Early life stress induces submissive behavior in adult rats

Behavioural Brain Research 372 (2019) 112025 Contents lists available at ScienceDirect Behavioural Brain Research journal homepage: www.elsevier.com/locate/bbr Research report Early life stress induces submissive behavior in adult rats a,1 a,1 b a c Dmitry Frank , Alexander Zlotnik , Ora Kofman , Julia Grinshpun , Olena Severynovska , ⁎ Evgeni Brotfaina, Ruslan Kuta, Dmitry Natanela, Israel Melamedd, Matthew Boykoa, T a Division of Anesthesiology and Critical Care, Soroka University Medical Center and the Faculty of Health Sciences, Ben-Gurion University of the Negev Beer-Sheba, 84101, Israel Psychology Department, Zlotowski Center for Neuroscience, Ben-Gurion University of the Negev, POB 653, Beer-Sheva, 84105 Israel c Department of Physiology, Faculty of Biology, Ecology and Medicine, Dnepropetrovsk State University, Dnepropetrovsk, Ukraine d Department of Neurosurgery, Soroka University Medical Center and the Faculty of Health Sciences, Ben-Gurion University of the Negev Beer-Sheba, 84101, Israel b A R TICL E INFO A BSTR A CT Keywords: maternal deprivation submissive behavior anxiety stress social dominance Maternal-deprivation of rodent pups is a relevant model of extreme early-life stress that can be relevant to the understanding of long-term effects of war, migration, parental loss and displacement. Although even mild stress during infancy affects brain development and behavior, the current study focused on the effects of six hour daily maternal-separation, a model that reflects the severe distress often experienced in those circumstances. This study emphasizes the effect of maternal separation on social behavior in the context of a variety of factors that measure cognitive and emotional behavior which were subject to principle component analysis. Sprague-Dawley pups were separated from the dam for 6 h each day during the first 3-weeks of life and underwent a battery of behavioral tests at 3-months of age. We found that rodents exposed to postnatal maternal deprivation displayed submissive behavior in residentintruder and dominant-submissive tests, as well as significantly more anxiety and anhedonia than control rats. The results of multivariate statistical analysis show that the dominant-submissive behavior correlates with depressive, anxiety and social behavior and can be predicted with an accuracy of 86.2%. The increased submissive behavior in male rats that had been subjected to severe postnatal stress suggests that exposure to stress during infancy and childhood could have long-term effects on social relationships. The mechanism of the longterm effects on depression, anxiety and submissive behavior requires further investigation. 1. Introduction amygdala connectivity pattern, indicating impaired ability to regulate amygdala activation to fear stimuli [12] and less reactivity of the nucleus accumbens to reward [13]. In preclinical research MS ranging from 15 min to 6 h daily induced long-term effects on measures related to depression and anxiety. The effects are variable and interact with genetic factors [14]. Six hours MS reduced stimulated dopamine release in the nucleus accumbens [15]. Three-hour daily MS reduced time in the open arms in the elevated plus maze (EPM) test for anxiety in both male and female rats [16,17] and male, but not female C57Bl/6 mice [16,18]. In other anxiety assays, male, but not female rats showed increased latency to reach the center of an open field [16], suggesting increased anxiety, whereas the female but not the males rats showed more anxiety as revealed by reduced time Maternal separation (MS) is widely used as a rat model for early life stress in the study of mood disorders, and long-term effects on cognition [1,2], memory [3,4] anxiety, depressive and social behavior [5–8]. In depressed women, early life stress predicted their response to psychotherapy, validating this model for preclinical studies [9]. Moreover, early life stress has intergenerational effects, as infants born to mothers who had themselves undergone childhood maltreatment had less grey matter in their brains and smaller intracranial volumes [10], and the male offspring of females who underwent maternal separation showed increased social contact [11]. Children who were institutionalized showed reduced maturation of the negative frontal lobe- ⁎ Corresponding author at: Brain Research Lab., Division of Anesthesiology and Critical Care, Soroka University Medical Center and the Faculty of Health Sciences, Ben-Gurion University of the Negev Beer-Sheba, P.O. Box 151, Beer-Sheba, 98105, Israel. E-mail addresses: frdima16@gmail.com (D. Frank), AleksZl@clalit.org.il (A. Zlotnik), kofman@bgu.ac.il (O. Kofman), juliag7648@gmail.com (J. Grinshpun), eseverinovskaya@gmail.com (O. Severynovska), bem1975@gmail.com (E. Brotfain), ruslanKo@clalit.org.il (R. Kut), dmitry.natanel@gmail.com (D. Natanel), IsraelMe@clalit.org.il (I. Melamed), matthewboykoresearch@gmail.com, matewboyko@gmail.com (M. Boyko). 