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. 2026 Mar 7;42:100788. doi: 10.1016/j.ynstr.2026.100788

Different early-life adversity paradigms have disparate effects on maternal care, neuronal activation, and social behavior in developing mice

Vibha A Bapat 1, Renée C Waters 1, Ja'nae K Gordon 1, Elizabeth Gould 1,⁎
PMCID: PMC13068839  PMID: 41969913

Abstract

In humans, early-life adversity (ELA) predisposes individuals to neuropsychiatric conditions, with different types of adverse experiences linked more strongly to certain maladaptive outcomes. Maternal separation with early weaning (MSEW) and limited bedding and nesting (LBN) are rodent models that expose litters to very different postnatal manipulations, but the effect of these paradigms on offspring through differences in maternal care and neuronal activation remains incompletely explored. To address this gap, we directly compared behavior of dams in MSEW and LBN conditions to control conditions. We found that LBN dams showed reduced duration of bouts on the nest, as well as increased exits from the nest, and attempts at nest maintenance, whereas MSEW dams demonstrate control-like caregiving aside from the daily separations from the litter. At P7, MSEW and LBN pups showed distinct neuronal activation patterns in hippocampal subregions associated with social recognition. Next, we assessed social recognition at different developmental timepoints and found impairments after both ELA paradigms in both sexes at P21. After puberty, at P45, ELA females had improved social recognition while males remained impaired. Hippocampal plasticity measures known to participate in social recognition function in adults revealed age-specific alterations in neural stem cell density in the dentate gyrus of MSEW offspring, as well as alterations in parvalbumin-positive interneurons and perineuronal nets in the dorsal CA2 region of LBN offspring. These findings highlight differences between two ELA paradigms in maternal care, as well as neuronal activation, hippocampal plasticity measures, and social recognition in offspring.

Keywords: Maternal separation with early weaning, Limited bedding and nesting, Postnatal stress, c-fos, Hippocampus

1. Introduction

Early life adversity (ELA) encompasses many negative experiences, such as physical and emotional abuse, neglect, poverty, and witnessing violence or natural disasters. These experiences can predispose individuals to develop adverse neuropsychiatric outcomes (Copeland et al., 2018; Dunn et al., 2018; Strathearn et al., 2020; McKay et al., 2022; Lian et al., 2024). Studies have shown that specific types of childhood maltreatment are more closely associated with certain maladaptive outcomes in later life. For example, parental loss has been linked to a higher risk of clinical depression (Berg et al., 2016; Burrell et al., 2022), physical abuse has been associated with a higher risk of alcohol use disorder (Wang et al., 2020), and physical/sexual abuse has been associated with a predisposition to posttraumatic stress disorder (PTSD) (Adams et al., 2018; Westermair et al., 2018; Boumpa et al., 2024). Studies directly comparing diverse neuropsychiatric patients with history of adverse childhood experiences (ACE) show that certain forms of ACE are strongly correlated with specific neuropsychiatric outcomes and can be predictive of certain diagnoses (Nikulina et al., 2011; Westermair et al., 2018; Strathearn et al., 2020; Alkema et al., 2024). Furthermore, among adults without neuropsychiatric diagnoses, a childhood history of neglect is associated with impaired face recognition (Iffland and Neuner, 2020) whereas a childhood history of physical and emotional abuse is associated with difficulty recognizing emotions on faces (Masten et al., 2008; Iffland and Neuner, 2020). Taken together, these findings suggest that in addition to having shared effects on stress systems, different types of ELA may affect disparate neural circuits and cellular mechanisms.

Several rodent paradigms have been designed to model different types of childhood adversity through alterations in maternal care. Beginning with the groundbreaking work of Levine almost 40 years ago (reviewed in Suchecki, 2018), and extending to the present day, the most widely used rodent ELA paradigm is maternal separation. Maternal separation is a general model of physical and emotional neglect; an extended version of this is maternal separation combined with early weaning (MSEW) (George et al., 2010; Murthy et al., 2019; Waters et al., 2022; Laham et al., 2022). More recently, the limited bedding and nesting (LBN) model was introduced and has been widely adopted for ELA research. LBN has been described as a model of scarce resources and, potentially, physical adversity (Ivy et al., 2008; Rice et al., 2008; Walker et al., 2017). Both rearing paradigms have been shown to cause maladaptive effects on social behavior, cognitive function, stress coping, and avoidance behaviors in adult offspring (Murthy et al., 2019; Laham et al., 2022; Xu et al., 2022; Waters et al., 2022, 2026; Baram and Birnie, 2024). Some of these studies have produced contradictory results, raising questions about whether different ELA paradigms produce different outcomes in rodents.

In previous work, we examined the effects of MSEW and LBN on social recognition in adult offspring and found impairments in males, but not females (Waters et al., 2022, Waters et al., 2026). Despite these similarities, disruptions of nonoverlapping plasticity mechanisms were observed in the adult hippocampus of MSEW versus LBN males (Waters et al., 2022, 2026). MSEW, but not LBN, produced diminished adult neurogenesis in the dentate gyrus of males, which was associated with social recognition impairment (Waters et al., 2022). Adult-born granule cells are necessary for social recognition and exert their influence through connections with the CA2 region of the hippocampus (Cope et al., 2020; Laham et al., 2024). By contrast, LBN, but not MSEW, males have excess CA2 perineuronal nets (PNNs), extracellular matrix structures known to inhibit plasticity. LBN-induced social recognition dysfunction can be reversed in males by reducing PNNs in the dorsal CA2 (Waters et al., 2026). These findings raise questions about how these two ELA paradigms are distinctly experienced by pups, and how they produce differential plasticity disruptions and social recognition dysfunction during development.

