Appetitive maternal behavior in mice is resistant to perinatal chronic variable stress and perinatal chronic corticosterone treatment

Abstract

Perinatal stress is a risk factor for emotional and cognitive disturbances in both caregivers and their infants. Here, we compared the impact of two stressors, perinatal chronic variable stress (CVS) and perinatal chronic corticosterone treatment (C-CORT), on maternal exploratory behavior, maternal caregiving, and hypothalamic neural activity. We stressed mouse dams with each paradigm for 13 days starting at gestational day 11 (GD11). We examined open field exploration behavior on day 12 of stress (or post-partum day 3, PP3) and pup-directed behavior on day 13 of stress (PP4). We collected brains and serum 30 minutes after pup exposure to study circulating maternal corticosterone and the expression of the immediate-early gene Fos in hypothalamic neuropeptidergic cell populations. We found that perinatal CVS reduced distance moved in the open field, suggestive of a depression-like phenotype, while perinatal C-CORT reduced time spent in the center of the open field, suggestive of an anxiety-like phenotype. Dams exposed to both stressors showed intact appetitive maternal behavior. Importantly, we found that CVS in mated, unpregnant females led to hypoactivity in the open field and significantly increased pup retrieval latency. Both stressors led to reduced Fos colocalization with corticotropin-releasing factor in the paraventricular nucleus of the hypothalamus (PVH^CRF) after pup exposure. Perinatal C-CORT treatment increased Fos colocalization with urocortin-3 in the perifornical area of the hypothalamus (PeFA^ucn3). Together, these results suggest that adaptations in maternal physiology and brain function contribute to stress resistance, thereby protecting appetitive maternal behavior.

Graphical Abstract

Introduction

During the perinatal period, the human maternal brain undergoes extensive structural and functional changes (Dufford et al., 2019; Hoekzema et al., 2017; Hoekzema et al., 2022; Kim, 2016; Kim et al., 2010; Paternina-Die et al., 2024; Pritschet et al., 2024). These changes are theorized in part to support behavioral and physiological processes associated with caregiving toward the infant (Brunton and Russell, 2008; Spalek et al., 2024). In particular, changes in hypothalamic volume are observed across studies and may correlate with maternal-infant bonding in humans (Kim et al., 2010; Spalek et al., 2024). Stress during this perinatal period disrupts these brain adaptations (Kim, 2021), with consequences for caregiving as well as mood and emotional regulation in the caregiver, including susceptibility to postpartum mental disorders such as depression and anxiety (Barba-Muller et al., 2019; Pawluski et al., 2017; Raver and Leadbeater, 1999; Simpson and Catling, 2016; Singer et al., 1999).

Adaptations in brain structure and function can also be observed in animal models during the transition to parenting (Lonstein et al., 2015; Numan and Insel, 2003). Recently, studies in laboratory mice have revealed structural, cellular, and molecular mechanisms involved in the functional plasticity in the female mouse brain that promote infant care during the postpartum period (Ammari et al., 2023; Barriere et al., 2021; Celik et al., 2022; Chaker et al., 2023). Furthermore, the hypothalamic-pituitary-adrenal and hypothalamic-pituitary-gonadal axes in mice and humans are anatomically and physiologically comparable (Kaprara and Huhtaniemi, 2018; Keller et al., 2019; Kohl et al., 2017; Packard et al., 2016; Plant, 2015; Russell and Lightman, 2019; Sheng et al., 2020). With respect to the impact of chronic stress, mice show behavioral, neurophysiological, and endocrine responses that are analogous to those in humans, including peripartum stress-induced changes in neural circuitry and infantdirected behaviors (Baram et al., 2012; Hillerer et al., 2012; Klampfl and Bosch, 2019; Slattery and Hillerer, 2016; Zoubovsky et al., 2020). However, relatively few studies have examined the effects of peripartum stressors on maternal caregiving behavior in mice in a systematic manner (Orso et al., 2019; Zoubovsky et al., 2020). Among studies that report infant-directed behavior, the evidence is conflicting on the impact of chronic stress on caregiving measures such as contact with pups, pup retrieval latency, and nest-building (Orso et al., 2019). This variability in outcomes could be attributed to the timing and severity of the stress, the variability in methods for measuring maternal caregiving, differences in susceptibility to stress among mouse strains and/or individual mice, or other factors. In our lab, we have previously observed that caregiving in C57/BL6J mouse dams is unaffected by chronic restraint stress, chronic social stress, and chronic variable stress in the postpartum period (Abdelmesih et al., 2023). This lack of effect may be attributed to HPA axis hyporesponsivity during the perinatal period (Douglas et al., 2003). We wondered if applying stress during pregnancy may reveal impacts of chronic stress on maternal caregiving.

In the present study, we observed the effects of two models of perinatal stress on mouse dam caregiving behavior. We used chronic variable stress (CVS) from gestational day 11 (GD11) to post-partum day 4 (PP4) as an unpredictable, ethologically relevant stress. Additionally, we used chronic corticosterone administration (C-CORT, 20 mg/kg s.c.) for the same peripartum period (GD11-PP4) in a separate cohort to upregulate stress hormones in a controlled manner. We found distinct impacts of these stressors on exploratory behavior, but appetitive maternal behavior was resistant to both. Perinatal C-CORT, but not CVS, led to reduced average pup weight at birth, which normalized by PP2. Dams with CVS did not show differences in circulating corticosterone levels, while C-CORT-treated dams showed reduced circulating corticosterone levels compared to control dams. Both stressors reduced activity in PVH^CRF neurons, while only C-CORT treatment significantly increased PeFA^ucn3 neuron activity after pup interaction. Overall, these results demonstrate that perinatal CVS and perinatal C-CORT affect maternal mood-related behavior while appetitive maternal behavior is resistant to these stressors.

Materials and Methods

Animals

Pregnant female (GD4) C57BL/6J mice age 8–10 weeks were ordered from Jackson Laboratories (Bar Harbor, ME). Animals received from Jackson were singly housed and habituated to our facility for 7 days prior to behavioral testing. Mice were maintained in a temperature and humidity-controlled room on a 12h:12h dark light cycle (10:30am-10:30 pm dark phase) with access to food and water ad libitum. Pups remained with the mother until the end of the experiment. Experimental animals were randomly allocated to experimental groups using an online random number generator. All experiments were performed following the ARRIVE ethical guidelines and in accordance with NIH guidelines and approved by the Albert Einstein College of Medicine Institutional Animal Care and Use Committee (IACUC; protocol 20180110; 20180111; 00001386). Dams were weighed every other day during the experimental period. Litters were counted and weighed on the day of birth (PP0) and PP2. No pups were experimentally culled in this study.