1 Equal contribution. https://doi.org/10.1016/j.bbr.2019.112025 Received 25 November 2018; Received in revised form 18 April 2019; Accepted 8 June 2019 0166-4328/ © 2019 Elsevier B.V. All rights reserved. Behavioural Brain Research 372 (2019) 112025 D. Frank, et al. in the light side of the light-dark box [17]. In contrast, 3 -h MS in pups actually reduced anxiety in the adult male mice in the elevated zero maze and had no effect in the EPM in male and female mice from several strains [14,19]. Parfitt et al. [19] pointed out that in contrast to the enhanced anxiety observed in mice that had undergone 3 h MS, brief daily handling of pups led to reduced behavioral and hormonal responses to stressful stimuli and to less [18,20] or no change [11] in open arm exploration in the EPM assay. In addition to modifying anxiety in a sex-dependent manner, MS was shown to reduce sweet preference in male, but not female adult rats [17], whereas regular chow intake was not affected. MS has also been used to explore the long-term effects of stress on gastrointestinal disease, suggesting that it can be adapted to disease models that are not primarily psychiatric, but are commonly exacerbated by stress [21]. In social groups a mood disorder of a member can affect a change in hierarchical relations [15,22]. Dominance and submissiveness are important functional elements in maintaining the social hierarchy which has the advantage of limiting the amount of violence in a group. Two forms of dominance hierarchies have been described: linear and despotic. In the linear dominance hierarchy, each individual dominates the individuals below him. In the hierarchical structure only one individual is dominant and all the others are submissive [23]. The hierarchy is beneficial in conquering and guarding territories, regardless of whether the dominant status is held by an individual or a group of members. The nature and stability of dominance relations within groups over time were studied by Blanchard and colleagues (1988), who established that dominant–submissive behavior in rats develops a few days after grouping and persists up to 21 days [24]. The social hierarchy helps to maintain order within a group, social defeat is stressful and can lead to the expression of depressive-like behavior in individuals [23,25]. Subordinate animals show helplessness, anhedonia, behavioral despair and other neurovegetative changes such as alterations in sleep and appetite [26]. Dominance-submissive hierarchies based on resource holding potential or age are central to the social structure of many group-living animals [27] and can be quantified by competitive tests in model experiments by the priority of access to these resources. Measurement of dominance as “winner” behavior and submissiveness as “loser” behavior in pairs or in triads of animals has better reproducibility compared to studies of large groups. In pairs or triads dominance can easily be determined by measuring time spent on a feeder [28,29] or number of sucrose pellets consumed in a competitive situation. Such social interactions lead to the formation of a hierarchy. Experiments designed to study social behavior in the rat are mainly concerned with dominance and aggression, gregariousness, social facilitation, competition and co-operation [30]. Currently, a number of different behavioral paradigms are employed to study mouse social behavior, such as the three-chamber social approach [31–34], resident–intruder [35–37], partition [38,39] and dominant–submissive relationship [28,29,40,41] tests. Social support dominance can be relevant to coping with stress as social defeat itself is stressful. Inducing a tendency to submissiveness following early maternal separation could negatively affect social interactions and prevent the individual from benefitting from post-traumatic social contact. The principal aim of this manuscript is to test the hypothesis that MS can induce long-term effects on social behavior, and in particular dominant-submissive behavior. The second goal of our study was to examine which behavioral traits correlated with dominant–submissive behavior, by exploring behavioral assays of anxiety, depression, social behavior, and learning. For this purpose, a statistical analysis of correlations was carried out and a model for predicting dominant–submissive behavior based on a battery behavioral tests was constructed. 2. Materials and methods The experiments were conducted in accordance with the recommendations of the Declarations of Helsinki and Tokyo and the Guidelines for the Use of Experimental Animals of the European Community. The experiments were approved by the Animal Care Committee of Ben-Gurion University of the Negev, Israel. Rats had unrestricted access to standard laboratory chow and water in a vivarium with a reversed light/dark cycle of 12:12-h (lights off at 8:00 a.m. and lights on at 8:00 p.m.) at a constant temperature of 23 °C with 35% humidity. All efforts were made to minimize the number of animals used and their suffering. Sprague-Dawley rats (12 females, 4 male) were purchased from Envigo, Israel. Mating was accomplished by housing 3 females with one male per cage. After birth, the sires were removed from the cage and the dam was housed with her litter. 