In this study, we compared differential rearing experiences during MSEW versus LBN, focusing on maternal caregiving patterns as well as ELA-induced c-fos labeling in stress-sensitive brain regions of offspring. We then investigated the development of hippocampus-dependent social recognition before and after puberty and examined maturation of hippocampal structural plasticity measures that have been linked to ELA and social recognition in adulthood.

2. Methods

2.1. Animals

Adult male and female C57BL/6J mice were obtained from Jackson Laboratories and bred in-house. Mice were housed in Optimice cages with standard bedding and nesting material (unless mentioned otherwise) on a 12 h reverse light-dark cycle, with unlimited access to food and water. Pups were weaned into same-sex groups on postnatal day 21 (P21) (unless mentioned otherwise). All animal procedures were approved by Princeton University's Institutional Animal Care and Use Committee and were in accordance with the guidelines of the National Research Council's Guide for the Care and Use of Laboratory Animals.

2.2. Early life adversity paradigms

On the day after birth (P1), pups were cross-fostered, and the dams and their litters were assigned to one of three groups: 1) control-rearing; 2) MSEW; or 3) LBN. Control-reared (CON) mice were left undisturbed until weaning on P21 and were housed under standard laboratory cage conditions.

2.2.1. MSEW

Pups were separated from their dam daily for 4 h per day from P3–P6 and for 8 h from P7–P16 according to a previously published protocol (Murthy et al., 2019; Laham et al., 2022). The separations were done during the ‘lights-off’ phase. The dam was removed from the home cage, and the cage containing the pups was placed on a thermal heating blanket maintained at 34°C. Pups remained with their littermates in a separate room from the dam for the entire period of separation after which the dam was returned to the home cage. These pups are weaned early at P17. After weaning, the pups were housed in groups of same sex littermates.

2.2.2. LBN

Pups and dams were placed in a new cage with a perforated metal floor, no bedding and minimal nesting material, where they remained from P4 to P11 following a previously published protocol (Waters et al., 2026). On P11, the pups and dams were returned to standard housing, with control-like bedding and nesting, which included corn cob or ALPHA-dri bedding, a paper nestlet and a cardboard house, and remained in the standard cage until weaning at P21. After weaning, the pups were housed in groups of same sex littermates.

2.3. Maternal care behavior

Pregnant female mice were housed in a satellite colony room in the standard Optimice cages with unlimited access to food and water. Pregnant dams were randomly allocated to control-rearing, MSEW, or LBN groups (N = 6 per rearing group). Home cage video recordings were made from the side view without disturbing the dams and their litters. The camera was set to take automated 1-h long video recordings at three equally spaced timepoints across 24 h, including two recordings with the lights off (9:00 – 10:00 a.m., 6:00 – 7:00 p.m.) and one recording with the lights on (2:00 – 3:00 a.m.) across five days P3, P4, P6, P10, and P13. The MSEW dams were present in the cages with the pups at all recording times (recordings were done either prior to separation or after reunion). From each of the 1-h long videos, three equally spaced 5-min-long videos were sampled for behavioral analysis using Behavioral Observation Research Interactive Software (BORIS v. 8.24.1).

The caregiving behaviors analyzed included time spent by the dam on the nest, which was defined as the dam in direct physical contact with the pups on the nest, or within the region of the home cage where the pups were huddled together (in the case of LBN litters where a well-defined nest is absent), time away from the nest, as well as entries and exits from nest, which were counted as entries to and exits from these defined areas. Additionally, arched back nursing and nest maintenance behaviors were analyzed. Nest maintenance was defined as attempts of the dam at organizing nesting materials using her nose and forelimbs. Pup stepping behavior was quantified when the dam stepped on a pup with at least one hind paw, and tail chasing was counted as any instance where the dam chased her tail in a circle more than once (Ward et al., 2013). Time spent on each behavior, number of occurrences, and bout duration of each of these behaviors were calculated using BORIS. All behaviors were compared across different time points of the day and across postnatal days in the control, MSEW, and LBN groups.

2.4. Pup body surface temperature measurements

Abdominal surface temperatures of all pups in each litter used for the maternal behavior measurements were recorded at P7 and P11 using an infrared temperature gun (Thermo Fisher Scientific #NC1854657). The temperature gun was pointed 5–10 mm above the pup's abdomen to obtain readings. The dam was briefly removed from the home cage during measurements, and temperatures were recorded immediately after the dam's removal, following which the pups and dam were returned to the cage. All measurements were taken during the lights-off phase (for MSEW, temperature measurements were taken while pups underwent maternal separations in their home cage which was placed on heated thermal blankets as per the MSEW paradigm).

