PPD28-32). However, the results of pup rescue tests on the postpartum weeks 2 and 4 will be reported elsewhere due to substantial differences in experimental design. We created three levels of adversity: 1) 0 mm, 2) 3 mm, and 3) 20 mm water depth in the pool, based on the results of a previous study\u0026nbsp;\u003csup\u003e21\u003c/sup\u003e. We used room temperature water (temperature range between +21.0\u0026deg;C and +23\u0026deg;C). The habituation and pup rescue experiments were structured in the following way. One subject (a mother or a virgin) was left in the experimental cage, while other animals, including pups, were temporarily transferred to a clean husbandry cage. The subject was habituated to the empty cage with no water in the pool for 5 min. After that, 3 healthy pups were placed behind the pool in the experimental area and the subject\u0026rsquo;s behavior was observed for 30 min. Next, the subject was habituated to the empty cage with 3mm-deep water in the pool\u0026nbsp;\u003csup\u003e21\u003c/sup\u003e and then 3 healthy pups were placed behind the pool in a similar way to observe subject\u0026rsquo;s behavior for 30 min. Finally, the same experiments were conducted with the 20mm-deep water in the pool. Upon the end of these experiments, all animals were reunited in the experimental cage. Next day, another subject underwent the experiments.\u0026nbsp;All experiments were recorded on videos and analyzed manually and automatically using DeepLabCut\u0026nbsp;\u003csup\u003e22\u003c/sup\u003e. Manual analysis was performed by several raters with exceptional inter-rater reliability (the absolute‑agreement, two‑way random‑effects Intraclass Correlation Coefficient (ICC) for k\u0026nbsp;=\u0026nbsp;4 rater ICC(A,4)\u0026nbsp;=\u0026nbsp;0.996 (95%\u0026nbsp;CI\u0026nbsp;0.995\u0026ndash;0.997; F(85,\u0026nbsp;257)\u0026nbsp;=\u0026nbsp;262, p\u0026nbsp;\u0026lt;\u0026nbsp;1\u0026nbsp;\u0026times;\u0026nbsp;10⁻\u0026sup2;\u0026sup1;⁰)).\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003cstrong\u003eAdversity-overcoming \u0026ldquo;trapped pup\u0026rdquo; rescue for c-Fos sampling.\u003c/strong\u003e To examine c-Fos activity during the pup rescue test, only virgin female mice were used. First, we trained subjects to retrieve pups as described above at each water depth for one day (max three tests per day). If the subject failed to retrieve pups at a particular water depth, we started from this water depth the next day and repeated the tasks for one more day. Next, we conducted similar experiments but placed pups in two 50 ml falcon tubes (2 pups/tube) closed with paper lids (\u0026ldquo;trapped pup\u0026rdquo;) to make pup rescue comparable with the one of adult conspecifics or older pups \u003csup\u003e1,2,28,29\u003c/sup\u003e and repeated these tests for up to two days. After that, each subject was isolated in the husbandry cage overnight and then underwent the \u0026ldquo;trapped pup\u0026rdquo; rescue test at the 3mm water depth in the pool. Mice were labeled as \u0026ldquo;rescuers\u0026rdquo; (= opened both tubes within 30 min) and \u0026ldquo;non-rescuers\u0026rdquo; (= did not open the tubes within 30 min), while subjects with intermediate performance were excluded from future studies. \u0026nbsp;Two hours from the start of the test, mice were perfused, and brains were sampled.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eBrain tissue processing.