Combined effect of gestational stress and postpartum stress on maternal care in rats

Physiology & Behavior 184 (2018) 172–178

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Combined effect of gestational stress and postpartum stress on maternal care in rats

T

Giorgia Boeroa,1, Francesca Biggioa,1, Maria Giuseppina Pisub, Valentina Loccic, Patrizia Porcub, ⁎ Mariangela Serraa,d, a

Department of Life and Environment Sciences, Section of Neuroscience and Anthropology, University of Cagliari, Cagliari, Italy Neuroscience Institute, National Research Council of Italy, Cagliari, Italy c Department of Psychiatry, University of Illinois at Chicago, Chicago, IL, USA d Center of Excellence for Neurobiology of Dependence, University of Cagliari, Cagliari, Italy b

A R T I C L E I N F O

A B S T R A C T

Keywords: Maternal care Intragastric intubation Pup separation Rats

Variations in maternal care in the rat influence the development of individual differences in behavioral and endocrine responses to stress. This study aimed to examine the interaction between intragastric intubation during late gestation and postpartum stress, induced by pup separation, on maternal behavior and on dams' emotional state and HPA axis function. Rats received intragastric intubation of water on days 12–20 of gestation or remained untreated in their home cage (naïve dams). Pup separation was used as a model of postpartum stress. The procedure consisted of a daily separation of the dam from its litter for 3 h from PND 3 until PND 15. Pup separation was carried out in both naïve and intubated dams. The behavioral results indicate that the association of these two stressors significantly decreased arched-back nursing (ABN) and licking and grooming (LG), behaviors considered important parameters to discriminate the high quality of maternal care. Moreover, dams that received both stressors displayed less nest building and blanket nursing behaviors; no effect on the frequency of passive and total nursing was recorded. The analysis of single effects on ABN and LG, revealed that dams that underwent gestational stress induced by intragastric intubation displayed less LG, but ABN was overall unchanged. On the contrary, pup separation stress significantly increased ABN and LG upon reunion of naïve dams with their pups. Treatments per se or the association of both induced modest changes in plasma levels of allopregnanolone and corticosterone that likely did not influence maternal care. These data show that the association of a mild stress during gestation with an unfavorable experience after parturition had a significant impact on maternal care. This effect seems independent from HPA axis activation or from changes in emotional state; further studies would be necessary to ascertain the neural changes that could contribute to altered maternal behavior in stressed mothers. Moreover, these results suggest that the use of intragastric intubation during gestation would interfere with measures of drug-induced changes in maternal behavior and likely their consequences on the offspring.

1. Introduction It is widely accepted that the maternal hypothalamic-pituitaryadrenal (HPA) axis undergoes adaptations through pregnancy essential to provide protection to negative effects of stress on pregnancy and parturition [1]. Stress hyporesponsiveness continues during lactation allowing the dam to effectively care for her developing offspring [2]. However, attenuation of HPA axis has a limited protective strength against adverse effects of stress. In fact, gestational stress has been linked to postpartum depression in humans [3,4], and to depressive-like

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behavior during postpartum in rats [5,6], which may be associated with a short breastfeeding duration [7] and a decrease in maternal behavior in dams [5,8]. Likewise, high levels of corticosterone (CORT) during pregnancy or the postpartum period induce depressive-like behavior and reduce maternal care in rodents [9]. These effects were observed in animal models of chronic strong stress such as 1 h restrain stress for 10 days [5] or following prolonged exposure to high levels of CORT [9]. Recently, it has been found that a mild stress during pregnancy (G1720), due to intragastric intubation of water, may influence maternal behavior [10]. Intragastric intubation, proposed by Majchrowicz [11]

Corresponding author at: Department of Life and Environment Sciences, Section of Neuroscience and Anthropology, University of Cagliari, Cagliari 09100, Italy. E-mail address: [email protected] (M. Serra). Drs. Boero and Biggio contributed equally to this work.

https://doi.org/10.1016/j.physbeh.2017.11.027 Received 11 August 2017; Received in revised form 20 October 2017; Accepted 23 November 2017 Available online 26 November 2017 0031-9384/ © 2017 Elsevier Inc. All rights reserved.