2.5. Direct social interaction test

The direct social interaction test (DSIT) was carried out on male and female offspring from a separate cohort of MSEW, LBN, and control rearing groups. DSIT was conducted twice on the same cohort, at P21 and P45 (N = 10 males, N = 10 females per rearing group). As described previously (Diethorn and Gould, 2023a), a 12″ x12” square acrylic arena with black walls and a clear base was used. The test was conducted under low light conditions, between 9 a.m. and 6 p.m. during the lights-off phase of the light cycle. The mice were habituated to the testing room for 15 min prior to behavior and then habituated to the testing arena for 5 min prior to the actual test. The DSIT consisted of two 5-min trials, separated by a 1h inter-trial interval (ITI). In trial 1 or ‘novel’ trial, the test mouse was paired with a novel stimulus, which was an age- and sex-matched control-reared conspecific and was allowed to interact freely with the stimulus in the arena for 5 min. During the ITI, the mice were returned to their home cages. Following the ITI, in trial 2 or the ‘familiar’ trial, the same two mice were paired again and were allowed to interact freely for 5 min. DSIT trials were recorded using a video camera placed above the arena. The time spent by the test mouse actively interacting with and investigating the stimulus conspecific in both trials was manually scored, with the experimenter blinded to the trial and experimental group of the mouse. Active social interaction and investigation by the test mouse included behaviors like anogenital sniffing and allogrooming. The events counted as social interactions were strictly restricted to interactions initiated by the test mouse and were defined as the events where the test mouse oriented its nose and touched the stimulus mouse to initiate social interaction. The DSIT arena was cleaned thoroughly with 70% ethanol between each trial and animal. The time spent in active social investigation in trial 1 (novel) was compared to trial 2 (familiar) for each mouse. A discrimination index was calculated for each mouse as follows: time spent investigating novel stimulus minus time spent investigating familiar stimulus divided by the total time spent investigating. A positive discrimination index, indicating higher time spent investigating a novel mouse compared to a familiar mouse is expected in healthy mice given previous studies that demonstrate a strong social novelty preference beginning in the postnatal period (Diethorn and Gould, 2023a) and extending into adulthood (Cope et al., 2023).

2.6. Histology

P7 mouse pups (1 male pup and 1 female pup of each litter from the maternal care experiment) were perfused for c-fos+ cell density quantification to analyze neuronal activation in response to MSEW, LBN, or control rearing. MSEW pups were perfused toward the end of the 8 h maternal separation on P7. For histological analysis of hippocampal plasticity measures associated with social recognition, mice from the social behavior study or from a separate cohort were perfused on P21 or P45.

Mice were deeply anesthetized with Euthasol (Virbac) and were transcardially perfused with cold 4% paraformaldehyde. The extracted brains were postfixed in 4% paraformaldehyde for 48 h, followed by cryoprotection in 30% sucrose in 0.1M PBS (10% for P7 brains), before being frozen and stored at −80°C until sectioning on the cryostat. Coronal sections (40 μm) through the hippocampus were collected using a Leica cryostat. Sections were blocked in a solution of 3% normal donkey serum in 0.1M PBS with 0.3% Triton X-100 for 1.5 h on a shaker at room temperature. Sections were then incubated on a shaker overnight in the cold, in a blocking solution containing combinations of the following antibodies or labeling reagents: rabbit anti-c-fos (used as an indirect label of neuronal activation; 1:1000, Synaptic Systems Cat # 226 008), mouse anti-Sox2 (used as a marker of neuronal stem cells; 1:500, Sigma Aldrich, Cat # SC1002), rabbit anti-Purkinje cell protein 4 (PCP4 used as a CA2 label; 1:1000, Sigma Aldrich, Cat# HPA005792), goat anti-parvalbumin (PV) used to label PV+ inhibitory interneurons; 1:2500, Swant, Cat# PVG213), and the plant lectin Wisteria floribunda agglutinin (WFA used to label PNNs; 1:1000, Sigma-Aldrich, Cat #L1516). Following the primary antibody, sections were washed and then incubated in a secondary antibody solution in 0.1M PBS with 0.3% Triton X-100 for 1.5 h on a shaker at room temperature. Combinations of the following secondaries were used: donkey anti rabbit Alexa Fluor 488 or 647 (1:1000, Invitrogen, Cat # A21206, A31573), donkey anti mouse Alexa Fluor 568 (1:500, Invitrogen, Cat# A10042), streptavidin Alexa Fluor 488 (1:1000, Invitrogen, Cat# S32354). The sections were then counterstained with Hoechst 33342 (1:5000, Molecular Probes, Cat# H3570), mounted onto slides, and cover slipped with Vectashield (Vector Laboratories).

2.7. Microscopy and image analysis

2.7.1. Density of PV+ cells, and optical intensity of WFA+ and PV+ cells

A Leica SP8 confocal microscope with LASX imaging software (version 35.6) was used to take high-resolution z-stack images of the dorsal CA2 of the hippocampus for PNN and PV+ cell analyses. For each label, 3 sections through the dorsal CA2 of P21 and P45 brains were imaged and analyzed. Z-stack images of PCP4+/WFA+ and PCP4+/PV+ labeling were obtained from the pyramidal cell layer of dorsal CA2, and were analyzed for optical intensity using Fiji (Image J) (NIH). The images were preprocessed using background subtraction (rolling ball radius = 50 pixels). Using the ROI functions, a perimeter was drawn around the pyramidal layer marked by PCP4+ cell bodies. For PNNs, the maximum intensity value of WFA stain within the ROI throughout the entire Z stack was identified and the mean gray value was averaged across all the sections per brain, to obtain a single value per mouse. For identifying PV+ cells, an initial threshold was applied using the thresholding function in imageJ, and cells were counted within the defined PCP4+ ROI using the particle analyzing tool. Overlay masks were generated to visualize the identified PV+ cells. The PV+ cell density was then obtained by dividing the number of PV+ cells by the volume of the total PCP4+ ROI in the dorsal CA2 (PV+ cells per mm3). Each of the identified PV+ cells were saved as ROIs and then an intensity analysis was done on each of these identified PV+ cells in the dorsal CA2. The maximum intensity value of PV stain within each PV+ cell's ROI throughout the entire Z stack was identified and the mean gray value was averaged across all the cells per section and then the sections were averaged to get a value per brain, to obtain a single value per mouse.