Perinatal chronic variable stress

Pregnant females were stressed starting from gestation day 11 (Figure 1A). Pregnant females underwent 1–2 stressors a day for 13 days. Both stressed and unstressed mice were brought to a test room under dim red light during their dark cycle. All animals were weighed, and females were placed back into their home cage and either returned to the housing rack or were stressed. The humidity and temperature in the test room were recorded each day. On the last day of stress (PP4), females remained in the test room for 1–2 hours before being exposed to foreign-born pups (see Appetitive Maternal Behavior). Stressors and behavior were conducted as follows:

Figure 1. Perinatal chronic variable stress and perinatal corticosterone treatment show distinct impacts on maternal weight and exploratory behavior.

A. Schematic showing experimental timeline for perinatal chronic variable stress (CVS). B. CVS shows no effect on maternal weight. CVS does not affect C. time in center or D. time in border in the open field test. However, open field E. distance moved (unpaired two-tailed t-test t=3.091, **p=0.0063), and F. velocity (unpaired two-tailed t-test t=2.289, *p=0.0344) are significantly reduced while G. immobility is significantly increased (unpaired two-tailed t-test t=3.122, **p=0.0059) in dams with CVS compared to control dams. H. Schematic depicting experimental timeline for perinatal chronic corticosterone (C-CORT) treatment. I. C-CORT treatment leads to reduced weight in the postpartum period (Two-way repeated measures ANOVA main effect of time x treatment interaction F_5–85=7.674, p=0.0001, Tukey’s multiple comparisons test postnatal day 1 **p=0.0064 and postnatal day 3 *p=0.0113). C-CORT treatment significantly reduced J. time in center (unpaired two-tailed t-test t=2.844, *p=0.0112) and K. time in border (Two-tailed Mann-Whitney test U=14, *p=0.0101) compared to control dams. However, C-CORT treatment did not affect L. distance moved, M. velocity, or N. immobility. Schematics developed in Biorender.com.

Stress Day 1 (GD11): overnight cage tilt (12h)

Stress Day 2 (GD12): morning 2h loud music, afternoon 2 h wet bedding

Stress Day 3 (GD13): morning 4h reverse light cycle, overnight (12h) food deprivation

Stress Day 4 (GD14): morning 5 min foot shock, afternoon 5 min tail suspension

Stress Day 5 (GD15): morning 2h no bedding, afternoon 1h restraint with shaking

Stress Day 6 (GD16): morning 2h loud music, overnight (12h) cage tilt

Stress Day 7 (GD17): morning 2h wet bedding, overnight (12h) reverse light cycle

Stress Day 8 (GD18): morning 2h rat bedding, afternoon 5 min tail suspension

Stress Day 9 (PP0): morning 2h no bedding, afternoon 5 min foot shock

Stress Day 10 (PP1): morning 2h wet bedding, afternoon 1h restraint with shaking

Stress Day 11 (PP2): morning 5 min swim stress, overnight (12h) cage tilt

Stress Day 12 (PP3): morning 5 min tail suspension, afternoon open field assay

Stress Day 13 (PP4): morning 5 min foot shock, afternoon pup exposure test and sacrifice

Perinatal Corticosterone Administration

Corticosterone (CORT; Sigma-Aldrich, St. Louis, MO, USA) was dissolved with 5% ethanol in sesame oil (Sigma-Aldrich, St. Louis, MO, USA) and administered at a dose of 20 mg/kg subcutaneously to the nape of the neck. Vehicle (Oil) mice were injected with sesame oil at the same volume. CORT dose was chosen based on previous studies that reported that 20mg/kg generated a depressive-like phenotype in C57BL/6J mice (Cameron et al., 2023; Yao et al., 2023) and Long-Evans rats (Hill et al., 2003). Mice were weighed and injections started on gestation day 11 and ended 4 days after birth for a total of 13 daily injections (Figure 1H). All injections were done within 2 hours of the start of the dark phase. Behavioral tests were performed 2 hours after the CORT administration.

Serum Corticosterone Measurement

Trunk blood samples were taken at the time of sacrifice after brief anesthetization with inhaled isoflurane and serum was isolated from whole blood by centrifugation (for adults, 1 mouse per replicate; for pups, 3 mice per replicate). A high-sensitivity corticosterone (CORT) enzyme immunoassay (EIA) was used and analyzed according to manufacturer’s instructions (Immunodiagnostic Systems Ltd, Fountain Hills, AZ, USA). Briefly, the percent binding (B/Bo%) of each calibrator, control, and sample was calculated by dividing the mean absorbance over the mean absorbance for ‘0’ calibrator and multiplied by 100. A calibration curve was used to plot B/Bo% on the ordinate against the concentration of corticosterone. A 4PL curve fit was applied.

Behavior Assays

Experiments were conducted during the dark phase under dim red light. Tests were recorded by Fly Capture cameras (Point Grey, Richmond, BC, Canada), and behaviors were scored by an observer blind to experimental conditions using Observer XT13 Software or Ethovision XT 13 or 18 (Noldus Information Technology, Leesburg, VA, USA).

Appetitive Maternal Behavior

Appetitive maternal behavior tests were conducted in the mouse’s home cage as previously described (Abdelmesih et al., 2023; Autry et al., 2021; Kohl et al., 2018; Wu et al., 2014). For dams with peripartum manipulations, the assay was conducted at least 2h after the final stress (5 min foot shock for CVS; injection for C-CORT). Mice were habituated to the testing room for 1–2 hours and then the lid of their cage was replaced with a clear Plexiglas lid with holes and were habituated for an additional 10 minutes. Two C57BL/6J pups 1–3 days old from a non-experimental C57BL/6J dam were presented in the cage in the opposite corner to the undisturbed home cage nest containing all of the dam’s own pups. Test sessions started at pup approach (female first touches the pup with its snout) and lasted for 10 minutes. The following behaviors were quantified: latency to retrieve, pup investigation (sniffing, close contact with snout), grooming (handling with forepaws and licking), nest building, time spent in the nest, crouching, rearing, digging, and self-grooming.

Behavioral sequences analysis

Behavioral sequences were quantified using several complexity metrics: Transition Entropy was computed as the Shannon entropy of transition probabilities between behaviors in ethogram sequences, reflecting the unpredictability of behavioral shifts; Normalized Transition Entropy divided this by the logarithm of unique behavior counts for sequence-length invariance; Switching Rate was calculated as the number of behavioral transitions per unit time, capturing dynamism; and LZC Normalized employed Lempel-Ziv complexity (LZC) to measure sequence compressibility normalized by sequence length to enable cross-comparison. Linear mixed-effects models (LMM) were fitted using statsmodels.mixedlm (Seabold and Perktold, 2010) with the formula Metric ~ C(Group)+C(Phase)+C(Group):C(Phase), where Group and Phase are categorical, incorporating random intercepts for mouse ID to account for repeated measures. Post-hoc simple effects analyses included Wilcoxon signed-rank tests for paired phase comparisons within groups (with rank-biserial correlation as effect size) and independent tests (t-test or Mann-Whitney U, based on normality via Shapiro-Wilcoxon) for group effects per phase, supplemented by permutation tests (10,000 iterations) and bootstrapped 95% confidence intervals (10,000 resamples) for robustness in small samples.

