Maternal-induced shifts in allostatic demands: Reproductive experience alters emotional and cognitive biobehavioral responses in rats (Rattus norvegicus)

Abstract

The arrival and subsequent care of offspring require abrupt shifts in biobehavioral responses in mammalian mothers. In the current study, female rats with one reproductive experience [primiparous (PRIM) rats, n = 8] or no reproductive experience [nulliparous (NULL) rats, n = 8] were assessed in a dry land maze to determine both learning acquisition and responses to uncertainty/prediction errors during the probe trial. Additionally, rats were observed in a swim task and an open field arena to assess responsiveness to varied environmental challenges. Results indicated that the PRIM rats investigated more previously baited wells during the probe trial (on-task behavior) whereas the NULL rats exhibited more peripheral-oriented rearing responses (off-task behavior). Further, a nonsignificant trend was observed indicating more dive responses in the PRIM animals. Focusing on endocrine markers, the PRIM animals had higher DHEA/CORT ratios than the NULL animals following the probe trial. Finally, PRIM animals had less hippocampal glucocorticoid receptor immunoreactivity and more hippocampal BDNF immunoreactivity than NULL animals. In sum, behavioral, endocrine and neural markers suggest that PRIM rats exhibit long-lasting modifications to stress responsivity.

1. Introduction

Outside of the predictable and safe conditions of the laboratory, the wild maternal rat faces many challenging situations requiring strategic responses to assure her survival, as well as the survival of her offspring [1,2]. Consequently, physiological functions are modified to accommodate maternal responses to environmental threats to prevent the female from experiencing allostatic load, a marker of an inefficient stress response [3], during the high energy expenditure climate surrounding the maternal experience. Accordingly, laboratory rats with maternal experience have been found to be more efficient hunters and foragers [4,5] and exhibit enhanced learning, persisting through the challenges of aging [6], stress exposure [7] kainic acid-induced neural insults [8], and social competition [9].

In addition to behavioral modifications, a well-established literature has emphasized alterations in the costly stress response in lactating rats [10,11]. Specifically, lactating females experience diminished HPA activity [12,13] and exhibit less anxiety in an open-field task [14]; further, older maternal rats exhibit less anxiety in an elevated-plus maze [9] and placement in restraint tubes [15]. Given that glucocorticoids are critical regulators of homeostasis and allostasis [16], if an animal fails to adapt to various stressors, allostatic load is experienced (e.g., psychiatric-like illness, cardiovascular disease) and the distribution of resources to the pups becomes compromised [17,18]. Alternatively, dehydroepiandrosterone (DHEA), released along with corticosterone from the adrenal gland, is associated with antiglucocorticoid and antiglutametergic effects in the brain and may provide neuroprotective effects [19,20]. The stress-activated endocrine response is also influenced by the activity of glucocorticoid (GR) and mineralocorticoid receptors (MR), especially in the hippocampus [21,22]. Specifically, hippocampal MR activity provides tonic inhibition of circulating corticosteroids that modulate excessive stress responsivity [23].

In keeping with the traditional definition of resilience referring to an animal’s achievement of successful outcomes during various adverse experiences [24], the resilient maternal rat must maintain efficient allostatic functioning to successfully raise her offspring. Consequently, the goal of the current study was to investigate relevant neurobiological and behavioral responses in maternal and virgin rats to evaluate the contributions of endocrine, behavioral and neural markers of allostatic responses. The assessments were conducted postweaning to avoid confounds related to the maternal rats being separated from their pups, an event that cannot be replicated in virgin animals. It was hypothe-sized that the maternal-experienced animals would exhibit more resilience-aligned responses (e.g., increased neuroplasticity markers, higher DHEA/corticosterone endocrine ratios and efficient behavioral responses) than their virgin counterparts.