2.7.2. Density of c-fos+ cells and Sox2+ cells

Quantification of c-fos+ and Sox2+ cell number was determined using an Olympus BX60 fluorescence microscope with the MBF Bioscience Stereo Investigator software v.11.03. Cell density was calculated in several brain regions in P7 c-fos labeled tissue, including hippocampal subregions, amygdala, and parietal cortex, which includes the somatosensory cortex. It should be noted that because CA2 region-specific markers are not expressed until the end of the second postnatal week (Diethorn and Gould, 2023a,b), it was not possible to separate the CA2 from the neighboring CA3 in this analysis. Thus, for P7 brains, CA2 and CA3 were analyzed together. All brain regions at P7 were delineated using a developing mouse atlas (Kronman et al., 2024). Cell density was calculated in the dentate gyrus in P21 and P45 Sox2 labeled tissue. Cell counts were expressed per volume (mm3) for each cell label and brain region.

2.8. Statistical analysis

Statistical analyses were conducted using GraphPad Prism 10.4.2. All graphs were prepared using GraphPad Prism 10.4.2. All data are presented as the mean ± standard error of mean (SEM), unless mentioned otherwise. All data were analyzed using mixed effects model, one-way or two-way ANOVAs in GraphPad Prism as appropriate. Tukey's post hoc tests were used to follow up any main effects or significant interactions detected with ANOVAs. Data sets that did not meet requirements of parametric statistics were analyzed using Kruskal-Wallis tests followed by false discovery rate (FDR) post hoc tests.

3. Results

3.1. LBN dams displayed many differences in caregiving behavior while MSEW dams were similar to control dams

To investigate maternal care during ELA rearing (Fig. 1A–B), we analyzed caregiving behaviors of dams from all groups across multiple postnatal days during the ELA period (Fig. 1C). MSEW litters were separated from their dams on an average of almost 30% of the time through ELA, during which the pups received no maternal care (Fig. 1D). Although MSEW dams did not show altered behaviors on multiple measures compared to control dams, this substantial absence of care during the postnatal period is important to note while comparing MSEW and LBN. During the time of LBN rearing (P4 - P11), LBN dams displayed shorter bouts of time on the nest overall (Fig. 1E). However, dams from all the groups (control, MSEW, and LBN) showed similarities in the percent time engaged in arched back nursing (Fig. 1F). LBN dams showed altered behavior patterns in the time spent on- and off-the nest. LBN dams spent proportionately less time off the nest than either control or MSEW dams during the lights-off phase (Fig. 1G). These differences in bout of time spent on nest appeared to result from frequent transitions to- and from-the nest shown by greater number of exits from the nest (Fig. 1H). LBN dams also showed more instances of attempted nest maintenance (Fig. 1I) compared to MSEW and control dams. These differences in caregiving behavior displayed by LBN dams compared to MSEW and control dams were more prominent during the lights-off phase than during the lights-on phase (Fig. 1G–I). Two-way ANOVAs for time off nest (Fig. 1G) and nest maintenance (Fig. 1I), but not exits from nest (Fig. 1H) revealed a significant interaction between light phase and group. Additionally, most of these altered behavior patterns shown by LBN dams were obvious when the pups were P4 and P6 but seemed to normalize by P10 despite remaining in LBN conditions (Fig. S1A–C). LBN dams also engaged in behaviors that are not directly tied to caregiving, including tail chasing and stepping on pups. These behaviors were higher in number compared to MSEW and control dams only during the lights-off phase (Fig. S1D and E).

Fig. 1.

Fig. 1

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MSEW and LBN dams exhibited different maternal caregiving patterns.(A) Schematic of the maternal separation with early weaning (MSEW) paradigm. (B) Schematic of the limited bedding and nesting (LBN) paradigm. (C) Timeline of the design for assessment of maternal care. (D) Proportion of time spent on-versus off-nest by dams, emphasizing the time spent away from home cage during maternal separations for MSEW. (E) Average bout of time spent on nest per entry to the nest by dam, showed shorter bouts by LBN dams compared to MSEW and CON. (F) MSEW and LBN dams spent comparable time to CON dams in arched back nursing. (G) LBN dams spent less time off-nest compared to CON dams during the lights-off phase. (H) LBN dams showed more exits from the nest during the lights-off phase compared to lights-on (although the number of exits was higher compared to MSEW and CON dams during both lights-off and-on). (I) LBN dams also displayed higher number of attempts of nest maintenance compared to MSEW and CON only during the lights-off phase. Data displayed in Fig. 1 were averaged across all recordings from all times of the day (or separated by light-phase) and postnatal days during the LBN manipulation (P4-P11). N = 6 dams per group. ∗p < 0.05 compared to all other groups on that measure. Refer to Table S1 for complete statistics.

Taken together, these findings suggest that, when they are allowed access to their pups, MSEW dams behave similarly to control dams on most caregiving measures, but LBN dams display many differences, including nest bout time, number of nest exits, and number of attempted nest maintenance events. Aberrant maternal behavior displayed by LBN dams is most prominent during the lights-off phase and is accompanied by higher amounts of tail-chasing and pup stepping behavior compared to the other rearing groups.