Transition Probability Analysis

Transition probability matrices were constructed for each mouse by tallying behavioral shifts in ethogram sequences, normalized by row sums to yield probabilities, and aggregated per group. Stationary distributions were derived via eigenvalue decomposition of smoothed matrices (epsilon=1e-9 added for stability), representing long-term behavior centrality. Group differences in node centrality were assessed using bootstrapped contrasts (1,000 resamples with replacement), computing p-values from the proportion of resamples exceeding observed differences. Edge (transition) probabilities were compared via Mann-Whitney U tests on per-mouse values, with two-tailed alternatives. Significant differences (p<0.05) informed network visualizations using NetworkX (Aric et al., 2008), where nodes were sized and colored by centrality shifts (red for increases, blue for decreases in the experimental group), and edges styled by probability differences (solid/thick for significant, dashed/thin for trends). A comprehensive summary table compiled means, differences, p-values, and significance for all nodes and edges across comparisons.

Open field

The open field assay started at least 2 hours after the stressor for Day 12 (5 min tail suspension for CVS; injection for C-CORT). Mice were assessed for activity in a 45cm × 45 cm open field at 40 lux for 5 min as previously described (Autry et al., 2009). The center was considered 15 cm × 15 cm, and the borders were 5 cm around the perimeter of the box. Time and frequency in the center and border, as well as distance moved and velocity were calculated using Ethovision XT13.

Fluorescence In Situ Hybridization

Fluorescence in situ hybridization (FISH) was performed as recommended by ACD Bio (Newark, CA, USA) using V2 RNAscope reagents. Briefly, fresh brain tissue was collected from animals 30 min after the end of a 10 min maternal behavior test for immediate early gene (Fos) studies after brief anesthetization by inhaled isoflurane as previously reported (Abdelmesih et al., 2023; Autry et al., 2021; Kohl et al., 2018; Wu et al., 2014). Brains were embedded in OCT (Tissue-Tek) and frozen with dry ice. Sections of 25 μm were prepared on a cryostat. Protease 3 was used for pretreatment. Fos (Cat No. 316921), Ucn3 (Cat No. 464861), and Crf (Cat No. 316091) probes were used as per the manufacturer’s instructions. Slides were mounted using Prolong Gold with DAPI. Four to seven brains per group were analyzed as in our previous histology experiments (Abdelmesih et al., 2023; Autry et al., 2021; Kohl et al., 2018; Wu et al., 2014). A Zeiss Axioscan was used for imaging at 10–20X magnification. The Zeiss AxioScan resolution for our experiments were 0.325 μm² per pixel with an effective NA of 0.45 using the 10X objective and 0.650 μm² with an effective NA of 0.8 using the 20X objective.

Image Analysis

Images were exported from Zen Blue software after spectral unmixing, and all CRF+ and Ucn3+ cells were counted and quantified for colocalization with Fos. Cells were manually counted in a region of interest (1.3mm × 0.75mm) around the 3^rd ventricle at Bregma AP −0.94 by an observer blinded to condition using FIJI Cell Counter. Two ROIs per mouse were quantified and averaged.

Data Analysis and Statistics

Data were analyzed in Graphpad Prism 10 with the investigator blinded to the experimental condition of the mice. Sample sizes were selected based on previous experiments (Autry et al., 2009; Autry et al., 2021; Kohl et al., 2018; Wu et al., 2014). We first determined if the data were normally distributed using a Shapiro-Wilk test. If data were normally distributed, we used an unpaired two-tailed t-test to compare two groups. For non-normally distributed data, we used a two-tailed Mann-Whitney U test. For repeated measures data, we used a Two-Way Repeated Measures ANOVA or Mixed Effects Repeated Measures test followed by Tukey or Fisher’s least significant difference post-hoc correction. Pup retrieval percentages are analyzed by Kolmogorov-Smirnov test. For colocalization experiments, we used Fisher’s exact test to compare the total number of Fos/marker double positive cells to the total number of Fos−/marker+ cell populations across all mice and expressed the data as proportional percentage in bars. P values reported as follows: * P<0.05, ** P<0.01, *** P<0.001, **** P<0.0001. All data are expressed as mean ± SEM. All source data with statistical test information, degrees of freedom, and effect size (Cohen’s d, KS D value, or partial eta squared) are reported in the Supplemental Source Data excel file.

Results

Peripartum CVS and peripartum C-CORT show distinct effects on maternal weight and exploratory behavior

We applied multimodal stressors on a variable schedule with different durations twice a day for 13 days (to avoid habituation) to stress females from GD11-PP4, or daily handling as a control (n=10 Control dams; n=10 CVS dams). We tested exploratory behavior in the open field assay on Day 12 of stress (PP3) followed by an appetitive maternal behavior assay on Day 13 (PP4) (Fig. 1A). We collected brain tissue and trunk blood 30 minutes after the behavior test. We found that maternal weight was not affected by peripartum CVS (Fig. 1B). Time spent in the center or border of an open field was also not affected by peripartum CVS (Fig. 1D–E). However, distance moved and velocity were reduced in dams with peripartum CVS compared to daily handled control dams (Fig. 1E–F). Conversely, immobility was significantly increased in dams with peripartum CVS (Fig. 1G). A small subset of females in our cohort was mated but did not subsequently become pregnant (n=5 daily handled control mated females; n=5 CVS mated females). We found that, like in dams, CVS did not affect weight or time spent in the center or borders of an open field, but did significantly reduce velocity in the open field in mated females compared to controls (Supp. Fig. 1A–F). These results suggest that CVS leads to hypoactivity in both dams and mated females, with dams additionally showing increased immobility.