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2.3. Postpartum stress

to induce physical dependence from alcohol in adult rodents, is widely employed during the perinatal period [12], being considered an effective tool for inducing and evaluating the consequences of alcohol consumption, since the intubation procedure per se is considered devoid of long-lasting anxiety-like effects [13]. Given that the long-term outcome of an individual is greatly influenced by stress during pre- or postnatal development [14–16], it is crucial to carefully assess the effect of mild stress during pregnancy on maternal behavior. Mothers exposed to stress during gestation are in general at higher risk of encountering a more stressful environment throughout the life span, including the postpartum period [17]. An animal model used as a stressor in the early postpartum is the repeated prolonged pup separation, a protocol mainly employed to study the effects of early life stress on the offspring. In agreement with other studies [18–20], we have shown a compensatory intensification of maternal care following reunion of dams and pups separated for 3 h during the first two weeks of life [21]. The principal aim of the present study was to investigate the potential interaction between two low-level stress exposures, the first during late gestation (intragastric intubation of water on days 12–20 of gestation), and the second during the postpartum period (pup separation for 3 h on days 3–15), on maternal care by performing a wide-ranging analysis of related behaviors. Moreover, given that maternal nurturing is influenced by the emotional state of the dam, at weaning we evaluated plasma levels of allopregnanolone (AP), a positive modulator of γaminobutyric acid type A (GABAA) receptors, and CORT, a marker of HPA axis function. We hypothesized that the pattern of secretion of these stress-sensitive neuroactive steroids during pregnancy and postpartum is altered by gestational intragastric intubation, and such alterations may influence the quality of maternal care. Indeed, in support to this hypothesis, it has been demonstrated that both AP and CORT affect maternal care [9,22].

Pup separation was used as a model of postpartum stress. The procedure was carried out according to Plotsky and Meaney [23] and consisted of a daily separation of the dam from its litter for 3 h (from 10:30 a.m. to 1:30 p.m.) from PND 3 until PND 15. During the 3 h separation, the pups were placed in a clean cage and transferred to another room (ambient temperature at 27–30 °C) to prevent vocal communication between mother and pups. The mothers were left undisturbed in their home cage until pup reunion. The procedure was performed every day by the same experimenter. After PND 15, dams and pups were left undisturbed until weaning. Pup separation was carried out in both naïve and intubated dams (10 rats per group, respectively); two separate groups of naive (10) and intubated (10) dams, not subjected to postpartum stress, were left undisturbed with the pups and served as control (Non Pup Separated; Fig. 1). 2.4. Maternal behavior Maternal behavior was carried out as previously described [22]. The behavior of each dam was observed for four daily 75-min observation sessions from PND 3 until PND 15. Observations occurred at a regular time each day with a session during the dark phase (8.00 a.m.) and three sessions during the light phase (9.15 a.m., 1.30 p.m. and 4.30 p.m.); thus, the first two observations took place before pup separation (which begins at 10.30 a.m.) and the last two observations were carried out after pup separation. The distribution of the observations was based on findings that nursing in rats occurs more frequently during the light phase. For each observation session, the behavior of each mother was observed for a brief period (approximately for 5 s) 25 times, once every 3 min. Thus, every dam was observed 100 times per day (25 observations per sessions × four sessions per day = 100 observation/mother/day). Data are presented as the average frequency of each behavior across the four daily observation periods and across the 13 days of postpartum period. The following behaviors were scored: (1) licking and grooming (LG) any pup, (2) nursing pups in passive and blanket postures (3) archedback nursing (ABN) posture, (4) building the nest, (5) self grooming, (6) staying in the nest in contact with the pups (in nest), (7) resting with no contact with the pups (resting) and (8) eating or drinking. A detailed description of these behaviors is provided in Myers et al. (1989). Note that the behavioral categories are not mutually exclusive. For example, LG often occurred while the mother was nursing the pups. Data were analyzed as the percentage of observations in which the animal engaged in the target behavior.