\u0026nbsp;\u003c/strong\u003eMice that underwent \u0026ldquo;trapped pup\u0026rdquo; rescue experiment were deeply anesthetized with 8% (v/v) Midazolam (Sandoz Group AG, Switzerland), 10% (v/v) Vetorphale (Meiji Animal Health Co., Ltd., Japan), 7.5% (v/v) Domitor (Nippon Zenyaku Kogyo Co., Ltd., Japan) in saline, then perfused transcardially with 4% (w/v) paraformaldehyde (PFA) in 1x phosphate buffered saline (PBS, pH 7.4). The brains were removed, immersed in the same fixative at 4\u0026deg;C overnight, followed by cryoprotection in the series of 20% and 30% (w/v) sucrose in PBS for two days, embedded in O.C.T. Compound (Sakura Finetek Japan, Tokyo, Japan), and stored at -80\u0026deg;C until cryosectioning. Brains were cryosectioned coronally at a thickness of 40 mm according to the mouse brain atlas\u0026nbsp;\u003csup\u003e38\u003c/sup\u003e. Every third section from the serial sections was used for immunohistochemistry.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eImmunohistochemistry (IHC).\u0026nbsp;\u003c/strong\u003eImmunohistochemical detection of c-Fos on free-floating sections was performed as described previously\u0026nbsp;\u003csup\u003e24\u003c/sup\u003e. The sections were washed with PBS containing 0.2% Triton-100 (PBST), incubated with 0.3% H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e in methanol for 5 minutes, washed with PBST, blocked with 0.8% Block Ace (Dainihon-Seiyaku, Osaka, Japan) in PBST, and incubated at 4\u0026deg;C overnight with rabbit primary antibody against c-Fos (1:5000, Cat# sc-52, RRID: AB_2106783, Santa Cruz Biotechnology, Inc., Dallas, TX, USA). The following morning, the sections were washed and incubated with biotin-conjugated horse anti-rabbit secondary antibody (1:2000, Cat# BA-1100, RRID: AB_2336201, Vector Laboratories, Inc., Burlingame, CA, USA) for 2 hours and then in ABC peroxidase reagent (Cat# PK-6100; Vectastain ABC Elite kit; Vector Laboratories) for 1 hour according to the manufacturer\u0026rsquo;s instructions. The labeling was visualized by incubation in 3,30\u0026rsquo;\u0026ndash;diaminobenzidine (DAB) solution with nickel intensification (DAB peroxidase substrate kit Cat#SK-4100, Vector Laboratories) for 5 minutes. Finally, Nissl staining using cresyl violet was performed.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eHistological analysis.\u003c/strong\u003e Obtaining brightfield photomicrophotographs, image processing, and calculation of c-Fos\u003csup\u003e+\u003c/sup\u003e cell density were performed as described elsewhere\u0026nbsp;\u003csup\u003e16,24,39\u003c/sup\u003e. Brain regions abbreviations are based on Paxinos G. and Franklin K. B. J. mouse brain atlas \u003csup\u003e38\u003c/sup\u003e with additional specifications of anterior (a) and posterior (p) parts. Due to ~60% success rate of the \u0026ldquo;trapped pup\u0026rdquo; rescue in virgin females, we counted c-Fos in both left and right hemispheres of \u0026ldquo;rescuer\u0026rdquo; and \u0026ldquo;non-rescuer\u0026rdquo; group brains (technical replicates) to increase the number of samples and reliability of results.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eStatistical analysis.\u003c/strong\u003e All statistical analyses were conducted using R v. 4.4.0 (R Development Core Team, 2024; https://www.r-project.org/). No statistical methods were used to predetermine sample sizes. For analyses of the latencies to the first pool entry and pool crossing (\u003cstrong\u003eFigs. 1c-d\u003c/strong\u003e), the numbers of pool entries and pool crossings (\u003cstrong\u003eFigs. 1f-g\u003c/strong\u003e), and the time spent in the pool \u003cstrong\u003e(Fig. i\u003c/strong\u003e) during the 5-min habituation test, as well as during the pup rescue test (\u003cstrong\u003eFigs. 2d-f\u003c/strong\u003e); for the analysis of multiple pup nurturing behaviors (\u003cstrong\u003eFig. 2c\u003c/strong\u003e); for the analysis of latency to each pup nurturing behavior (\u003cstrong\u003eFigs. 