In a second set of experiments, we chronically treated females from GD11-PP4, following the same behavioral timeline as peripartum CVS, with a moderate dose of corticosterone (C-CORT, 20 mg/kg s.c.), or vehicle (sesame oil) as a control (n=10 vehicle dams; n=9 C-CORT dams) (Fig. 1H). We found that C-CORT treatment significantly reduced dam weight at postpartum days 1 and 3 compared to vehicle-treated dams (Fig. 1I), suggesting a minor impact of C-CORT on body weight attributable to altered body composition, metabolism, and/or fluid retention as has been reported previously (Brummelte et al., 2012; Mishima et al., 2015). We found that C-CORT-treated dams spent less time in the center and more time in the borders of the open field compared to vehicle-treated dams (Fig. 1J–K–J). This difference in exploration was not accompanied by changes in distance moved, velocity, or immobility in the open field (Fig. 1L–N). These results suggest that peripartum C-CORT treatment leads to increased preference for the border of the open arena. Taken together, the results of both experiments indicate that peripartum chronic stressors differentially affect exploratory behavior: peripartum CVS induces depression-like hypoactivity and immobility, whereas peripartum C-CORT promotes anxiety-like, safety-seeking behavior.

Maternal behavior is resistant to both peripartum CVS and peripartum C-CORT

Next, we analyzed maternal care in both experiments by introducing two foreign newborn pups (1–2 days old) into the far corner of the dam’s undisturbed home cage. We quantified the percentage of pups retrieved, the latency to retrieve each introduced pup, the time spent investigating, grooming, crouching over pups, the time spent in the nest with the pups, as well as rearing, self-grooming, and digging. For the maternal behavior experiments, we included an additional cohort of females for the peripartum C-CORT treatment (total of n=11 vehicle-treated dams; n=18 C-CORT-treated dams). We found that peripartum CVS impacted the pattern of caregiving across the 10-minute trial (Fig. 2A). There was a leftward trend in pup retrieval in the CVS females (Fig. 2B) that resulted in a trend toward decreased retrieval latency of the 1^st (Fig. 2C) and second (Fig. 2D) pup. We observed a significantly reduced duration of retrieval compared to control dams (Fig. 2E), suggestive of more efficient retrieval. Other caregiving behaviors were not affected by peripartum CVS (Fig. 2F–J). We additionally studied the behavioral patterning of caregiving between control and CVS dams using behavioral syntax analysis to, the normalized Lempel-Ziv complexity (LZC) metric, switching rate, and transition entropy. For LZC normalized, switching rate, and transition entropy, we divided the trial into a retrieval phase (first 125 seconds) and a caregiving phase (the remaining 425 seconds of the 10-minute trial) because we reasoned that different behavioral strategies may be used before and after pup retrieval. Behavior syntax shift analysis identified no significant node centrality shifts or edge probabilities, implying preserved behavioral syntax under CVS in dams (Fig. 2K). LZC Normalized, a measure of sequence complexity, showed a significant phase reduction (main effect, p<0.001), suggesting simplified patterns in caregiving, but no group or interaction effects (p>0.3). Post-hoc analyses confirmed these phase-driven changes without group-specific differences (Fig. 2L). Switching rate exhibited a significant phase effect (main effect, p=0.004), with behaviors switching less frequently during caregiving across both groups (Fig. 2M). We observed no significant group or interaction effects on transition entropy (Fig. 2N). Together, these results suggest that caregiving in dams is resistant to perinatal CVS, with CVS dams retrieving more efficiently compared to control dams.

Figure 2. Appetitive maternal behavior is resistant to perinatal CVS.

A. Ethograms depicting pup-directed behavior across the 10 minutes trial are shown for (top) Control and (bottom) CVS dams (each row is one mouse). B. Retrieval latencies over the trial are similar between control and CVS dams. Latency to retrieve the C. first or D. second pup is not different between groups. E. Retrieving duration is significantly reduced in CVS dams (Welch’s t-test t=3.496 df=11.04 p=0.005 Cohen’s d=1.563). We observed no significant differences in F. pup investigation, G. pup grooming, H. crouching over pups, or I. time in the nest between groups. J. Rearing is not different significantly between groups. K. Behavioral syntax shift comparing control dams and CVS dams reveals no significant changes among nodes. There were no significant differences in behavioral caregiving patterning between control and CVS dams during the retrieval or caregiving phases using metrics L. normalized LCZ, M., switching rate, or N., transition entropy.

On the other hand, we found that CVS in mated nonpregnant females significantly reduced the percentage of pups retrieved (Supp. Fig. 2A). Mated nonpregnant females with CVS showed a significant rightward shift in retrieval (Supp. Fig. 2A–B) compared to control mated nonpregnant females, accompanied by trends toward increased latencies to retrieve the first and second pup (Supp. Fig. 2C–D). We did not observe an impact of CVS on other caregiving parameters in nonpregnant females (Supp. Fig. 2E–I). However, mated nonpregnant females with CVS displayed significantly more rearing behavior (Supp. 2J), consistent with typical home cage stress response behavior (Boulle et al., 2014; Fuzesi et al., 2016). Behavioral syntax shift analysis uncovered significant edges: increased retrieving->pup grooming probability in CVS Mated (+36.67%, p=0.009) and decreased nest building->in nest (−46.54%, p=0.010), alongside a rearing centrality trend (+4.84%, p=0.058), suggesting that CVS increases pup-directed syntax in mated females (Supp. Fig. 2K), potentially reflective of the longer latency to retrieve. We observed no notable effects on LZC normalized (Supp. Fig. 2L). Switching rate showed a robust phase decrease (main effect, p<0.001) in both mated female groups (Supp. Fig. 2M), similar to the effect in dams. In this experiment, transition entropy displayed a significant phase effect (main effect, p=0.006) and interaction effect (main effect, p=0.031) (Supp. Fig. 2N). These data reveal that mated females show greater susceptibility to the impact of CVS on caregiving relative to dams.

We found that peripartum C-CORT also impacted the pattern of caregiving across the 10-minute trial (Fig. 3A). We did not observe effects on retrieval behaviors (Fig. 3B–E), or other caregiving behaviors (Fig. 3F–H), but we saw a trend toward C-CORT-treated dams spending more time in the nest compared to vehicle-treated dams (Fig. 2I). We did not observe an effect of C-CORT on rearing (Fig. 3J). Behavioral syntax shift analysis highlighted one significant edge: decreased rearing->nest building in CORT (−0.91%, p=0.029), with a retrieving centrality trend (+0.89%, p=0.052), pointing to C-CORT-induced shifts toward retrieval-focused behaviors (Fig. 3K). LZC Normalized revealed treatment (main effect, p=0.036) and phase effects (main effect, p<0.001), with post-hoc analysis showing a marked phase reduction in C-CORT treated dams (p<0.001, effect size=1.0) but not vehicle-treated dams (p=0.320) (Fig. 3L). Switching rate exhibited phase (main effect, p<0.001) and interaction effects (main effect, p=0.037), with post-hoc analysis confirming a significant retrieval-to-caregiving reduction only in CORT (p<0.001, effect size=1.0; Vehicle p=0.278) (Fig. 3M). For Vehicle versus C-CORT treatments, transition entropy showed a strong phase effect (main effect, p<0.001) without group or interaction influences (p>0.1). Overall, stress paradigms (CVS, C-CORT) amplified phase-dependent reductions in behavioral complexity, particularly in mated females and C-CORT-treated groups. These findings underscore context-specific disruptions in maternal syntax, with C-CORT eliciting more pronounced simplification during caregiving. Node and edge analyses further reveal subtle reallocations toward pup engagement under stress in dams, without broad syntax overhaul. Altogether, our results suggest that appetitive maternal behavior is resistant to peripartum CVS and peripartum C-CORT treatment, while alloparental pup retrieval behavior is susceptible to CVS.