2. Material and methods 2.1. Animals The study was performed using males and females Sprague-Dawley rats (Charles River, Calco, Italy). All animals were housed under an artificial 12 h light (9:15 a.m.–9:15 p.m.), 12 h dark cycle (9:15 p.m.–9:15 a.m.) at a constant temperature of 23 ± 2 °C and 65% humidity. Food and water were available ad libitum. Adequate measures were taken to minimize pain or discomfort of animals, whose care and handling throughout the experimental procedures were in accordance with the European Parliament and the Council Directive of 22 September 2010 (2010/63/UE) and were approved by the Italian Ministry of Health (685/2015-PR) according to the Italian Legislative Decree no. 26 of 4 March 2014. To obtain pregnant rats, one male 150 days old was paired with one female 120 days old. Mating was verified by the presence of the sperm cap (plug); this day was considered as gestational day 0 (GD 0). After breeding all animals were housed in group until GD 20.

2.5. Measurement of hormones levels Within 3 h from weaning, dams were sacrificed (between 10:00 a.m. and 12:00 p.m.) by decapitation. Blood was collected from the trunk into K3-EDTA tubes, then centrifuged at 900 x g for 10 min at 4 °C; the resulting plasma was frozen at −80 °C until use. Levels of AP and CORT were assayed in plasma from the same rats. AP was extracted from plasma as previously described [24]. The combined organic phases were dried under vacuum. The recovery (70–80%) through the extraction procedure was monitored by addition of a trace amount (6000 to 8000 cmp; 20–80 Ci/mmol) of [3H]-AP (Perkin Elmer Italia, Monza) to the plasma samples. AP levels were quantified by radioimmunoassay with a specific antibody generated in rabbit as previously described [24,25]. The limit of detection for the AP radioimmunoassay, expressed as the minimal amount of steroid distinguishable from the blank sample, was 0.01 ng. Intra- and interassay coefficients of variation ranged between 5 and 7% and between 9 and 11%, respectively. The enzyme-linked immunosorbent assay (ELISA) was used to quantify plasma levels of CORT. ELISA was performed according to the manufacturer's instruction (CORT ELISA, IBL International, Germany)

2.2. Gestational stress Twenty rats underwent gestational stress from GD 12 to GD 20 (intubated dams); once daily an intragastric cannula, connected to a 2.5 cm3 syringe filled with water, was introduced into the stomach through the oral cavity. The control group (20 rats) was left undisturbed in their home cage (naive dams) (see Fig. 1 for a schematic of the experimental design). On GD 20 each pregnant dam was individually housed in a sawdust cage (40 cm × 60 cm × 20 cm) for parturition. All intubated females gave birth. Starting from postnatal day 2 (PND 2), all litters born within a 24 h period from mothers belonging to the same experimental group were equally distributed between males and females (5–6 for each sex). Pups were weaned at PND 21. 173

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Fig. 1. Timeline of the experimental procedures. Panel A: naive dams; panel B: intragastric intubation-treated dams.

Two-way ANOVA revealed a significant effect of intubation [F(1,36) = 24.898, p = 0.0001], no significant effect of separation [F(1,36) = 3.21, p = 0.081], and no significant interaction between factors [F (1,36) = 0.525, p = 0.473]. Overall passive and total nursing frequencies were not affected (Fig. 3B and C).

using a 96-well plate that was precoated with a polyclonal antibody against an antigenic site on the CORT molecule. The kit also provided a seven-point standard curve using two-fold serial dilutions. Each sample was run in duplicate. The limit of detection for the CORT ELISA, expressed as the minimal amount of steroid distinguishable from the blank sample, was 1.63 nmol/l. Intra- and interassay coefficients of variation ranged between 2.77 and 4.08% and between 6.14 and 5.54%, respectively.