2.1. Maternal separation On the day after birth, maternal separation was conducted in 6 litters by separating the pups from their dams in a separate room in the animal housing facility from 8 a.m. to 2 p.m. each day. Control litters were not handled. Following the separation the litter was returned to the dam who had ad lib access to food and water, similar to the method of Veenema & Neurmann [42]. On postnatal day 21, the pups were weaned. At the age of 3 months the male offspring (28 MS and 30 control) underwent a battery of behavioral tests [43,44]. All the tests were conducted in the dark phase between 8 a.m.–4 p.m. 2.2. Open field test (OFT) The open field boxes were round black plastic arenas 2 m in diameter, 60 cm high walls situated in a darkened room. For analysis, the arena was divided into a central zone and the surrounding border zone (20 cm from the wall) and was cleaned with 5% ethanol after each behavioral recording. The OFT was performed by placing the rat into the apparatus facing the wall. The rat was allowed to explore the arena for 5 min. Locomotor activity was recorded with a video camera (CC TV Panasonic, Japan) and subsequently analyzed using Ethovision XT software (Noldus, Wageningen, Netherlands) [45,46] in this and in subsequent experiments that were filmed. Specifically, the following parameters were analyzed: total travel distance, travel distance in central part of the field, entries in center zone and time spent in central part of the field. 2.3. The sucrose preference test The test was performed as described previously [45]. After an adaptation period, the rats were allowed to consume 1% (w/v) sucrose solution by placing two bottles in each cage for 24 h in order to overcome neophobia. Then one of the two bottles was filled with water for 24 h. During the test the rats were housed in individual cages with free access to two bottles, containing 100 mL of the sucrose solution and 100 mL of water. After 4 h, the consumed volume from each of the bottles was recorded. The sucrose preference is then calculated from the expression: sucrose preference(%) = sucrose consumption (ml) × 100% sucrose consumption (ml) + water consumption(ml) 2.4. Elevated plus maze The plus maze was situated in a darkened room and consisted of a plus shaped black Plexiglas construction positioned 50 cm above the 2 Behavioural Brain Research 372 (2019) 112025 D. Frank, et al. day for four days. For the first three days (trials 1–6) the location of the escape box remained the same. On the fourth day (trials 7 and 8) a different escape box, located at a 135 degrees angle to the original box was used. Trials 1–6 were subsequently referred to as habituation (trials or days), and trials 7 and 8 on day four of testing were subsequently referred to as test (trials or days). The following dependent variables were measured and recorded: 1) The latency to find the escape box, defined as the rat putting its entire head into the correct hole, 2) the number of errors before finding the correct hole, defined as investigations (nose-head deflections) into any hole that was not the one with the escape box underneath, and 3) time spent in the quadrant with the correct hole, measured as the time spent in a defined quadrant of interest divided by the total time spent in the maze during the trial. A quadrant was represented by one quarter of the circular platform (containing 4 ½ holes). Note that the quadrant in which the first escape box (trials 1–6) was located, was referred to as the original quadrant fraction and was measured for all 8 trials (1–8). For trials 7–8 the time spent in the quadrant in which that box was located was measured and referred to as the new quadrant fraction. floor, having two open and two closed arms (each of dimensions 16 × 46 cm). The closed arms, opposite to one another, had walls 10 cm. high. Experiments were filmed and analyzed using Ethovision XT software [47]. The apparatus was cleaned with 5% ethanol prior to the introduction of each animal. Rats were tested on the maze in a randomized order. Each rat was placed in the center of the plus maze facing one of the open arms, and allowed to explore the maze for 5 min. The number of entries and time spent in open and closed arms and time in the center of the elevated plus maze were analyzed. 2.5. Light-dark box Behavior assessment was conducted using a black-white box (60 × 60 × 60 cm, with each side measuring 30 cm in width, custommade of laminated wood in the workshop of the university). An aperture (10 × 12 cm) between the black and white chambers allowed rats to pass between black and white compartments. The black chamber was illuminated with infrared light, while the white chamber was illuminated by bright white light. Rats were placed into the middle of the white compartment at the start of the trial and left in the box for 15 min [48]. The behavior of rodents was recorded by the camera and the time spent in the light and dark chambers was analyzed using Ethovision XT software. 