Figure 3. Appetitive maternal behavior is resistant to perinatal chronic corticosterone.

A. Ethograms depicting pup-directed behavior across the 10 minutes trial are shown for (top) Vehicle and (bottom) C-CORT treated dams (each row is one mouse). B. Retrieval latencies over the trial are similar between vehicle and CORT treated dams. Latency to retrieve the C. first or D. second pup, or E. time spent retrieving is not different between groups. We observed no significant differences in F. pup investigation, G. pup grooming, H. crouching over pups or I. time in the nest (although there is a trend toward increased time in nest in C-CORT treated dams: two-tailed Mann-Whitney U test U=64, p=0.0520) between groups. J. There is no difference in rearing between the groups. K. Behavioral syntax shift analysis reveals that transitioning from rearing to nest building is significantly less likely in C-CORT dams compared to vehicle dams. L. Normalized LCZ revealed significantly higher behavioral complexity in C-CORT treated dams during the caregiving phase, and M., significantly reduced switching rate in C-CORT treated dams during the caregiving phase. N. There were no significant differences in transition entropy between vehicle and C-CORT treated groups.

Pup weight is affected by maternal C-CORT treatment, but not peripartum CVS

We tracked litter size and weight at PP0 and PP2 for both HPA axis challenge groups. Litter size was not affected by peripartum CVS (Fig. 4A) or by peripartum C-CORT (Fig. 4C). However, from birth (PP0) to PP2, we did observe a significant loss of an average of ~1.5 pups per litter in the control handled group (Fig. 4A), as well as both vehicle and C-CORT-treated groups (Fig. 4C). CVS dams had slightly fewer pups per litter at birth (PP0), so the effect of pup loss at PP2 was not significant. Despite a minor impact on number of pups, peripartum CVS did not affect average pup weight, with both control handled and CVS dams’ litters gaining weight over the postpartum period (Fig. 4B). On the other hand, maternal C-CORT treatment led to lower average pup weight compared to offspring from vehicle treated dams at birth (PP0), though this difference was recovered by PP2 (Fig. 4D). This lower birth weight in maternal C-CORT-treated offspring mirrors the lower maternal weight observed in C-CORT-treated dams (Fig. 1I). Together, these data suggest that chronic perinatal C-CORT treatment impacts both maternal and offspring weight, and while offspring weight recovers by PP2, maternal weight remains low.

Figure 4. Dams lose pups, but their litters gain weight from birth to PP2.

A. Control and CVS dams had comparable litter sizes, but control handled dams lost an average of 1.5 pups from PP0 to PP2 (Mixed effects repeated measures ANOVA, significant main effect of time p=0.0035, post-hoc Fisher’s least significant difference comparisons significant for control PP0 vs. PP2, p=0.0062). B. Control and CVS dams have pups with comparable average weight, with litters gaining weight over time (Mixed effects repeated measures ANOVA, significant main effect of time p=0.0003, post-hoc Fisher’s least significant difference comparisons significant for control PP0 vs. PP2, p=0.0005 and CVS PP0 vs. PP2, p=0.0441). C. Vehicle and C-CORT treated dams have comparable litter sizes, with both groups losing an average of 1.5 pups from PP0 to PP2 (Mixed effects repeated measures ANOVA, significant main effect of time p=0.0001, post-hoc Fisher’s least significant difference comparisons significant for vehicle PP0 vs. PP2, p=0.0003 and C-CORT PP0 vs. PP2, p=0.0003). D. C-CORT treated dams give birth to pups that are on average smaller than Vehicle treated dams, but this weight difference in recovered by PP2 (Mixed effects repeated measures ANOVA, significant main effect of time p=0.0001 and significant main interaction effect of time x treatment p=0.0485, post-hoc Fisher’s least significant difference comparisons significant for vehicle vs. C-CORT PP0 p=0.0311, vehicle PP0 vs. PP2, p=0.0025 and C-CORT PP0 vs. PP2, p=0.0001).

Circulating corticosterone levels in dams and offspring are affected by peripartum C-CORT

To assess the impact of the stressors using a physiological readout, we analyzed circulating corticosterone levels in dams from both experiments. We found that peripartum CVS did not significantly alter serum corticosterone levels (Fig. 5A). Similarly, mated nonpregnant females that experienced CVS did not show altered circulating corticosterone levels (Supp. Fig. 3). In dams with peripartum C-CORT treatment, we found significantly reduced circulating corticosterone levels compared to vehicle-treated dams (Fig. 5B). This result suggests that the exogenously introduced corticosterone suppressed activity of the dam’s hypothalamic-pituitary-adrenal (HPA) axis (Andrews et al., 2012; Kinlein et al., 2015).

Figure 5. Perinatal C-CORT, but not CVS, alters maternal corticosterone.

A. Serum corticosterone is not significantly different between control and CVS dams. B. Circulating corticosterone is significantly reduced in dams treated with C-CORT perinatally (two-tailed unpaired t-test t=3.547, p=0.0014). C. Serum corticosterone is significantly increased in 4-day-old offspring of dams treated with perinatal C-CORT (two-tailed unpaired t-test t=4.356, p=0.0014).

Because maternal corticosterone can be passed to the offspring through the blood-placental barrier during gestation or through breastmilk postnatally (Brummelte et al., 2010; Chatuphonprasert et al., 2018; Hollanders et al., 2017), we also examined offspring corticosterone levels at PP4 (n=6 pooled litters from vehicle-treated dams; n=6 pooled litters from C-CORT-treated dams). We found that offspring from C-CORT-treated dams showed significantly increased serum corticosterone levels compared to offspring from vehicle-treated dams (Fig. 5C), confirming that C-CORT-treated dams passed maternal corticosterone on to the pups either during pregnancy or through breastmilk.