2. Method and materials

2.1. Animals

Upon the arrival of 16 four-month-old female Long Evans rats, 8 primiparous (PRIM) rats with one pregnancy/lactation experience and 8 nulliparous rats (NULL; Envigo, Frederick, MD) were pair-housed in 48 × 26 × 21 cm cages with another animal from the same experimental group. Except for brief periods prior to behavioral assessments, food and water were provided ad libitum (Teklad Lab, Envigo). When mild food deprivation was necessary for behavioral assessments, food was removed three hours prior to testing and was returned directly after the assessment was completed. The colony room was maintained on a 12 h light/dark cycle with lights on at 0800 h. The current study was approved by the Randolph-Macon College Institutional and Animal Care and Use Committee.

2.2. Novel environment task

One week following their arrival to the laboratory, all rats were assessed in a novel environment task in a 122 cm × 91 × 51 cm arena with a novel wire mesh container (13 cm diameter × 9 cm high) containing five plastic cat-ball toys. Each session was videotaped so that the following behaviors could be recorded: latency to approach the object, frequency of object approaches, latency to contact the object, duration of contact with the object, number of freeze responses, number of grooming responses, number of interrupted grooming responses and number of fecal boli.

2.3. Dry land maze (DLM)

Two days after the novel environment assessment, habituation for the DLM foraging task commenced in a 124.5 cm diameter circular arena with 40.5 cm walls. The arena floor was covered in corncob bedding and had eight small food wells located at equidistant positions around the periphery [25]. As depicted in Fig. 1, during the first day of habituation, each of the eight wells was baited with a food reward (piece of Frosted Cheerios^™ cereal), on the second day, every other food well was baited and, on the third day of habituation, two of the previously baited wells were baited. For each trial, corncob bedding was redistributed between animals to minimize any cues from the previous trial. During habituation trials, the bedding was pushed away from the food wells and the rats had six minutes to retrieve the food rewards while the number of wells visited and number of food rewards consumed were recorded. Animals were removed from the task if all rewards were consumed prior to the end of the trial. The next day, during the acquisition trial, only one of the previously baited wells was baited so that the animal could learn the specific well that would be baited throughout all testing trials. Finally, following the test trials, the probe trial, in which no food reward was present, was conducted so that the animals’ problem-solving strategy in response to a prediction error (i.e., no food reward where it was expected to be) could be recorded. During the test trials and probe trial, the following behaviors were recorded: latency to approach the wells, number of wells visited prior to receiving the reward (in test trials), number of fecal boli, number of rearing responses and frequency of interrupted grooming bouts (i.e., grooming bouts that did not continue through to completion).

Fig. 1.

Dry Land Maze: Training consisted of three days of habituation (HAB 1–3) training, one day of training for the acquisition (ACQ) trial indicating the permanent position of the food reward, and three days of testing (TEST 1–3). Following training and testing, animals were assessed in the DLM arena with no baited well (PROBE).

2.4. Swim task

Each animal was exposed to a three-minute swim task in a large aquarium (60 × 30 × 50 cm) to provide an assessment of behavior in a stressful context. During this time the following behaviors were observed: latency to swim, duration of floats, swim and treading (i.e., only moving hind limbs) bouts and frequency of full and half-dives (i.e., not going to the bottom of the tank).

2.5. Vaginal cytology

All rats were vaginally smeared prior to the probe trial with calcium alginate swabs (Puritan Medical Products Company, LLC, Guilford, Maine) that were immersed in sterile saline solution before insertion. The cells were visualized with Cresyl Violet stain and observed at 10x and 20x magnification to determine the estrous cycle. Animals were assessed in the probe trial when they were in diestrus to avoid endocrine-related effects on behavioral outcomes.