3.1.3. Non pup-directed behaviors As showed in Fig. 4A, compared to the respective naive dams, intragastric intubation reduced nest building in both non pup separated (− 75%, p < 0.01) and pup separated dams (− 63%; p < 0.01). Intragastric intubation also prevented the increase in the frequency of this behavior induced by pup separation in naive dams (+35%, p < 0.05). Two-way ANOVA revealed a significant effect of intubation [F(1,36) = 59.79, p = 0.0001], a significant effect of separation [F(1,36) = 8.536, p = 0.0056], but no significant interaction between factors [F (1,36) = 0.204, p = 0.654]. Gestational stress did not alter the frequency of self-grooming (Fig. 4B) and resting (Fig. 4C) in both pup separated and non pup separated dams. Finally, intragastric intubation did not affect eating/drinking frequency in non pup separated dams. However, in pup separated dams, intragastric intubation prevented the reduction (− 27%, p < 0.05) in eating/drinking frequency observed in naive dams (+ 26%, p < 0.05; Fig. 4D). Two-way ANOVA revealed no significant effect of intubation [F(1,36) = 3.198, p = 0.081], a significant effect of separation [F (1,36) = 8.752, p = 0.005], but no significant interaction between factors [F(1,36) = 1.54, p = 0.221].

2.6. Statistical analysis Quantitative data are presented as means ± SEM of 10 animals for each experimental group and were compared by analysis of variance (ANOVA) followed by Newman-Keuls post-hoc test (Statistica 6.0, StatSoft Inc.). A p < 0.05 value was considered statistically significant. 3. Results 3.1. Effects of gestational intragastric intubation and postpartum pup separation on maternal care 3.1.1. Arched-back nursing and licking-grooming We investigated the effect of intragastric intubation and pup separation on the quality of maternal care by analyzing ABN and LG frequency. For ABN, two-way ANOVA revealed no significant effect of intubation [F(1,36) = 0.69; p = 0.42], a significant effect of separation [F(1,36) = 57.89; p = 0.0001], and a significant interaction between factors [F(1,36) = 5.97; p = 0.019]. For LG, two-way ANOVA revealed a significant effect of intubation [F(1,36) = 14.126; p = 0.0005], a significant effect of separation [F(1,36) = 12.996; p = 0.0008], but no significant interaction between factors [F(1,36) = 0.091; p = 0.764]. Post hoc test revealed that in non pup separated dams intragastric intubation did not influence ABN frequency (Fig. 2A), while it decreased LG frequency (−19%; p < 0.05; Fig. 2B). As expected [21], in naive dams pup separation increased the frequency of both behaviors (ABN: + 241%, p < 0.01; LG: + 16%, p < 0.05; Fig. 2A and B). In pup separated dams, intragastric intubation significantly decreased both ABN frequency (− 23%, p < 0.05) and LG frequency (− 14%, p < 0.05), although these behaviors are still significantly increased vs. intubated non pup separated dams (ABN: +89%, p < 0.01; LG +23%, p < 0.01).

3.2. Plasma CORT and AP concentrations At weaning, the outcome of gestational stress due to intragastric intubation per se and in combination with postpartum stress due to pup separation was evaluated by measuring plasma levels of CORT and AP. The results are depicted in Fig. 5. For CORT, two-way ANOVA revealed no significant effect of intubation [F(1,36) = 0.298; p = 0.587], no significant effect of separation [F(1,36) = 0.127; p = 0.722], but a significant interaction between factors [F(1,36) = 25.019; p = 0.001]. Intragastric intubation increased CORT concentrations in non pup separated dams, (+227%, p < 0.01). As expected [21], in naive dams, pup separation increased CORT levels (+ 219%, p < 0.01), while in intubated dams the combination of both stressors significantly decreased CORT levels (−58% vs. naïve NPS dams, p < 0.01; − 57% vs. naive PS dams, p < 0.01; Fig. 5A). For AP, two-way ANOVA revealed a significant effect of intubation [F(1,36) = 21.584; p = 0.001], a significant effect of separation [F

3.1.2. Nursing posture Gestational stress due to intragastric intubation decreased blanket nursing (Fig. 3A) both in non pup separated (−20% vs. naive-NPS, p < 0.01) and pup separated dams (− 14% vs. naive-PS, p < 0.05). 174

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Fig. 2. Effect of gestational stress and postpartum stress on arched-back nursing (A) and licking grooming (B) behaviors. Maternal care was observed from the 3rd to the 15th postpartum day in naive and intubated dams. Behaviors are expressed as a percentage of the number of observations for each behavior/total observations over time (frequency %) across all days of the observation period and are means ± SEM of 10 dams per group. Data were analyzed by two way ANOVA, followed by Newman-Keuls post hoc test. ap < 0.01, bp < 0.05 vs. the respective NPS dams; cp < 0.05 vs. the respective naive dams. Group symbols: NPS, non pup separated dams; PS, pup separated dams.

