Sprague-Dawley rats (Charles River Inc., Kingston, NY, USA) in this study were maintained in accordance with the guidelines of the Committee of the Care and Use of Laboratory Animals Resources, National Research Council, and the research protocol was approved by the Tufts Institutional Animal Care and Use Committee. “CSS dams” refers to the adult females exposed to CSS during lactation (F0), “CSS F1 dams” refers to the maternal adult female offspring of the CSS F0 dams, which were treated with chronic saline, AVP, or OXT, and “CSS F2 juveniles” refers to female juvenile F2 offspring, which are the focus of the present study. The five experimental groups consisted of control (CON), control + intranasal saline (CON SAL), CSS + intranasal saline (CSS SAL), CSS + intranasal AVP (CSS AVP), and CSS + intranasal OXT (CSS OXT). Open-field and social behavior were tested on days 37–38, the marble burying test was done on days 38–39, and all rats were euthanized on days 39–40 (the day following the marble burying test) between 0900 and 1100 hours.

The CSS dams were subjected to the CSS protocol from days 2 to 16 of lactation as reported (14, 15). This procedure consisted of placing a similarly sized (220–300 g) novel male intruder into a lactating female’s home cage for 1 h from days 2 to 16 of lactation. CON dams were not exposed to the CSS protocol. The pups were left in the cage during the novel male intruder presentation, and the CSS exposure results in reduced maternal care (pup grooming and nursing) and increased anxiety-related behavior and maternal aggression in F0 dams (14), creating an early-life stress of depressed maternal care and social conflict for the F1 generation.

The CON and early-life CSS F1 females were the offspring of the F0 CON and CSS dams; the differences between the treatments of the CON and early-life CSS F1 females consisted of the exposure of the CSS F1 females to attenuated maternal care and conflict between their F0 mothers and the male intruders during age 2–16 days. The F1 CON and early-life CSS animals were treated identically after the age of 16 days. After weaning all F1 pups on day 23, the female offspring from the 12 CON and 12 CSS dams were housed in groups of four until 70 days of age, when 1–2 females from CON and CSS litters were mated with breeder males to obtain 12 CON and 18 CSS F1 females.

All experimental animals were euthanized within 3 min of entering the animal room between 0900 and 1100 hours, the day following social behavior testing, and trunk blood was collected for the analysis of TNFα and IFNγ. These were measured by individual rat ELISAs (R&D Systems, USA). Samples were run in duplicate in an individual assay to eliminate interassay variation, and intraassay variability was 4%.

Basal cytokine and behavior levels were analyzed with one-way ANOVA (on all five treatment groups and four groups with the two control groups combined) as well as t-tests of the combined control groups compared to the combined CSS groups to assess the overall effect of CSS (Table 1). These CSS-focused tests were followed by additional t-tests comparing individual treatment groups if there were significant treatment effects with ANOVA or the combined CON vs. combined CSS t-test. There were no differences between the CON and CON SAL groups with any of the variables, so these groups were combined to compare to individual CSS groups. All graphical results are presented as mean + SEM, significance was denoted as p ≤ 0.05, and p values refer to two-tailed tests unless noted. The use of one-tailed tests was justified for use with IFNγ based on previous findings of its importance in neuronal connectivity and social behavior (10) and recently published data on impaired functional connectivity in the CSS model (26). The use of one-tailed tests was justified for use with behavioral data by initial effects of CSS in the present study or previous reports of increased anxiety and decreased social behavior in the F2 generation of CSS model (5). Cohen’s d effect size tests were used to assess effect sizes, with 0.2–0.5 considered a small effect, 0.5–0.8 a medium sized effect, and values greater than 0.8 a large effect. Pearson correlations were used to test for significant IFNγ-behavior associations.

Basal plasma levels of TNFα were undetectable. There were no significant differences in IFNγ following one-way ANOVA (F_4,38 = 1.0, p = 0.4, F_3,38 = 1.3, p = 0.3, Figure 2). Basal plasma IFNγ levels were increased more than two-fold in combined CSS F2 offspring compared to combined CON F2 offspring (Table 1). This difference was driven by the CSS OXT group (22.7 ± 6.0 ng/ml, Figure 2), which had higher levels than the combined control groups (t = 2.5, p = 0.02, Cohen’s d = 1.1) and when compared only with the CON SAL group (one-tailed t = 2.0, p = 0.03, Cohen’s d = 0.3).

There were no significant differences in marble burying following one-way ANOVA (F_4,38 = 1.5, p = 0.2, F_3,38 = 1.5, p = 0.2, Figure 3). CSS-exposed F2 female juveniles buried more marbles compared to CON animals during the 15-min test (Table 1). When comparing individual treatment groups, both the CSS SAL (3.9 ± 0.5 s, one-tailed t = 1.8, p < 0.05, Cohen’s d = 1.0) and CSS AVP (3.8 ± 0.4 s, one-tailed t = 2.0, p = 0.03, Cohen’s d = 1.0) groups buried more marbles than the CON SAL group (2.5 ± 0.6 s, Figure 3). CSS F1 dam treatment with OXT decreased the number of marbles buried by CSS F2 female juveniles to a level similar to controls.