Perinatal CVS and perinatal C-CORT have distinct effects on neural activation of hypothalamic cell populations after pup exposure

Previous studies suggest that the impact of stress is mediated by hypothalamic cell populations, including corticotropin-releasing factor-expressing neurons in the paraventricular hypothalamus (PVH^CRF) as well as urocortin-3-expressing neurons of the perifornical area of the hypothalamus (PeFA^ucn3) (Abdelmesih et al., 2023; Daviu et al., 2020; Henckens et al., 2016; Jiang et al., 2019; Venihaki et al., 2004). We examined the colocalization among Fos, Ucn3, and CRF, in the PVH and PeFA 30 minutes after exposure to pups using fluorescent in situ hybridization in both the perinatal CVS (Fig. 6A, Supp. Fig. 4A, B) and C-CORT (Fig. 6F, Supp. Fig. 4C, D) experiments on postnatal day 4. For the CVS experiment, we found no significant difference in the percentage of PeFA^ucn3 or PVH^CRF neurons colocalized with Fos between groups (Fig. 6B, D). In examining the proportional percentage Fos+ versus Fos− neurons, we observed no difference in the PeFA^ucn3 neurons (Fig. 4C), but a significant reduction in PVH^CRF Fos+ versus Fos− neurons in CVS dams compared to control dams (Fig. 6E).

Figure 6. Perinatal CVS and perinatal CORT have distinct effects on neural activation of hypothalamic cell populations after pup exposure.

A. Representative image of the paraventricular nucleus and perifornical area of hypothalamus from a Control dam (top) and CVS dam (bottom) (DAPI, blue; CRF, green; Ucn3, magenta; Fos, orange; scale bar: 200 μm; 3v: 3^rd ventricle) with insets of the 1. PVH^CRF neurons and 2. PeFA^ucn3 neurons colocalized with fos (colocalization indicated by yellow arrows; scale bar: 20 μm; 20X magnification). B. Quantification of the percentage of Fos and Ucn3 colocalization in the PeFA in control versus CVS dams reveals no significant differences. C. Proportional percentage of PeFA Ucn3+/Fos+ neurons is not significantly different between control and CVS dams (Control n = 720 cells; CVS n = 400 cells). D. Quantification of the percentage of Fos and CRF colocalization in the PVH in control versus CVS dams reveals no significant differences. E. Proportional percentage of PVH CRF+/Fos+ neurons is reduced in CVS dams compared to control dams (two-sided Fisher’s exact test: Control n = 1805 cells; CVS n = 730 cells; p = 0.0014; represented in bars). F. Representative image of the paraventricular nucleus and perifornical area of hypothalamus from a Vehicle-treated dam (top) and C-CORT-treated dam (bottom) (DAPI, blue; CRF, green; Ucn3, magenta; Fos, orange; scale bar: 200 μm) with insets of the 1. PVH^CRF neurons and 2. PeFA^ucn3 neurons colocalized with fos (colocalization indicated by yellow arrows; scale bar: 20 μm; 10X magnification). G. Quantification of percentage of Fos and Ucn3 colocalization in the PeFA reveals a trend toward an increase in C-CORT-treated dams compared to vehicle (Two-tailed Welch’s t-test t = 2.021, p = 0.0842). H. Proportional percentage PeFA Ucn3+/Fos+ neurons is significantly increased in C-CORT-treated dams compared to vehicle-treated dams (two-sided Fisher’s exact test: Vehicle n = 717 cells; C-CORT n = 736 cells; p = 0.0001; represented in bars). I. Quantification of the percentage of Fos and CRF colocalization in the PVH shows a significant decrease in C-CORT-treated versus vehicle-treated dams (unpaired two-tailed t-test t = 2.563, p = 0.0282). J. Proportional percentage of PVH CRF+/Fos+ neurons is increased in C-CORT-treated dams compared to vehicle-treated dams (two-sided Fisher’s exact test: vehicle n = 1066 cells; C-CORT n = 1509 cells; p = 0.0001; represented in bars).

In the C-CORT experiment, we saw a trend toward an increase in the percentage of Fos+ PeFA^ucn3 neurons (Fig. 6G), and a significant reduction in the percentage of Fos+ PVH^CRF neurons between groups (Fig. 6I). Consistent with these results, the proportional percentage of Fos+ PeFA^ucn3 cells was significantly increased (Fig. 6H), while the proportional percentage of PVH^CRF Fos+ neurons was significantly decreased in C-CORT-treated compared to vehicle-treated dams (Fig. 6J). Our observation of decreased activity in PVH^CRF neurons with both perinatal CVS and perinatal C-CORT treatment is consistent with previous studies showing reduced neuronal excitability and Fos expression in PVH^CRF neurons after chronic stressors (Girotti et al., 2006; Matovic et al., 2020; Morrison et al., 2017; Radley and Sawchenko, 2015). However, we find that perinatal CVS and perinatal C-CORT treatment have distinct effects on activity in the PeFA^ucn3 neurons after pup exposure, suggesting a role for PeFA^ucn3 neuron activity in stress-coping behaviors with respect to appetitive maternal care as we have previously observed with other stress paradigms (Abdelmesih et al., 2023).

Discussion

In the present study, we compared the impact of chronic variable stress and chronic corticosterone administration in the perinatal period on maternal caregiving behavior in mice. These stressors had distinct effects on exploratory behavior, with perinatal CVS leading to a depressive-like hypoactive phenotype characterized by reduced activity and increased immobility, and perinatal C-CORT leading to an anxiety-like phenotype characterized by no change in activity but reduced time in the center of the open field. Despite observing alterations in mood-related behavior, we found only subtle differences in appetitive caregiving behavior in dams exposed to either perinatal CVS or perinatal C-CORT, even with some trends toward improved caregiving in certain measures with these stressors. However, we observed a reduction in alloparental care in mated nonpregnant females exposed to chronic CVS, suggesting that post-partum physiological effects may buffer the impact of stress on caregiving behavior in dams. We observed that both stress paradigms reduced activity in the PVH^CRF neurons after pup exposure, while C-CORT treatment increased activity in the PeFA^ucn3 neurons after pup exposure. These results suggest that hypothalamic cell activity in response to pup-directed behavior is affected in specific ways by perinatal CVS and perinatal C-CORT to buffer appetitive maternal behavior against the impact of stress.