separation, on maternal behavior and on dams' emotional state and HPA axis function. The behavioral results indicate that the association of gestational stress and pup separation stress significantly decreased ABN and LG, two behaviors that are considered important parameters to discriminate the high quality of maternal care [26–28]. Remarkably, the analysis of single effects on ABN and LG, revealed that dams that underwent gestational stress induced by intragastric intubation displayed less LG, but ABN was overall unchanged. In contrast, Popoola et al. [10] found an intragastric intubation-induced decrease only on ABN3 frequency. A direct comparison between these and our results cannot be assessed; in fact, these authors divided ABN into three categories, where the ABN2 and ABN3 categories (in both of

(1,36) = 5.012; p = 0.031], but no significant interaction between factors [F(1,36) = 1.089; p = 0.3]. AP levels were not altered by intragastric intubation in non pup separated dams (Fig. 5B). In naïve dams pup separation increased AP levels (+ 39%, p < 0.05; Fig. 5B), effect that was absent in intragastric intubated dams; as a result AP levels in intubated dams subjected to pup separation were decreased compared to the respective naïve dams (−49%, p < 0.01). 4. Discussion This study aimed to examine the interaction between intragastric intubation during late gestation and postpartum stress, induced by pup

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Fig. 3. Effect of gestational stress and postpartum stress on nursing posture: blanket nursing (A), passive nursing (B) and total nursing (C). Maternal care was observed from the 3rd to the 15th postpartum day in naive and intubated dams. Behaviors are expressed as a percentage of the number of observations for each behavior/total observations over time (frequency %) across all days of the observation period and are means ± SEM of 10 dams per group. Data were analyzed by two way ANOVA, followed by Newman-Keuls post hoc test. ap < 0.01 vs. the respective NPS dams; bp < 0.05 vs. the respective naive dams. Group symbols: NPS, non pup separated dams; PS, pup separated dams.

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Fig. 4. Effect of gestational stress and postpartum stress on non-pup directed behaviors: nest building (A), self-grooming (B), resting (C) and eating/drinking (D). Maternal care was observed from the 3rd to the 15th postpartum day in naive and intubated dams. Behaviors are expressed as a percentage of the number of observations for each behavior/total observations over time (frequency %) across all days of the observation period and are means ± SEM of 10 dams per group. Data were analyzed by two way ANOVA, followed by Newman-Keuls post hoc test. ap < 0.05 vs. the respective NPS dams; bp < 0.01 and cp < 0.05 vs. the respective naive dams. Group symbols: NPS, non pup separated dams; PS, pup separated dams.

of intragastric intubation on maternal care may be attributable to the change in CORT levels, due to a persistent effect of the stressful procedure on HPA axis activity. Accordingly, an effect of intubation per se on the expression of three genes involved in epigenetic regulation has been reported in the hippocampus of controls [31]. It is postulated that the increase in HPA axis activity mediates the decrease in maternal care [9,32]. However, in rats, only a high dose of CORT (40 mg/kg), administered during the postpartum period, which raised serum CORT levels above 300 ng/ml, increased depressive-like behavior and altered maternal care [9]. Since the physiological increase in basal HPA activity, necessary to keep the HPA axis stress responsiveness low, is maintained constant during lactation by the suckling stimulus [33], the

which the mother's back was arched) correspond to the ABN position reported in our study. In addition, the observation periods differed: in the Popoola et al. [10] study maternal behavior was recorded for the first 6 postnatal days, while in our study it was conducted for 15 days starting from postnatal day 3 (see methods for further details). Although based on different parameters, our results are in agreement with the conclusion of Popoola et al. [10] that the experimental procedure during gestation may induce important effects on maternal behavior. It is worth noting that decreased maternal LG has been associated with increased behavioral responses to stress in the offspring, consequence of an alteration of the epigenome at the glucocorticoid receptor gene promoter in the offspring hippocampus [14,29,30]. The negative effect