2.6. Endocrine samples and assays

Four days after arrival to the laboratory and cage assignment, individual rats were placed in a clean cage for the baseline fecal sample collection for baseline corticosterone and DHEA metabolites. A second fecal sample was collected at the same time of day as the first sample (2100 h) but 12 h following the probe trial, the necessary time for endocrine metabolites to be present in the fecal sample [26]. Following storage in a −80 freezer, fecal samples were homogenized in 100% methanol, vortexed for 1 min and then centrifuged for 15 min. A 1:20 dilution was made using assay buffer (Enzo Scientific, Farmingdale, NY) with a sensitivity of 27 pg/ml and a range of 32–20,000 pg/ml. As per ELISA kit instructions, density readings were determined using an automated microplate reader (BioTek, Winooski, VT) and read with the Gen5 software (BioTek) at a wavelength of 405λ with correction at 490 λ. The cross-reactivity of the assay was 100% with corticosterone and DHEA. The intra-assay % coefficients of variation (CV) for corticosterone was 6.6 and 8.0 for low and high concentrations, respectively, while the comparable inter-assay % CV values were 7.8 and 13.1. The intra-assay %CV for DHEA was 4.8 (high) and 6.4 (low) while the inter-assay %CV was 6.5 (high) and 8.4 (low). The corticosterone kit cross-reactivity for other steroids determined by Enzo was 0.28% for tetra-hydrocorticosterone and a < 0.03% for 11-dehydrocorticosterone acetate, two steroids found in fecal matter.

2.7. Immunohistochemistry and neural quantification

Animals were perfused transcardially with phosphate buffered saline (PBS) and 4% paraformaldehyde. Brains were post-fixed in 4% paraformaldehyde, ending with 20% sucrose solution. Subsequently, brains were sectioned with a cryostat at 20 μm through the dorsal hippocampus starting at −3 mm from bregma (plate #58; [27]). Prior to antibody exposure, the brains were washed in PBS and incubated for two hours in PBS containing 2% BSA and 3% Triton-X, followed by PBS washes. For the GR histological assessment, brain sections were exposed to primary GR antibody at a dilution of 1:5000 (Santa Cruz Biotechnology, Dallas, TX). Following a 48 h incubation at 4 °C, the brains were washed in 3% H2O2/PBS solution, washed in PBS and exposed to 10% normal goat serum. The secondary antibody (GR goat-anti-rabbit, Vector, Burlingame, CA) was applied in a dilution of 1:200, followed by PBS washes and Avidin-Biotin Complex (Vector). For BDNF-ir, following PBS washes and a 30 min incubation in PBS-Triton-X, sections were incubated overnight in the PBS-TX-rabbit anti-BDNF antibody at a dilution of 1:1000 (Abcam, Cambridge, MA). For both antibodies, a pre-incubation with 3,3′-diaminobenzidine (DAB) substrate occurred prior to 10-minute incubation with DAB. Following staining and three additional PBS washes, brain sections were mounted on to microscope slides for further analysis.

For neural quantification, the GR-immunoreactive cells were located in the CA1 and CA3 regions of the hippocampus and counted in a 300 × 300 um visual field at 40X magnification with Neurolucida (Microbrightfield; Williston, VT). Similarly, the BDNF-immunoreactive cells were counted in CA1 and CA3 hippocampal regions. See Fig. 2 for representative photomicrographs of GR- and BDNF-positively stained cells in the targeted hippocampal areas. Only cell bodies were counted for the neural quantification data from 6 sections for each immunological marker (i.e., BDNF and GR). To avoid double counting of cells in continuous sections, every third section was used for quantification of each marker.

Fig. 2.

GR and BDNF Immunoreactive Cells. Representative GR- and BDNF-positively stained cells (as indicated by arrows) in the hippocampal CA3 and CA1 areas, respectively. Larger hippocampal section represents a sample cresyl-violet stained section from a previous study used for depicting targeted hippocampal areas.

2.8. Statistical analysis

The mean values for each dependent variable were calculated for each group and a one-way analysis of variance (ANOVA) was used to determine the effects of reproductive experience. Further, correlations for relevant comparisons were calculated for the variables. SPSS 25 was used for all statistical analyses.