There were no significant differences between CON and CSS-treated F2 female juvenile between any treatment groups in durations or frequencies of moving along the edge of the open field, stationary along the edge, rearing along the edge, and moving in the center of the open field (one-way ANOVA, all p’s > 0.3). Durations and frequencies for stationary and rearing in the center were too low for statistical comparison. There were no significant differences in self-grooming in the open field following one-way ANOVA with all five treatment groups (F_4,38 = 2.6, p = 0.06), but there was a significant effect of treatment when the two control groups were combined (F_3,38 = 3.5, p = 0.03, Figure 4). CSS SAL juveniles expressed an almost three-fold increase in mean duration of self-grooming during open-field testing compared to the combined control groups (Table 1). Mean self-grooming durations in both the CSS AVP (2.3 ± 1.5 s) and CSS OXT (1.5 ± 1.0 s) groups were lower than grooming in the CSS SAL group (both one-tailed t tests = 2.1, p = 0.03, Cohen’s d = 1.1, Figure 4), and similar to control values.

There were no significant differences in social interaction following one-way ANOVA (F_4,38 = 1.7, p = 0.2, F_3,38 = 2.4, p = 0.09, Figure 5A). Combined juvenile female F2 CSS rats spent 25% less time displaying social behavior during the 10-min social interaction test compared to combined CON rats (Table 1). This overall difference was due to significant differences between the combined controls and the CSS SAL (37.4 ± 5.2 s, one-tailed t = 1.9, p = 0.04, Cohen’s d = 0.7) and CSS OXT (29.9 ± 7.3 s, t = 2.6, p = 0.02, Cohen’s d = 1.1, Figure 5A)-treated groups. Social contact duration in CSS F2 juvenile offspring of AVP-treated F1 dams was similar to control values.

There were no significant differences in allogrooming following one-way ANOVA (F_4,38 = 1.2, p = 0.3, F_3,38 = 1.5, p = 0.2, Figure 5B). When CON and CSS groups are combined, durations of allogrooming were decreased in CSS animals compared to controls (Table 1).

There were no significant correlations between basal peripheral IFNγ levels and durations of marble burying, self-grooming, social contact, or allogrooming (all p’s > 0.1).

Vasopressin and OXT have substantial immune functions and modulate the immune system during its development, homeostasis, and in response to injury and stress. AVP is emerging as a critical immunoregulatory peptide, capable of maintaining immune function. This is due to the ability of AVP and OXT to stimulate both the HPA axis and prolactin release (27). The genes for OXT and OXTR are expressed in the thymus (28) and monocytes and macrophages (29). OXT is the target of immunological cytokines (e.g., prostaglandin E2, IL2, and IL6) and prolactin, which can promote its secretion into the blood (30–33). Johnson et al. (34) demonstrated that AVP and OXT were able to replace the IL2 requirement for IFNγ production by T-helper cells from mouse spleen cultures. Importantly, this did not involve cell proliferation, suggesting that these neuropeptides possess cytokine activity supporting a relationship between neuroendocrine and immune systems (34). Further work demonstrated that AVP and OXT can replace IL2 for T cell mitogen induction by IFNγ in mouse spleen cells (12), and OXT significantly increases peripheral blood mononuclear cell blastic response to phytohemagglutinin (35). Based on these findings and the current data, it is hypothesized that AVP and OXT act as regulators of immune cells.

Elevated basal IFNγ in CSS F2 juvenile females was driven by the maternal OXT treatment, suggesting a specific effect of this neuropeptide on IFNγ and general immune functioning. The developmental importance of IFNγ (10) suggests that there may be substantial neuroanatomical changes in CSS F2 offspring. Robust changes in resting-state functional connectivity have been documented in studies of CSS F1 offspring (26), and detailed neuroanatomical and neuroimmunological investigation of the CSS F2 generation and similar populations would be valuable.

The overall increase in CSS F2 marble burying augments our previous report of social anxiety in this generation (5), supporting the hypothesis that CSS has transgenerational effects on both social anxiety and repetitive/perseverative behavior. In a natural setting, increased perseverative behavior could lead to decreased expression of critical behaviors such as foraging, sexual behaviors, and/or parental care. The CSS model is characterized by the increased expression of inappropriate behaviors, such as excessive nesting, locomotor activity, and unnecessary pup retrieval when maternal care is depressed in F0 and F1 dams. These behaviors have been referred to as maternal anxiety, but they may also reflect increased perseveration in stressed dams. A lack of a significant increase in marble burying in the CSS OXT group compared to significant increases in the CSS SAL and CSS AVP groups suggests that OXT may be specifically effective at preventing perseveration in the offspring of mothers exposed to early-life social stress. While AVP and OXT have been reported to have similar effects on anxiety and social behavior in some species (20, 36), the present results support the hypothesis that they have unique, behaviorally specific programing effects on offspring.