3.2. LBN pups had lower body surface temperature than MSEW or control-reared pups

On both P7 and P11, MSEW and control-reared pups exhibited expected healthy mouse pup body temperatures (Nagy, 1993) that did not differ statistically between groups. By contrast, LBN pups had statistically lower body temperatures on both P7 and P11 (Fig. S2). These findings suggest that LBN conditions produce lower body temperature of pups beyond the time when aberrant behavior of LBN dams normalizes, while MSEW and control pup temperatures remain similar.

3.3. MSEW and LBN pups had differential c-fos+ cell densities in multiple brain regions

By assessing cells labeled for c-fos, the protein product of the immediate early gene Fos, as an indirect measure of neuronal activation in multiple stress-linked brain regions (Fig. 2A–C), we found that LBN pups had significantly greater c-fos+ cell density in the amygdala compared to MSEW pups, with no significant differences of either ELA group compared to control pups (Fig. 2D). By contrast, we found lower c-fos+ cell density in the parietal cortex of LBN pups compared to MSEW and control-reared pups with no differences observed in MSEW pups compared to controls (Fig. 2E). In the overall dorsal hippocampus, both LBN and MSEW pups showed statistically lower c-fos+ cell density compared to controls, whereas in the overall ventral hippocampus, only LBN pups showed statistically lower c-fos+ cell density (Fig. 2F). Next, we examined hippocampal subregions specifically associated with social recognition and found clear differences between c-fos+ cell density in the MSEW and LBN pups. The dorsal dentate gyrus (Fig. 2G) showed significantly lower values in MSEW pups compared to control-reared pups whereas dorsal CA2/CA3 (Fig. 2H) showed significantly lower values in LBN pups compared to control-reared pups. The ventral CA1 showed significantly lower values in MSEW pups compared to control-reared pups (Fig. 2I). There were additional differences across groups in hippocampal subregions not directly linked to social recognition, including dorsal CA1, which had lower c-fos+ cell density in MSEW pups compared to other groups (Fig. S3A), and ventral dentate gyrus (Fig. S3B), which had lower c-fos+ cell density in LBN pups compared to other groups. No differences were observed in the ventral CA2/CA3 (Fig. S3C).

Fig. 2.

Fig. 2

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MSEW and LBN pups had different c-fos+ cell densities across brain regions, including hippocampal subregions associated with social recognition. (A–C) Representative confocal images of c-fos+ cells, counterstained with the DNA dye Hoechst 33342 (blue). (D) Higher c-fos+ cell density was observed in the amygdala of LBN pups compared to MSEW pups. (E) Lower c-fos+ cell density was observed in the parietal cortex of LBN pups compared to MSEW and CON pups. (F) In the dorsal hippocampus, both MSEW and LBN pups had lower c-fos+ cell density compared to CON pups, while in the ventral hippocampus, only LBN pups had lower c-fos+ cell density compared to CON pups. (G) In the dorsal dentate gyrus, MSEW, but not LBN, pups showed lower c-fos+ cell density compared to CON pups. (H) In the dorsal CA2-CA3 region, LBN, but not MSEW pups showed lower c-fos+ cell density, and in the (I) ventral CA1, MSEW pups showed lower c-fos+ cell density compared to CON pups. N = 7-12 per group. ∗p < 0.05 compared to group indicated by horizontal brackets. Scale bars equal 50 μm and pertain to A-C. Refer to Table S1 for complete statistics.

3.4. MSEW and LBN pups of both sexes had impaired social recognition with ELA-reared females showing recovery after puberty

We tested mice on a two-trial direct social interaction test involving free interaction with a novel stimulus conspecific in trial 1 (novel), followed by an ITI of 60 min and subsequent interaction with the same now familiar stimulus mouse in trial 2 (familiar) at P21 and P45 (Fig. 3A and B). At P21, we found that control-reared male pups showed the expected decrease in investigation time between exposure to a novel social stimulus (trial1) and exposure to the now familiar social stimulus (trial 2). However, at this age, both MSEW and LBN male pups did not show a decrease in investigation times between trial 1 and trial 2 (Fig. 3C), an effect that remained at P45 in males (Fig. 3D) and was observed with examining the discrimination indices, measures that control for the overall amount of time spent investigating (Fig. 3E). At P21, MSEW males showed higher overall investigation times compared to other rearing groups, and by P45, LBN males had significantly lower investigation times (Fig. 3F). At P21, we found that control-reared female pups also showed the expected decrease in investigation time between exposure to a novel social stimulus (trial 1) and exposure to the now familiar social stimulus (trial 2). Both MSEW and LBN female pups did not show a decrease in investigation times between trial 1 and trial 2 (Fig. 3G), but their behavioral responses were recovered by P45 (Fig. 3H). Analyzing sex as a variable at P21, there were no significant effects of sex or the interaction between sex and rearing condition in the discrimination indices; both males and females from MSEW and LBN groups had lower scores indicating impaired social recognition (Fig. 3E and I). By P45, however, a significant main effect of sex as well as rearing condition was seen, revealing the emergence of sex differences in social discrimination in MSEW and LBN (Fig. 3E and I). Females in both MSEW and LBN groups had not only recovered discrimination indices, but overall control-like investigation times by P45 (Fig. 3J).

Fig. 3.