2g-l\u003c/strong\u003e), the generalized linear mixed model (GLMM) from glmmTMB package (v1.1.10) was used \u003csup\u003e40\u003c/sup\u003e. For analysis of correlation between latencies to pool entry and pool crossing, as well as between numbers of pool entry and pool crossing, Pearson correlation and Intraclass Correlation Coefficient (ICC) from the irr package (v0.84.1;\u0026nbsp;https://cran.r-project.org/web/packages/irr/index.html) were used. For the analysis of density c-Fos-positive neurons (\u003cstrong\u003eFig. 3c\u003c/strong\u003e), Welch`s t-test was used. Normality was assessed using the Kolmogorov\u0026ndash;Smirnov test. For the analysis of correlation between the latencies to rescue behaviors and density of c-Fos-positive neurons (\u003cstrong\u003eFig. 3e\u003c/strong\u003e), tydiverse (v2.0.0) \u003csup\u003e41\u003c/sup\u003e, ggdendro (v0.2.0;\u0026nbsp;https://cran.r-project.org/web/packages/ggdendro/index.html), and corr (v0.4.4;\u0026nbsp;https://cran.r-project.org/web/packages/corrr/index.html) packages were used. Detailed information about statistical tests is provided in corresponding figure legend.\u003c/p\u003e\n\u003cp\u003eError bars represent mean \u0026plusmn; standard error of mean (S.E.M.). Data from censored observations were replaced by the maximum observation time (1800 s (=30 min) for all mice). The sample size is the same as the number of animals (biological replicate) in all cases, except for the c-Fos\u003csup\u003e+\u003c/sup\u003e cell density data where technical replicates (left and right hemispheres) were utilized together with biological replicates.\u003c/p\u003e"},{"header":"Declarations","content":"\u003ch2\u003e\u003cstrong\u003eAuthor contributions statement\u003c/strong\u003e\u003c/h2\u003e\n\u003cp\u003eK.P. and K.O.K. designed research with help from M.S. and R.K; K.P., M.S., and R.K. performed the experiments with help from C.Y. and K.O.K.; K.P., M.S., R.K., and K.O.K analyzed data; K.P., M.S., and R.K. wrote the first draft of the paper; all authors reviewed and edited the paper; K.O.K. conceived the research; C.Y. and K.O.K. contributed unpublished reagents/analytic tools.\u003c/p\u003e\n\u003ch2\u003e\u003cstrong\u003eAdditional Information\u0026nbsp;\u003c/strong\u003e\u003c/h2\u003e\n\u003cp\u003eAuthors declare no competing interests.\u003c/p\u003e\n\u003ch2\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/h2\u003e\n\u003cp\u003eWe thank Eri Miyazawa, Gulimire Yilihan, and Haruto Ijiri for technical assistance, Dr. Hiroshi Ueno for fruitful discussion,\u0026nbsp;the Center for Integrative Biosciences of Institute of Science Tokyo for animal husbandry.\u003c/p\u003e\n\u003ch2\u003e\u003cstrong\u003eFunding Declaration\u003c/strong\u003e\u003c/h2\u003e\n\u003cp\u003eThis work was supported by Japan Society for the Promotion of Science KAKENHI Grant-in-Aid for Scientific Research (B) (JP23K23927), the Bioscience Research Grant of Takeda Science Foundation to K.O.K, Japan Society for the Promotion of Science KAKENHI Grant-in-Aid for Early-Career Scientists (JP25K18581) to K. P., Japan Society for the Promotion of Science KAKENHI Grant-in-Aid for Scientific Research (C) (JP22K12236) to C.Y., Mitsubishi Electric Software Scholarship to M. S.\u003c/p\u003e\u003ch2\u003eData Availability\u003c/h2\u003e\u003cp\u003eThe datasets used and/or analyzed during the current study are available from the corresponding authors on reasonable request. Codes for behavioral analysis after DLC tracking and Spearman correlation matrix are available on GitHub (https://github.com/k-prokofeva/Prokofeva_Shibamiya_et_al_2025_code).\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eBartal, I. B. A., Decety, J. \u0026amp; Mason, P. 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E. \u003cem\u003eet al.\u003c/em\u003e glmmTMB balances speed and flexibility among packages for zero-inflated generalized linear mixed modeling. \u003cem\u003eR Journal\u003c/em\u003e \u003cstrong\u003e9\u003c/strong\u003e, 378\u0026ndash;400 (2017).