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Fig. 5. Effect of gestational stress and postpartum stress on plasma CORT (A) and AP (B) concentrations. (A) CORT and (B) AP were measured in plasma samples of dams after weaning. Data are expressed as ng/ml and are means ± SEM of values from 10 rats per group. Data were analyzed by two-way ANOVA, followed by Newman-Keuls post hoc test. ap < 0.01, bp < 0.05 vs. the respective NPS dams; cp < 0.01 vs. the respective naive dams. Group symbols: NPS, non pup separated dams; PS, pup separated dams.

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5. Conclusions

levels of CORT measured within 1 h from weaning likely reflect those of lactating dams [34–35]. We found a modest increase in plasma CORT levels in intragastric intubated dams, which likely did not influence maternal care. It has been demonstrated that stress across the postpartum period induced structural changes in brain regions such as medial prefrontal cortex and nucleus accumbens linked to anxiety and maternal care [6,36]. In rats, anxiety-like symptoms are inversely correlated with maternal behavior and with circulating levels of AP [5,22,32,37]. AP is one of the most potent and efficacious positive allosteric modulators of GABAA receptor function [38], and its administration to animals induces marked anxiolytic and antidepressant effects [39,40]. In humans, reduced levels of AP in the peripheral blood or cerebrospinal fluid were found to be associated with major depression and anxiety disorders, also during pregnancy [41,42]. In rats, anxietylike states are induced by chronic stress (social isolation at weaning) [24], or by treatment with finasteride (5α-reductase inhibitor) [43], two conditions that also result in a significant decrease in AP levels [24,44]. Likewise, reduced AP concentrations induced in dams by juvenile social isolation or by administration of finasteride, induced a decrease in ABN frequency but not in LG [22]. The present results show that intragastric intubation induced a very modest reduction in plasma levels of AP, which is probably not sufficient to influence the emotional state in these animals, and, consequently, affect the frequency of ABN. Although a direct measurement of the emotional state of intragastric intubated dams during lactation would be needed to confirm this hypothesis, a lack of anxiety-like effects produced by the intubation procedure has been previously demonstrated in male rats [13]. Moreover, the evidence that locomotor activity in the open field did not differ in control male and female rats exposed to ethanol by intragastric intubation [45], further supports our hypothesis. LG behavior is triggered by sensory stimuli from the pups [46]. Studies directed to link variation in gene expression and differences in LG pattern have pointed out the attention to several neuromodulators such as dopamine, prolactin and estrogen and found that mothers with a high liking frequency had higher gene expression in the olfactory bulb [47]. Rodents' maternal behavior is contingent on olfactory information from the pups and requires both the mothers' ability to discriminate pup odors, and the motherhood-induced enhancement in odor memory which is dependent on neurogenesis [48]. The decrease in LG frequency observed in intragastric intubated dams may be due to an altered plasticity in the olfactory bulb. Fittingly, it has been demonstrated that gestational restrain stress (on GD 12–20) alters maternal behavior and changes dendritic morphology of adult-born granule cells in the olfactory bulb [49]. In agreement with previous data [18–21], pup separation stress significantly increased ABN and LG upon reunion of naïve dams with their pups. It is well established that the quality of maternal care, provided to offspring early in life, is a direct determinant of developmental programming and calibration of the HPA axis [14,16]. Thus, the compensatory intensification of maternal care following reunion of pups and dams seems to act as a potential buffer, provided by the mother to the offspring, against an adverse environment later in life. In fact, animals that underwent maternal separation showed increased resilience to adverse effects induced by social isolation during adolescence [21]. The association of intragastric intubation and pup separation induced a significant decrease in ABN and LG frequencies, compared to the non-intubated dams, demonstrating that a stressful experience during pregnancy is able to partially eliminate a compensatory intensification of maternal care triggered by pup separation stress. Fittingly, adult offspring of intragastric intubated and pup separated mothers are more sensitive to the effect of acute stress, compared to offspring of dams that underwent pup separation only, without an additional stress during gestation (Biggio et al., Neuropharmacology, submitted).

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