Fig. 3

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ELA induced sex- and age-specific social recognition impairments in offspring. (A) Timeline of longitudinal assessment of social recognition at P21 and P45. (B) Schematic of direct social interaction test used to assess social recognition. (C) At P21, MSEW and LBN males did not show the expected decrease in investigation time from the novel to familiar trial, which is observed in CON males. (D) At P45, LBN males continued to lack a decrease in investigation time between trials, while MSEW males showed a slight improvement in ability to discriminate. (E) MSEW and LBN males showed lower discrimination indices compared to CON males at P21 and P45, reflecting the persistent social recognition impairment. (F) MSEW males showed higher total investigation times compared to LBN and CON at P21, whereas at P45, LBN males showed lower total investigation times compared to MSEW and CON males. (G) At P21, MSEW and LBN females did not show the expected decrease in investigation time from the novel to familiar trial, as observed in CON females. (H) However, at P45, all the female groups showed decreases in investigation times from the novel to familiar trial. (I) This impairment at P21 and recovery by P45 in MSEW and LBN females was reflected through the low discrimination indices compared to CON females at P21, and comparable discrimination indices at P45. (J) MSEW females showed higher total investigation times compared to LBN and CON females at P21, but at P45 all groups had comparable total investigation times. N = 9−10 males and females per group. ∗p < 0.05 compared to group indicated by horizontal brackets. Refer to Table S1 for complete statistics.

3.5. MSEW and LBN resulted in different age-, and sex-specific effects on hippocampal plasticity measures associated with social recognition

We next investigated several plasticity measures in the hippocampus. At P21, we found that the density of neural stem cells marked by the transcription factor Sox2 in the dorsal dentate gyrus was not different between groups or across sexes (Fig. 4A and B). However, we found that male, but not female, P21 LBN pups had lower WFA intensity in the dorsal CA2 region compared to both other rearing groups at the same age (Fig. 4C and D). We also found overall decreased PV+ cell density with no changes in PV intensity in the dorsal CA2 of LBN males, but not in females compared to other rearing groups (Fig. 4E and F). At P45, there was a main effect of group where MSEW males and females showed higher Sox2+ cell density compared to controls but not with LBN males and females, and no sex differences were seen (Fig. 4G and H). Further, analyses of PNNs in the dorsal CA2 at P45 revealed no differences in WFA intensity across groups or sexes (Fig. 4I and J). Lastly, investigation of PV+ cell density and PV intensity at P45 revealed no differences in PV+ cell density, but a significant interaction between sex and group in PV intensity, with control females showing higher PV intensity than control males (Fig. 4K and L).

Fig. 4.

Fig. 4

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ELA induced age-specific effects on hippocampal stem cells, PNNs, and PV+ cells. Representative confocal images of Sox2+ labeling in dorsal DG counterstained with the DNA dye Hoechst 33342 (blue) at P21 (A). No differences were observed in the Sox2+ cell density among the groups or between sexes (B). Representative confocal images of WFA staining in dorsal CA2 pyramidal cell layer marked by PCP4+ labeling at P21 (C). LBN male pups showed lower WFA intensity compared to CON and MSEW male pups (main effect of group only) (D). Representative confocal images of PV+ labeling in the dorsal CA2 at P21 (E). LBN male pups showed lower PV+ cell density compared to CON and MSEW male pups (main effect of group only), but no differences were seen in PV intensity among groups or sexes (F). Representative confocal images of Sox2+ labeling in dorsal DG at P45 (G). MSEW males and females showed higher Sox2+ cell density in the dorsal DG compared to CON (H). Representative confocal images of WFA staining in dorsal CA2 at P45 (I). No differences were seen in WFA intensity across groups in males or females (J). Representative confocal images of PV+ labeling in dorsal CA2 at P45 (K). No differences were found across groups in PV+ cell density in males or females, but a significant interaction between group and sex was seen in PV intensity, with CON females showing higher PV intensity than CON males (L). N = 8-11 per group. ∗p < 0.05 comparisons between specific groups indicated by horizontal brackets. Scale bars equal 50 μm. Refer to Table S1 for complete statistics.

4. Discussion

Our results show that MSEW and LBN paradigms have differential effects on maternal care, as well as on neuronal activation, social recognition, and hippocampal plasticity measures in offspring. Compared to control dams, LBN dams had shorter bouts on the nest, with more exits from the nest, and more attempted nest maintenance events. By contrast, behavior of MSEW dams, when in contact with their pups, was more like control dams, although their pups were completely deprived of maternal contact during the separation periods. In addition to differences in maternal care, LBN also resulted in the pups exhibiting lower abdominal surface temperatures than MSEW and control-reared pups. Pups in the three rearing groups showed different patterns of neuronal activation, with LBN pups having higher c-fos+ cell density in the amygdala than MSEW pups, as well as lower c-fos+ cell density in the parietal cortex, overall hippocampus, and the dorsal CA2/CA3 hippocampal regions compared to MSEW and control-reared pups. By contrast, MSEW pups exhibited lower c-fos+ cell density in the dorsal dentate gyrus and ventral CA1 region. Offspring from both MSEW and LBN groups showed deficits in social recognition that lasted beyond the end of the ELA paradigms although MSEW and LBN females showed signs of functional recovery after puberty, while the deficits in males persisted. Lastly, analyses of hippocampal plasticity measures linked to social recognition in adulthood showed that P21 LBN pups had altered PNNs and PV+ interneurons, while after puberty MSEW offspring had altered neural stem cell density. These findings suggest that MSEW and LBN differentially shape the early development of social memory circuits and potentially drive long-term, sex-specific functional outcomes through distinct hippocampal plasticity mechanisms.