3. Results

3.1. Dry land maze

DLM behaviors were separated into the following phases: habituation, testing, and probe test. No significant differences were observed during habituation training. Across both groups, females exhibited learning by demonstrating a decrease in errors as they transitioned from habituation and acquisition to test trials (Fig. 3A). Averaging across all three test days, NULL rats exhibited significantly more wall contact rearing responses than PRIM animals (oriented away from the task; F_1,14 = 5.992; p = 0.028; Fig. 3B). However, no significant differences were observed in the remaining dependent variables during DLM testing.

Fig. 3.

Dry Land Maze Behavior: (A) Descriptive statistics indicating that animals exhibited more errors during habituation and acquisition trials than during the test trials; (B) NULL females exhibited significantly more wall-contact rears than PRIM females across the three days of testing (p = 0.028); (C) During the probe trial, NULL females exhibited significantly more wall-contact rears than PRIM females (P = 0.003); (D) During the probe trial, NULL females excreted more fecal boli than PRIM females (p = 0.007); (E) The PRIM females visited more wells during the probe trial than the NULL females (p = 0.007).

During the probe test, NULLs, once again, exhibited significantly more wall-contact rearing responses (F_1,14 = 12.838; p = 0.003) (Fig. 3C) and also excreted more fecal boli (F_1,14 = 10.072; p = 0.007) (Fig. 3D). PRIM females visited significantly more wells than NULL animals (F_1,14 = 9.887; p = 0.007) (Fig. 3E). No significant differences were observed in the remaining dependent variables.

3.2. Novel environment task

Several behaviors were measured in the novel environment task but no significant differences between groups were observed. Regardless of group, females interacted with the novel object the same amount of time and neither group showed increased levels of anxiety-like behavior.

3.3. Swim task

During the swim task, NULL rats spent more time treading water (defined as only moving back legs) compared to their PRIM counterparts (F_1,14 = 4.710; p = 0.048) (Fig. 4A). Latency to float, number of dives and frequency/duration of floating did not differ between groups. However, PRIM rats exhibited a nonsignificant trend toward more ½ dives than the NULLs, (F_1,14 = 3.431; p = 0.085) (Fig. 4B).

Fig. 4.

Swim Task: (A) NULL females spent more time treading water compared to PRIM females (p = 0.048); (B) A nonsignificant trend was observed indicating that the PRIM females exhibited half dives more than NULL females (p = 0.085).

3.4. Endocrine markers

Although no effects were observed in baseline endocrine values, after the probe test, trends were observed in CORT levels (F_1,14 = 4.013; p = 0.065) and DHEA levels (F_1,14 = 3.243; p = 0.093). Specifically, NULLs had higher CORT levels while PRIMs had higher DHEA levels (Figs. 5A, B). The DHEA to CORT ratio revealed a significant difference between groups (F_1,14 = 4.49; p = 0.05), with PRIMs exhibiting significantly higher ratios (Fig. 5C). A positive correlation between number of fecal boli excreted during the probe test and CORT levels (r₁₄ = 0.62; p = 0.01) after the probe test was observed (Fig. 5D) and a negative correlation was observed between fecal boli and probe test DHEA levels (r₁₄=−0.66; p = 0.0001 (Fig. 5E). Finally, probe test DHEA levels were positively correlated with the number of ½ dives observed during the swim task (r₁₄ = 0.52; p = 0.037; Fig. 5F).

Fig. 5.

Endocrine Markers: (A) Following the probe test, a nonsignificant trend was observed indicating that NULL females had higher levels of CORT than PRIM rats (p = 0.065); (B) Similarly, a nonsignificant trend was observed after the probe test, indicating that PRIM rats had higher levels than NULL rats (p = 0.093); (C) The DHEA to CORT ratio was significantly higher in PRIM rats than their NULL counterparts (p = 0.05). (D) The correlation between CORT metabolites following the probe trial and number of fecal boli during the probe trial were positively correlated (p = 0.01); (E) DHEA metabolites following the probe trial were negatively correlated with the number of fecal boli emitted during the probe trial (p = 0.0001); (F) DHEA metabolites during the probe trial were positively correlated with the frequency of half dives observed during the swim task.