\u003c/li\u003e\n\u003cli\u003eWickham, H. \u003cem\u003eet al.\u003c/em\u003e Welcome to the Tidyverse. \u003cem\u003eJ Open Source Softw\u003c/em\u003e \u003cstrong\u003e4\u003c/strong\u003e, 1686 (2019).\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Alloparental care, Pup retrieval, rescue, c-Fos, altruism","lastPublishedDoi":"10.21203/rs.3.rs-7240012/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7240012/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eRescuing infants under threat is fundamental parental behavior in mammals. However, the behavioral expression and neural correlates of adversity-overcoming infant rescue in non-parental female individuals remain poorly understood. In this study, we first established a novel pup rescue paradigm with scalable adversity, in which mothers and virgin female mice have to cross a water pool of varying depths (0, 3, or 20 mm) to retrieve pups into the nest. We unexpectedly found that virgin females were less averse to water and retrieved pups faster than mothers. Next, we implemented an additional hurdle by trapping pups into a tube, so that female mice had to cross the pool and open the tubes to rescue pups. The rescuer virgin females in this \u0026ldquo;trapped pup\u0026rdquo; rescue task showed increased neuronal activity in the anterior cingulate cortex, lateral septum, anterior commissural nucleus, basolateral amygdala, and dorsal raphe nucleus, compared with non-rescuers. The c-Fos\u003csup\u003e+\u003c/sup\u003e cell densities in these regions showed significant negative correlations with the latencies to rescue behaviors suggesting their positive impact on rescue. Given that the virgin females do not have genetic relations to the rescuee pups, our findings provide a basis of further analyses of adversity-overcoming altruistic behavior and its neural correlates.\u003c/p\u003e","manuscriptTitle":"Neural correlates of adversity-overcoming pup rescue behavior in female mice","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-09-12 14:58:47","doi":"10.21203/rs.3.rs-7240012/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-10-06T07:24:00+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-09-28T22:34:43+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-09-24T19:52:13+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"58112641681167318219691888983673019713","date":"2025-09-17T21:22:31+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"67582998653562448598510806220930764952","date":"2025-09-14T23:17:02+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-09-06T19:53:22+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-09-01T12:01:43+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-08-14T01:11:07+00:00","index":"","fulltext":""},{"type":"submitted","content":"Scientific Reports","date":"2025-08-14T01:07:50+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"d040438c-9d3c-403c-be65-aac6a80420af","owner":[],"postedDate":"September 12th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":54306648,"name":"Biological sciences/Evolution"},{"id":54306649,"name":"Biological sciences/Neuroscience"},{"id":54306650,"name":"Biological sciences/Zoology"}],"tags":[],"updatedAt":"2026-03-09T16:00:47+00:00","versionOfRecord":{"articleIdentity":"rs-7240012","link":"https://doi.org/10.1038/s41598-026-35639-7","journal":{"identity":"scientific-reports","isVorOnly":false,"title":"Scientific Reports"},"publishedOn":"2026-03-03 15:57:32","publishedOnDateReadable":"March 3rd, 2026"},"versionCreatedAt":"2025-09-12 14:58:47","video":"","vorDoi":"10.1038/s41598-026-35639-7","vorDoiUrl":"https://doi.org/10.1038/s41598-026-35639-7","workflowStages":[]},"version":"v1","identity":"rs-7240012","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7240012","identity":"rs-7240012","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}