Parental care is important for healthy development (Levine, 2005; Kaffman and Meaney, 2007; Curley and Champagne, 2016; Raineki et al., 2019), and for most mammals, the mother provides most early caregiving (Leuner et al., 2010; Numan, 2020). Unpredictable and disrupted maternal care has been linked to altered neurodevelopment and behavioral dysfunction in later life (Glynn and Baram, 2019). Our findings complement previous studies showing that mouse mothers subjected to the LBN paradigm exhibit fragmented maternal care (Gallo et al., 2019; Pardo et al., 2023). We found reduced duration of bouts on the nest, increased exits from the nest, and increased attempts at nest building, which occurred primarily during the lights-off phase when mice are most active. During this time, we also found that compared to controls, LBN dams were more likely to step on their pups and engage in self-tail-chasing behavior. Stepping on pups has been previously interpreted as rough or abusive behavior (Walker et al., 2017), while tail chasing may be linked to stress (Ward et al., 2013) and has been reported in LBN dams (O'Neill et al., 2025). Taken together, these findings suggest that the LBN paradigm may be particularly stressful to dams, likely because they are unable to successfully engage in the adaptive behavior of nest building, and as a result, they provide less consistent care to their pups. Despite these findings, LBN dams did not differ from controls on several important measures of caregiving, including overall time spent on the nest and arched back nursing. Even for the maternal behaviors that are atypical in the LBN paradigm, these abnormalities peaked toward the end of the first postnatal week and returned to control levels by P10, a day before the end of the LBN period, suggesting the dams habituated to the condition. By contrast to the behavior of LBN dams, we did not see any differences in caregiving behaviors in MSEW dams compared to controls. Some studies report compensatory caregiving, primarily in the form of higher licking and grooming behavior, in maternal separation models (Kosten and Kehoe, 2010; Own and Patel, 2013; Orso et al., 2018), which we did not assess in our study. Despite the similarities we noted in caregiving behaviors between MSEW and control dams, it is important to emphasize that MSEW pups are deprived of maternal contact for almost 30% of the time from P3-P16 and are weaned early on P17 after which they are deprived of maternal contact 100% of the time 4 days prior to normal weaning. Additionally, our MSEW paradigm spans a longer postnatal period than our LBN paradigm, so pups experience absence of maternal care for a longer period compared to the altered LBN caregiving patterns, most of which are induced at P4 and return to control values by P10. Thus, differences in the timelines of the models could interact with developmental neural trajectories to produce disparate outcomes.

In addition to differences in parental care, the physical environment itself can be an important component of the rearing experience. Poor nest quality due to LBN rearing is known to affect thermoregulation of pups (Lapp et al., 2020). Our results corroborate previous reports of lower body temperatures in LBN pups (Shupe and Clinton, 2021). We found that lower body temperatures persist beyond the time when maternal behavior normalizes for LBN dams, suggesting that the physical environment, which includes metal flooring instead of plastic flooring covered with bedding, is likely responsible.

To further assess differences between effects of the two ELA paradigms on the offspring, we examined labeling with the immediate early gene c-fos on P7, a time when both MSEW and LBN were ongoing. This analysis revealed differential effects on c-fos+ cell densities in the amygdala, parietal cortex, and hippocampus of MSEW and LBN pups. Higher densities of c-fos+ cells were observed in the amygdala of LBN compared to MSEW pups. Altered activation of the amygdala has been linked to maladaptive effects of ELA (Johnson et al., 2018, Manzano Nieves et al., 2020) and increased neuronal activation in LBN pups may be related to the fragmented maternal care and harsh physical environment they experienced. Lower c-fos+ cell densities were observed in the parietal cortex, which includes the somatosensory cortex, of LBN pups. Reduced neuronal activation in this region could be due to altered sensory experiences (e.g., pain and cold) due to the cage environment for the LBN pups. Lower c-fos+ cell densities were also observed in the hippocampus, which undergoes a protracted period of postnatal development (Cohen et al., 2000; Esther et al., 2013; Zeiss, 2021; Diethorn and Gould, 2023b), of both MSEW and LBN pups. Despite this commonality between ELA paradigms, differential effects of MSEW and LBN were observed in hippocampal subregions including the dentate gyrus, dorsal CA2/CA3, and ventral CA1, all of which are crucial for regulating social recognition (Hitti and Siegelbaum, 2014; Okuyama et al., 2016; Deng et al., 2019; Cope et al., 2020).

Given the diminished c-fos+ cell density in hippocampal subregions associated with social recognition on P7, it is perhaps not surprising that both ELA paradigms led to impairments in this function at P21 although MSEW pups of both sexes investigated stimulus mice more than controls, an effect not observed in LBN pups. However, at the post-puberty adolescent timepoint, P45, a sex difference emerged where ELA females showed restored social recognition while the ELA males remained impaired regardless of paradigm. Studies suggest that effects of ELA in females can vary across age and hormonal states, and that major hormonal changes such as those seen over puberty could unmask resilience or susceptibility to certain ELA effects (Hodes and Shors, 2005; Hodes and Epperson, 2019). Future studies involving perturbations during the peripubertal period are needed to determine the role of puberty in the emergence of sex-specific effects in social recognition. These would provide more insights into the mechanisms that protect females from social recognition impairment post-puberty.