Augmented self-grooming during open-field testing indicates novelty-induced anxiety where juvenile CSS F2 females increased self-grooming when placed in a novel environment. In contrast to the nanopeptide-specific differences in burying behavior, both AVP and OXT treatments prevented the CSS-induced increase in self-grooming during open-field testing. This may suggest context- or behavior-specific effects of AVP and OXT. The lack of effect on general locomotor parameters during open-field testing indicates that the behavioral effects were not mediated by changes in activity levels, consistent with all previous studies of the CSS model.

In direct support of our previous work on CSS F2 female juveniles, CSS decreased social interaction. Individual group comparisons indicate that AVP, but not OXT, treatment of F1 dams has protective effects on F2 social behavior. This protective effect may be specific to investigatory activities since treatment effects of CSS on F2 allogrooming were similar. AVP is an established mediator of social recognition (37), which may explain its effectiveness in ameliorating the social stress-induced deficits in social interaction through changes in investigatory behaviors, but not allogrooming. In maternal rats, AVP is a key mediator of maternal care, maternal aggression, maternal memory, and self-grooming (38–44), and chronic central infusion of AVP enhances maternal care in CSS-exposed F0 dams (19). The preventative effects of AVP on F2 juveniles indicate that it may be an effective treatment for both depressed maternal care and the adverse effects of deficient care on offspring. Reinforcing the specificity findings in the burying and self-grooming data, AVP and OXT have differential preventative effects on the negative impact of CSS on the F2 social behavior, with AVP having protective effects on overall social contact, yet no effect of either peptide on the CSS-induced decrease in allogrooming.

Studies in voles suggest that that chronic OXT may have adverse long-term effects on social behavior (22), and the present data could represent a similar phenomenon that is transmitted to offspring through changes in maternal care. The lack of preventative effects of F1 dam OXT on social behavior could be related to observations of elevated plasma OXT in CSS F2 juvenile females, which exhibit social deficits (5), and the present findings support related work in humans and rodents on OXT-associated disruptions in social functioning (45–47). Female rodents exposed to different social stressors exhibit long-lasting changes in the OXT system (48, 49). IN OXT is unable to ameliorate stress-induced deficits in mouse social behavior, and OXT decreased social interaction in a control population (49). It is postulated that exogenous OXT treatment of CSS-exposed F2 females may not be effective and could impair social behavior in mothers or their offspring due to context dependent adverse effects (38, 49) on F1 dam maternal care during early lactation when maternal anxiety is elevated (4). Beneficial effects of OXT on social behavior may require more positive social interactions where OXT can enhance the salience of these encounters (50–53).

In addition to the current juvenile social testing paradigm, it is possible that CSS could negatively impact several other forms of social interaction at later life history stages, such as mating, alloparental care (48, 54), parental care (55), and aggression (56, 57) in the F2 generation. The reported changes in juvenile behavior, particularly the decrease in allogrooming, may be predictive of future deficiencies in social bonding and mating behaviors. One possible mechanism supported by the allogrooming data is that depressed F1 maternal care mediates decreased F2 juvenile allogrooming, leading to general deficits in social functioning in the F2 offspring at multiple life history stages. In support of this hypothesis, similarly treated CSS F2 dams exhibit deficits in maternal care (manuscript in review), and additional CSS F2 investigations of mating, alloparental, and parental behavior are warranted.

The hypothesis that the effects of chronic F1 IN OXT and AVP treatment on F2 behavior are mediated through changes in F1 maternal care is supported by a wealth of literature on their roles in the establishment and expression of maternal care (20, 58). OXT’s role in maternal behavior has been studied for almost 40 years (59), and there is growing evidence for its role in pathologies that adversely impact parental care. Changes in OXT and dopamine mediate the adverse effects of parental deprivation on the parental care of vole offspring (60), and OXT has been clinically implicated in depression and anxiety (46, 61–63). AVP is also a critical mediator of maternal behavior (38, 64, 65) and the rationale for the current IN AVP treatment was directly supported by increased maternal care in CSS-exposed dams following central AVP infusion (19). Recent clinical evidence supports a key role for AVP V1a receptors in maternal social cognition (66), and animal studies indicate that this role of the V1a receptor may be mediated through hypothalamic nuclei (67). Considering that the F2 females were not treated themselves, there are several possible mechanisms for the effects of the treatments, including changes in F1 maternal care and effects on neuropeptide, inflammatory, or nutritional factors in milk. While IFNγ levels were not correlated with F2 behavior, F1 dam AVP and OXT treatments may have altered IFNγ in milk and F2 offspring and induced immune-mediated neurodevelopmental effects, which mediated the behavioral changes. Key questions to address in exploring AVP- and OXT-induced changes in F1 maternal care are how do both acute and chronic treatments affect F1 maternal care and what are the behavioral effects on F2 offspring. Cross fostering and artificial grooming manipulations may be particularly valuable in these efforts, and changes occurring during early lactation are likely to be critical. Ongoing studies of the CSS model will explore the role of maternal care in the effects of IN AVP and OXT on offspring behavior and immune function.