Appetitve maternal behavior is resistant to a variety of stressors

Several studies have investigated the effects of chronic perinatal stressors including limited bedding and nesting, maternal separation and early weaning, chronic social stress, and others, on maternal caregiving behavior (Orso et al., 2019). Taken together, the results do not reveal a consensus on whether these stressors impact maternal care and in which direction. Potential reasons for these disparate results may be attributed to the postnatal time at which the behavior is assessed, method of testing caregiving behavior (undisturbed observation vs. pup scattering), and pre- or postnatal time window/duration of the chronic stress. Prior research shows that rodent maternal behaviors adjust over the postnatal period to adapt to their pups’ needs, and that earlier postnatal timepoints (i.e. PP1-PP6) reveal greater differences in caregiving behavior of stressed dams (Curley et al., 2009; Franks et al., 2015; Rickenbacher et al., 2017; Rombaut et al., 2023). For caregiving assessments, most studies observe undisturbed care with a focus on behaviors such as licking, grooming, nesting, and time spent in the nest. Pup retrieval cannot typically be assessed without scattering pups away from the nest. Pup retrieval relies on auditory and olfactory cues to locate the pups and return them to the nest, and is sensitive to circuit and molecular manipulations in several brain areas (Autry et al., 2021; Fang et al., 2018; Kohl et al., 2018; Krishnan et al., 2017; Marlin et al., 2015; Morrison et al., 2017; Stagkourakis et al., 2020; Wu et al., 2014); thus, it is a useful measure for assessing changes in maternal responsivity. Finally, studies suggest that starting stress before parturition or even prior to pregnancy may contribute to greater behavioral susceptibility to the impact of stress in mice and humans (Coussons-Read, 2013; Kim et al., 2010; Morrison et al., 2017; Rinne et al., 2023; Wriedt et al., 2024).

In the present study, we compared an ethological stressor, chronic variable stress, with a controlled increase in HPA axis load using chronic corticosterone administration, using the same perinatal time window from GD11-PP4. We assessed exploratory behavior and caregiving behaviors with the same timeline as well. In addition, we used the introduction of foreign pups to the home cage to test pup retrieval behavior without disturbing the dam or the litter. The choice to use foreign pups was in part to keep the home cage undisturbed prior to the Fos experiment and also to have data that could be compared to previously collected datasets for alloparental/paternal behavior and/or datasets in which the dam’s offspring would be tested in subsequent tests, so that they did not get separated from their mother. Exposure to foreign pups is frequently used in studies examining the neurobiological mechanisms underlying caregiving behavior (Abdelmesih et al., 2023; Autry et al., 2021; Carta et al., 2025; Kohl et al., 2018; Marlin et al., 2015; Mei et al., 2023; Mogi et al., 2017; Tachikawa et al., 2013; Wang and Storm, 2011; Wu et al., 2014).

We collected trunk blood and brains 30 minutes after the introduction of foreign pups on the same early postnatal day (PP4) in both sets of experiments. In this way, we could compare the impact of the two types of chronic HPA axis challenges, ethological and pharmacological, on all behavioral, hormonal, and circuit activity parameters collected. In this study, we found that appetitive maternal behavior was resistant to both types of chronic stress despite significant impacts to mood-related behavior. These data are in line with our previous findings that maternal care in C57/BL6 dams assessed using foreign pup introduction was resilient to post-partum CVS (PP2-PP12), post-partum chronic restraint stress (PP2-PP18), and post-partum chronic intruder stress (PP2-PP16) (Abdelmesih et al., 2023). Altogether, these findings suggest that appetitive maternal behavior may be resistant to a variety of chronic peripartum stressors. We postulate that physiological and brain structure function adaptations that subserve caregiving may also impart stress resilience to protect maternal caregiving and in turn benefit offspring development.

Hypothalamic circuit adaptations in the perinatal period

Imaging studies in humans are revealing region-specific anatomical differences that are associated with caregiving behaviors (Kim, 2016; Kim et al., 2010; Spalek et al., 2024). The hypothalamus is known to undergo adaptations in the human brain during pregnancy and postpartum (Kim et al., 2010; Spalek et al., 2024) and peripartum stress can disrupt these adaptations (Kim, 2021). Preclinical studies have highlighted specific peripartum molecular adaptations in the hypothalamus that may mediate structural/functional plasticity in the maternal brain. Research has shown that regulation of inhibition in PVH^CRF neurons via the K+/Cl co-transporter KCC2 during pregnancy and postpartum is required to suppress HPA axis responses during pregnancy that promote caregiving (Melon et al., 2018). Recent studies have highlighted the critical role for estrogen, progesterone, and prolactin receptor function to refine medial preoptic area galanin-expressing neuron activity underlying caregiving in dams and sires (Ammari et al., 2023; Stagkourakis et al., 2020). A study examining changes in the aging female mouse hypothalamus revealed dynamic transcriptional changes across the lifespan in neuropeptide-expressing cell populations, suggesting that these cell types are a key substrate in mediating adaptive physiological and behavioral processes (Hajdarovic et al., 2022).

In the present study, we examined the function of two neuropeptidergic cell populations in the hypothalamus, the PVH^CRF and PeFA^ucn3 expressing neurons, in response to pup-directed behavior. As previously mentioned, decreased activity of PVH^CRF neurons during pregnancy and postpartum is thought to dampen maternal stress and support caregiving behavior (Melon et al., 2018). Indeed, fiber photometry recordings in virgin female mice reveal that activity in PVH^CRF neurons is decreased during pup-directed behavior (Kim et al., 2019). In our hands, we see that maternal caregiving is not disrupted by either peripartum CVS or peripartum C-CORT, and this preservation of caregiving in the face of stress could be in part attributed to our observation that activity of PVH^CRF neurons decreased in stressed dams after pup exposure. While acute stress has been shown to increase Fos expression in CRF neurons, repeated stress, even when followed by a novel stress, does not in male or female mice (Abdelmesih et al., 2023; Bonaz and Rivest, 1998; Matovic et al., 2020; Walker et al., 2019). This is consistent with evidence showing that chronic stress reduces Fos expression in the paraventricular nucleus in rats (Ostrander et al., 2009).

Peripartum CVS had no impact on pup-related activity in the PeFA^ucn3 neurons. In virgin males and females, activity of these neurons is associated with infant-directed neglect and aggression (Autry et al., 2021; Inada et al., 2022), and inhibition of PeFA^ucn3 neurons can block the effect of chronic restraint stress on infant-directed neglect in virgin female mice (Abdelmesih et al., 2023). On the other hand, in dams with postnatal CVS, we previously observed that higher activity in PeFA^ucn3 neurons was correlated with increased nest-building behavior. Here, we did not observe increased PeFA^ucn3 neuronal activity with perinatal CVS, but instead with perinatal C-CORT treatment. This increase in PeFA^ucn3 neuron activity after pup interaction could be a stress-coping mechanism to preserve maternal care, as we observed in our postnatal CVS experiment (Abdelmesih et al., 2023), because we did observe a trend toward more time spent in the nest in dams with perinatal C-CORT treatment, in line with our finding that postpartum CVS was correlated with increased nest-building behavior. Alternatively, the increased PeFA^ucn3 neuron activity could be attributed to cell-intrinsic changes due to expression of glucocorticoid receptors, mineralocorticoid receptors, or CRF receptors 1 and 2, given that all these factors may be regulated by corticosterone. In the future, it will be important to determine how gene expression changes in PeFA^ucn3 neurons after acute or chronic stress, after the transition to parenthood, and at the intersection of these physiological states.