The dorsal CA2 region receives inputs from multiple areas including the dentate gyrus (Kohara et al., 2014). Adult neurogenesis takes place in the dentate gyrus, where adult-born neurons, which arise from stem cell proliferation, actively integrate into the circuitry and project to the CA2 region (Song et al., 2012; Llorens-Martín et al., 2015). These neurons are known to modulate social recognition (Cope et al., 2020) through their influence on CA2 network activity (Laham et al., 2024). Depletion of the neural stem cell population and reduced adult neurogenesis coincides with the social recognition impairment in adult MSEW males (Waters et al., 2022). Although we did not see any differences in Sox2+ cell density at P21, any difference might be obscured by the overall higher number of stem cells at early ages. However, by P45, we found that MSEW offspring had higher Sox2+ cell density compared to control offspring. Since adult MSEW male, but not female, offspring showed depleted Sox2+ cells compared to other males (Waters et al., 2022), this could reflect dysregulation of neurogenesis, as neurogenesis is higher during adolescence than adulthood (He and Crews, 2007; Curlik et al., 2014), raising the possibility that MSEW potentially exhausts the stem cell pool earlier than other groups by the time offspring reach adulthood. However, when the sex difference arises remains an open question.

PNNs in the dorsal CA2 play important roles in social recognition in adult mice (Cope et al., 2020, 2022; Laham et al., 2024; Alexander et al., 2025). The emergence of PNNs in development has been associated with closure of critical periods in the second postnatal week (Wang and Fawcett, 2012; Carulli and Verhaagen, 2021). This timeline coincides with the postnatal rearing manipulations in ELA models, thus making altered PNNs a potential candidate mechanism underlying social recognition dysfunction. Excess PNNs in adult LBN male offspring have been reported, and their reduction was sufficient to rescue an LBN-induced social recognition impairment (Waters et al., 2026). Furthermore, fast-spiking PV+ interneurons, which are highly concentrated in the dorsal CA2, also have a protracted postnatal development in rodents (Nitsch et al., 1990; Lopez-Tellez et al., 2004). We found that PNNs and PV+ interneurons were affected selectively by LBN in an age-and sex-dependent manner. At P21, LBN pups show lower PNN levels and lower PV+ cell density in the dorsal CA2. These alterations may underlie social recognition impairment in LBN pups. However, by P45, LBN offspring had comparable PNNs to controls. Since higher PNN intensity in adult LBN males is known to mediate the social memory impairment (Waters et al., 2026), this could suggest a delayed developmental trajectory of PNNs in LBN males. Since PV+ cell activity has been linked to PNN intensity (Cisneros-Franco and Villers-Sidani, 2019), there could be an interplay between these components that are known to be involved in social recognition. These findings suggest that LBN alters the developmental trajectory of CA2 inhibitory circuitry. Shifts in timing might be an important mechanism through which ELA leads to sex-specific disruptions in social memory later in life.

Although establishing causal links between these ELA-induced differences in plasticity mechanisms and developmental social recognition dysfunction will require additional experimentation, it is interesting to note that MSEW, but not LBN, P7 pups showed lower c-fos+ cell density in the dentate gyrus, as well as elevated stem cells in the dentate gyrus at P45. Additionally, LBN, but not MSEW, offspring showed lower c-fos+ cell density in the dorsal CA2/CA3, as well as differences in PNNs and PV+ cells in the dorsal CA2 region. These findings suggest that subregion-specific changes in neuronal activation during ELA may set in motion developmental trajectories that evolve over time, thus producing social dysfunction through mechanisms that differ depending on specific types of adverse experience.

4.1. Conclusions

Our findings contribute to the growing body of evidence showing model-specific effects of ELA in rodents (Köhler et al., 2019; Catale et al., 2020; Demaestri et al., 2020; Waters and Gould, 2022). We found major differences in caregiving experience between MSEW and LBN models, as well as in effects of these models on neuronal activation in pups. We also identified social recognition impairments at early developmental timepoints in both MSEW and LBN models and found that sex-specific effects of ELA on social recognition emerge after puberty. These age- or sex-specific effects were also seen in dorsal hippocampal plasticity measures associated with regulating social memory in previous studies, including adult neurogenesis, PNNs, and PV+ interneuron intensity. Taken together, these findings suggest that these plasticity measures could be differentially involved in mediating the developmental effects of MSEW versus LBN. Future studies involving manipulation of these measures to characterize development of social memory circuits will better inform us about the differential mechanisms involved, which could enable identification of early interventions to rescue altered development post-ELA to prevent long-term maladaptive effects in adults.

CRediT authorship contribution statement

Vibha A. Bapat: Conceptualization, Data curation, Investigation, Supervision, Writing – original draft, Writing – review & editing. Renée C. Waters: Conceptualization, Investigation, Supervision, Writing – review & editing. Ja'nae K. Gordon: Investigation, Writing – review & editing. Elizabeth Gould: Conceptualization, Funding acquisition, Resources, Supervision, Writing – original draft, Writing – review & editing.

Declaration of competing interest

The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Elizabeth Gould reports financial support was provided by National Institute of Mental Health. Renée C Waters reports financial support was provided by National Science Foundation. Other authors declare they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgments

The authors thank Casey J. Brown for technical assistance. Research reported in this publication was supported by National Institute of Mental Health of the National Institutes of Health under grant number R01MH117459 (EG) and the National Science Foundation under award number GRFP 2021318039 (RCW). The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health nor the National Science Foundation. The authors acknowledge BioRender for figure schematics.

Footnotes

This article is part of a special issue entitled: Gig Levine Special Issue published in Neurobiology of Stress.

Appendix A

Supplementary data to this article can be found online at https://doi.org/10.1016/j.ynstr.2026.100788.

Appendix A. Supplementary data

The following is the Supplementary data to this article.

Multimedia component 1
mmc1.pdf (439.2KB, pdf)

Data availability

Data will be made available on request.

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