3.5. Neural markers

Significant differences were observed in hippocampal CA1 and CA3 areas assessed for GR-ir (Fig. 6A, B, C, D) with NULLs exhibiting significantly higher number of GR-ir cells in both CA1 (F_1,14 = 8.881; p = 0.01; Fig. 6A) and CA3 (F_1,14 = 52.029; p < 0.0005; Fig. 6B) hippocampal areas. Further, GR-ir in CA1 was positively correlated with probe test CORT metabolite levels (r₁₄ = 0.69; p = 0.003; Fig. 6C) and negatively correlated with the probe test DHEA/CORT ratio (r₁₄=−0.692; p = 0.009; Figure 6DE). GR-ir in CA3 was negatively correlated with number of wells visited during the probe task (r₁₄=−0.591; p = 0.016; Fig. 6E) and positively correlated with number of wall-contact rear responses during the probe task (r₁₄ = 0.652; p = 0.006; Fig. 6F).

Fig. 6.

Glucocorticoid-immunoreactivity: (A) NULL rats had significantly more GR-immunoreactive cells than PRIM females in the CA1 area of the hippocampus (p = 0.01); (B) NULLs also had significantly more CA3 g-immunoreactive cells than PRIM females (p = 0.0005); (C) GR-ir in CA1 was positively correlated with probe test CORT levels (r₁₄ = 0.69; p = 0.003 and (D) negatively correlated with the probe test DHEA/CORT ratio (r₁₄=−0.692; p = 0.009) (E) GR-ir in CA3 was negatively correlated with number of wells visited during the probe task (r₁₄=−0.591; p = 0.016 and (F) positively correlated with number of wall-contact rear responses during the probe task (r₁₄ = 0.652; p = 0.006).

A nonsignificant trend was observed for BDNF-ir in the hippocampus CA3 area (F_1,14 = 4.184; p = 0.06) with PRIMs exhibiting higher numbers of BDNF-ir compared to the NULLs (Fig. 7A). CA1 BDNF-ir was negatively correlated with the duration of water treading during the swim task (r₁₄=−0.545; p = 0.029; Fig. 7B) as well as with the number of wall-contact rearing responses during the probe task (r₁₄=−0.539; p = 0.031; Fig. 7C).

Fig. 7.

BDNF-immunoreactivity: (A) A nonsignificant trend was observed between the CA3 BDNF-immunoreactive cells in the PRIM and NULL females (p = 0.06); (B) A significant negative correlation was observed between the duration of treading in the swim task and CA3 BDNF-immunoreactivity (p= −0.029); (C) A significant negative correlation was observed between wall-contact rear responses and CA1 BDNF during the probe trial (p= −0.031).

4. Discussion

The results of the current study suggest an efficiency of energy expenditure in maternal rats (PRIM) when compared to their non-reproductive (NULL) counterparts. Specifically, more task-oriented behaviors in the maternal group during the DLM probe trial were recorded. In the swim task, PRIM animals exhibited more diving responses, viewed as targeted toward escaping from the energy-demanding swim task, whereas the NULL females exhibited more energy expenditure directed toward treading water. Compared to baseline values, the maternal animals had higher DHEA to CORT ratios, viewed as a marker of resilience in our laboratory that has also been observed in reproductively experienced owl monkeys [28]. Further, NULLs emitted more fecal boli during the uncertainty-generating probe trial of the DLM, a response traditionally interpreted as a heightened stress response [29]. Additionally, fewer GR-positive cells were observed in the PRIMs’ hippocampal CA1 and CA3 areas, an effect indicating a modified HPA axis response, and the CA3 BDNF-immunoreactivity data indicated heightened neuroplasticity in the PRIMs. In future assessments, neuroplasticity effects in the dentate gyrus of maternal-experienced females would be informative. Altogether, these findings suggest the existence of more streamlined and efficient neurobiological responses in the PRIM animals, responses consistent with an enhanced allostatic response in the maternal-experienced rats.