2.8. Vogel conflict test Rats were water-deprived for 24 h before testing. Food was available in the home cage at all times. Rats were habituated to the testing cages to assess licking behavior [52] and allowed to drink for 15 min (training session), with an additional 15 min drinking in their home cages. Water deprivation then continued for another 24 h. On the test day, rats were placed in the testing cages. When the rats licked the water spigot on the bottle, they receiving an electric shock of 0.5 mA with pulse duration of 0.2 s. The number of punished responses was automatically recorded for each rat during 5 min of testing. 2.6. Odor associated avoidance behavior The cat odor exposure paradigm is based on defensive behavior displayed by rodents when confronted by a predator or its odor. The test chamber was a box 60-cm long, 40-cm wide, and 50-cm high. The apparatus was divided into 2 sections by a partition. An opening hole (10 × 10 cm) allowed the rat to pass between compartments. A collar that was worn by a domestic cat for 3 weeks (‘worn cat collar’) was used as a cue for predator odor. The rat was allowed to explore the entire box for 5 min without the collar and 5 min after placing the collar on one side of the box [49]. The dependent variables were number of hiding responses, number of approaches and locomotor activity. 2.9. Shock-probe defensive burying test The rats were tested according to the protocol of Bondi et al., [53] in a polystyrene cage, 26 × 48 × 21 cm, identical to the rat's home cage. Fresh bedding lined the cage to a depth of 5 cm. The shock probe was a glass rod, 1.0 cm. wrapped with two alternating, non-touching 18 ga copper wires, spaced 5 loops cm. The probe protruded 6 cm into one end of the cage, 2 cm above the surface of the bedding. The wires were attached to a shock generator set to deliver 2 mA DC current when the probe was touched. A rat was introduced into the cage at the end opposite the shock probe, facing away from the probe. Rats typically approached the probe to investigate within 10–15 s, making contact with their paw or snout. Electric current was administered through two metal wires wrapped around the probe. Shock intensity was adjusted with a variable resistor in series with a 1800 V shock source and set at 2 mA. Upon contacting the probe, the current was turned off so that only a single shock was delivered, and the 15 min test period began. After withdrawing from the probe, rats typically showed a variable period of inactivity before beginning to bury, usually within 5–8 min. “Burying” consisted of burrowing into the bedding with their snout and upper body, then “plowing” the bedding toward the probe, and also flicking bedding toward the probe with the dorsal surface of the forepaws. After each test, the cage was washed with a wet sponge, and the bedding replaced with fresh bedding before testing the next rat. Behavioral scoring for defensive burying time and reaction to shock was scored manually offline by a blind observer using a four-point scale: 0 – no reaction, 1- weak response, 2-intermediate response, 3strong response, 4- strongest response. 2.7. The Barnes maze The Barnes Maze, a negative reinforcement paradigm used to test spatial memory, was a modified version based on that described by Fox et al. [50] and Barnes [51]. The circular platform, made of white acrylic, used for the task was 3 m in diameter, 0.64 cm thick, and 110 in. above the ground. A 500-watt halogen spot-light was hung from the ceiling 131 cm directly above the platform and served as the aversive stimulus. There were 12 holes around the perimeter of the platform, each 8 cm in diameter. Beneath one of the holes was an escape box 18 cm deep, 20 cm long and 14.6 cm wide. Rats were given an acclimation period, during which rodents were placed in the escape box with the aversive stimulus (the spot light) on for five minutes. Then, in the same day, at least 2 h after this adaptation period, testing began. Rats were first placed under a black, opaque, plastic start box 16 × 21 × 16 cm), attached to a wire and pulley system, in the dark testing room. After one minute in the start box, recording began, the spot-light was turned on and the start box was lifted. Rats were given a maximum of five minutes to locate and climb into the escape box under one of the holes of the platform. If the rat was unable to find the escape box after five minutes, it was put into the escape box by the experimenter. After freely entering or being placed into the escape box, rats were allowed one minute in the escape box with the spot light still on to establish the escape box as a safe environment. The rats were then taken out of the escape box and replaced in their home cage for five minutes. After this rest period, the rats were placed in the start box again, and the same procedure for testing described above was followed. The platform and the escape box were cleaned with 5% ethanol solution between every trial. Each rat was tested in this manner twice a 2.10. Dominant-submissive behavior Before the test each rat was habituated for two days, 15 min each session, to the apparatus that consisted of two transparent Plexiglas boxes (30 cm × 20 cm × 20 cm) connected by a narrow passage 3 Behavioural Brain Research 372 (2019) 112025 D. Frank, et al. (15 cm × 15 cm × 60 cm). A feeder containing sweetened milk was placed in the middle of the passage [54]. On the third day, rats were randomly paired: one animal from the control group, and the second from the group of animals that underwent MS. Paired rats were placed at an equal distance from the feeder and behavior was filmed for 5 min and scored offline. The time spent at the feeder for each rat and the first rat to arrive at the feeder was scored. 