Assessing alloparental and maternal care in mice

In the present study, we assessed appetitive maternal behavior by introducing 2 foreign pups to the dam’s undisturbed home cage. Previous research has shown that mouse dams and sires do not recognize individual pups until near weaning (PP19–21) and will accept and care for foreign pups (Abdelmesih et al., 2023; Autry et al., 2021; Kohl et al., 2018; Mak and Weiss, 2010; Wu et al., 2014). The utility of this assay is that we can compare caregiving behavior in virgin, mated, pregnant, and postpartum females or virgin, mated, and sire males under similar experimental conditions. However, most studies of maternal care study dam behavior with their own pups using naturalistic observations or by scattering the pups to observe pup retrieval (Krishnan et al., 2017; Morrison et al., 2017; Orso et al., 2019; Zoubovsky et al., 2020). This difference in method for testing maternal behavior could underlie disparate results on the impact of stress on caregiving. In the future, including tests of naturalistic behavior over long periods of time, combined with acute pup retrieval assays with both scattering of a dam’s own pups as well as introduction of foreign pups to the dam’s undisturbed cage, could provide a more comprehensive view of how caregiving is affected by experimental manipulations.

Using chronic corticosterone as an HPA axis challenge in the perinatal period

In the present study, we sought to overcome individual variability in dams’ responses to stress by subcutaneous administration of a controlled dose of corticosterone to increase HPA axis load chronically. Subcutaneous administration of CORT reliably leads to elevated plasma CORT levels in rodents (Kott et al., 2016). Prenatal CORT has previously been shown to reduce maternal care and affect offspring behavior in rodents (Brummelte and Galea, 2010; Catalani et al., 2011). Our chosen dose, 20 mg/kg, is moderate and has previously been used in mice and rats to induce anxiety and depression-like behavior (Cameron et al., 2023; Gregus et al., 2005; Hill et al., 2003; Johnson et al., 2006). Guided by this prior evidence, we performed a pilot study in a small cohort of mice. Based on our monitoring of weight and exploratory behavior, we decided to move forward with this dose in a larger cohort. Exogenously applied CORT is rapidly cleared from the bloodstream within minutes of application in rodents (Osterlund and Spencer, 2011; Osterlund et al., 2016; Waters and McCormick, 2011). In addition, chronic administration of CORT is known to suppress endogenous HPA axis function (George et al., 2017; Kinlein et al., 2019). A study in lactating mice showed that around 120 minutes after a dose of methylprednisone, a corticosterone analog, circulating CORT was decreased below baseline (Windle et al., 2013). In our study, we measured serum corticosterone around 2.5 hrs after the final administration of CORT. Thus, the significant decrease we observe in circulating CORT in C-CORT-treated dams is likely due to the timepoint of sampling after the final CORT dose together with the chronic suppression of the HPA axis. We observed a significant increase in serum corticosterone levels in the offspring, suggesting that our dosing leads to short-term increases of maternal CORT that are passed either through the placenta or through breastmilk to the pups. A caveat to our results is that we did not include multiple doses of CORT to compare the severity of effects on maternal care. However, higher doses of CORT (i.e. 40 mg/kg) have been reported to more significantly affect weight and fluid intake and lead to reduced litter size in post-partum rats (Brummelte and Galea, 2010). Indeed, even with the 20 mg/kg dose, we had a few dams lose their litter (5 dams across both cohorts). In the future, using multiple doses could help uncover how HPA axis load in the perinatal period affects maternal behavior in mice.

Conclusions

Altogether, our results support the idea that adaptations in maternal physiology and brain function contribute to stress resistance, thereby protecting caregiving behavior. In the future, we will focus on uncovering circuit and molecular adaptations in the hypothalamus and other brain regions that confer stress resistance.

Supplementary Material

MMC1

Supplemental Figure 1. Mated nonpregnant females that went through CVS show similar weight and exploratory behavior phenotypes as CVS dams. A. CVS does not affect weight in mated, non-pregnant females compared to controls. CVS in mated females does not affect B. time in the center, C. time in the border, or D. distance moved in the open field arena. We observed that mated females exposed to CVS show reduced velocity in the open field (unpaired two-tailed t-test t=2.774, *p=0.0241) compared to controls, similar to the phenotype in dams with CVS. F. Immobility was not significantly affected by CVS in mated nonpregnant females.

MMC4

Supplemental figure 2. Mated females with CVS are slower to retrieve than unstressed mated females. A. Ethograms depicting pup-directed behavior across the 10 minutes trial are shown for (top) Control-Mated and (bottom) CVS-Mated females (each row is one mouse). B. Retrieval latencies over the trial are shifted to the left in mated females with CVS compared to controls (Kolmogorov-Smirnov test D=0.4483, **p=0.0059). Latency to retrieve the C. first or D. second pup, or E. time spent retrieving is not significantly different between groups. We observed no significant differences in F. pup investigation, G. pup grooming, H. crouching over pups or I. time in the nest between groups. J. CVS-Mated females spent significantly more time rearing than control females (two-tailed unpaired t-test t=2.847, df=8, Cohen’s d=−1.801, p=0.0216). K. Behavioral syntax shift analysis reveals that transitioning from retrieving to pup grooming is significantly more likely, but transitioning from nest building to in nest is significantly less likely in CVS-mated compared to Control-Mated females. There were no significant differences in behavioral caregiving patterning between control-mated and CVS-mated females during the retrieval or caregiving phases using metrics L. normalized LCZ, M., switching rate, or N., transition entropy.

MMC2

Supplemental figure 3. Mated nonpregnant females with CVS have similar circulating corticosterone to control mated females. Serum corticosterone is not significantly different between groups in mated females with or without CVS.

MMC5

Supplemental figure. Colocalization of PeFA^Ucn3 and PVH^CRF neurons with Fos single channel images. A-D. Insets from Figure 4 displayed with single channel images (DAPI, blue; CRF, green; Ucn3, magenta; Fos, orange; colocalization indicated by yellow arrows; scale bar: 20 μm). A. Insets associated with Figure 4A (top). B. Insets associated with Figure 4A (bottom). C. Insets associated with Figure 4F (top). D. Insets associated with Figure 4F (bottom).

Highlights.

• Perinatal CVS and perinatal C-CORT differentially impact mood-related behavior in mouse dams

• Pup retrieval is intact in dams exposed to both stressors, but reduced in mated females

• Perinatal C-CORT treatment decreased circulating CORT in dams, but increased it in pups

• Both stressors decreased Fos expression in CRF neurons of the PVH after pup interaction

• Perinatal C-CORT increased Fos expression in Ucn3 neurons of the PeFA after pup interaction

Data availability

Source data will be made available upon publication.