Focusing more specifically on behavior, the PRIMs exhibited more efficient behavioral responses that were aligned with the acquisition of food rewards (i.e., searched more wells that were previously baited during habituation trials). This response may be construed as increased errors in some contexts; however, the number of wells visited during the DLM probe trial is interpreted as an adaptive shift to informed searching within the task arena considering that all wells were baited during habituation. The increased frequency of wall-contact rearing responses, potentially associated with a motivation to escape the arena, observed in the NULL females throughout the test and the probe trials are not associated with increased likelihood of food acquisition. In all, these results confirm past findings of efficient foraging strategies in animals with reproductive experience [4,5,28].

Associated with altered cognitive strategies, the increased density of immunoreactive BDNF cells in the PRIMs corroborates previous findings of neuroplasticity measures in reproductively experienced animals. Neurogenesis, a hallmark indicator of neuroplasticity, is complex across pregnancy and lactation; generally, decreased cell proliferation and survival have been observed throughout the postnatal period in various rodent species [30]. Alternatively, increased dendritic hippocampal spines and medial preoptic area hypothalamic cellular complexity have been observed in maternal rats [1,31]. Focusing on humans, neural restructuring in the form of reduced cortical gray matter in brain areas associated with emotional and social functions (e.g., temporal medial and inferior frontal cortex), as well as the hippocampus, has been observed post-pregnancy [32].

The shift from self-to other-attentiveness necessary for successful parenting requires varied types of neural responses [33]. One interesting potential mechanism, for example, underlying the shift to pup attentiveness in rats and enhanced empathic responses in humans could be the process of neural fetal microchimerism, the transplantation of fetal cells to the maternal brain during pregnancy [34]. In general, however, it has been difficult to determine the precise “net effect” of any specific neuroplasticity-related response in maternal animals [35]. Perhaps more important than the quantification of specific markers of neuroplasticity, more global factors may also represent a critical parenting-induced neural modification. Harkening back to Donald Hebb’s classic proposal of functional cell assemblies, more streamlined neural networks are likely necessary to sustain the complex and varied shifts in attention, motivation and sensorimotor circuits accompanying motherhood [36,37].

Because the stress response is metabolically costly, diminished HPA activity during lactation may conserve much-needed energy required for nurturing offspring [38]. The current finding that PRIMs had fewer hippocampal GR-immunoreactive cells indicates a modified HPA system; however, these results require a thorough contextual analysis to fully understand the effects. Although high hippocampal GR mRNA markers have been associated with more efficient negative feedback in the stress response [39]), the current finding that hippocampal GR-immunoreactivity was positively correlated with CORT levels assessed following the probe trial suggests that, in this case, the NULLs with higher GR-immunoreactivity also exhibited heightened stress responsiveness. Previous research in our laboratory also suggests that lower GR-immunoreactivity is a marker of enhanced resilience [40]. Future research evaluating GR and MR activity is necessary to understand more about corticosteroid sensitivity and its role in maintaining adaptive allostasis [22].

In agreement with past studies, the current findings provide evidence of biobehavioral modifications associated with adaptive responses related to enhanced offspring survival [15]. This late-onset developmental shift in stress responsivity may provide an important experimental portal to understanding more about the roles of behavioral, physiological and environmental factors in the energy-demanding context of motherhood, providing new insights into treatments for symptoms associated with allostatic load (e.g., postpartum depression, obsessive-compulsive disorder) [41,42].

5. Conclusions

In conclusion, these results provide additional evidence that reproductive experience alters stress-related biobehavioral responses in a trajectory that enhances the efficiency of energy expenditure during pregnancy and lactation. This shift in stress responsivity likely protects against maternal allostatic load when the maternal rat faces heightened energy demands to care for her pups.