2.11. The resident-intruder paradigm: a standardized test for aggression, violence and social stress Fig. 1. Sucrose preference for rats exposed to MS compared to control rats. Each male resident (control rat or MS) was housed with a nonstressed companion female which had not undergone MS and was not a sibling. They were housed in a polycarbonate cage with a floor space of about half a square meter to which they were habituated (7 days) prior to testing with ad libitum food and water. Bedding was not changed during that week and during testing. One hour before the test the companion female was removed from the residential cage and an unfamiliar male was introduced into the home cage of the resident. The interactions (the duration and frequency of behavioral parameters) of the two rats were recorded for 10 min as described by Koolhaas et al., [55]. After completion of the test, the male intruder was removed from the cage and the resident male was reunited with its companion female. (56) = 11, p < 0.001, d = 2.88 (see Fig. 1).The data are expressed as percent of control rats and presented as mean ± SEM. 3.2. Anxiety-like behavior 3.2.1. Models based upon spontaneous responses (unconditioned) 3.2.1.1. Exploratory behaviors 3.2.1.1.1. The elevated plus maze. An independent-samples t-test indicated that time spent in open arms of the EPM was significantly lower in the MS than in the Control group 25.7 s. ± 3.2 s. vs. 61.6 s. ± 6.1 s, t (44.6) = 5.1, p < 0.001, d = -1.3 Levene’s test indicated unequal variances (F = 7.4, p < .05), so degrees of freedom were adjusted from 56 to 44.6. (see Fig. 2a). An independent-samples t-test indicated that in the time on the middle platform of the EPM was significantly lower in the MS group compared to the Control group 54.4 s. ± 4.9 s. vs. 129.5 s. ± 4.6 s, t (56) = 11.1, p < 0.001, d = -2.9 (see Fig. 2b). An independent-samples t-test indicated that number platform entries in the EPM was significantly lower for MS than for the Control group 14.1 ± 0.9 vs. 22.3 ± 0.8, t (56) = 6.6, p < 0.001, d = -1.7 (see Fig. 2c). 3.2.1.1.2. Open field test for assessment anxiety and general assessment of animal basal locomotor activity and exploration.. For each variable, the results of the independent-samples t-test are presented, followed by the Levene test for homogeneity of variance. Total Distance Traveled in open field was significantly lower in the MS group than in control group, 5705 cm. ± 313 cm. vs. 8219 cm. ± 164 cm, t(46.1) = -7.6, p < 0.001, d = -2. Because the Levene’s test indicated unequal variances (F = 2, p < .05), the degrees of freedom were adjusted from 56 to 46.1. (see Fig. 2e). Distance Traveled in the Center Zone in the open field test was significantly lower in the MS group compared to the control group, 763 cm. ± 137 cm. vs. 2795 cm. ± 214 cm, t(50) = -7.8, p < 0.001, d = -2. The Levene’s test indicated unequal variances (F = 8, p < .05), so degrees of freedom were adjusted from 56 to 50. (see Fig. 2f). The number of entries in the Center Zone in the open field test was significantly lower in the MS group compared to the control group 11.2 ± 1.1vs. 27.3 ± 1.4, t(54.3) = -8.8, p < 0.001, d = -2.3 Levene’s test indicated unequal variances (F = 3.1, p < .05), so degrees of freedom were adjusted from 56 to 54.3. (see Fig. 2g). The duration of time spent in the Center Zone in open field test for the MS group was significantly lower than for control group 33.3 s. ± 3.9 s. vs. 90.6 s. ± 5.6 s, t(51.5) = -8.3, p < 0.001, d = -2.2 The Levene’s test indicated unequal variances (F = 4.2, p < .05), so degrees of freedom were adjusted from 56 to 51.5. (see Fig. 2h). The data are measured as sec or counts and presented as mean ± SEM. 3.2.1.1.3. Light-Dark test. The MS group spent significantly more time in the dark zone compared to the control group 250 s. ± 10 s. vs. 232 s. ± 5.2 s, t(56) = 3.4, p < 0.001, d = 0.92. (see Fig. 2f). The data are measured as sec and presented as mean ± SEM. 2.12. Statistical analysis Statistical evaluation of the results was performed with the SPSS 22 package (SPSS Inc., Chicago, IL). The significance of comparisons between groups was determined using the Mann-Whitney U test (for nonparametric data) or t-test (for parametric data). The number of rats who came first to the feeder in Dominant–Submissive behavior test and results of successfully locating the escape box in Barnes maze were tested using the chi-square, Fisher’s exact test. To study the correlations between variables and to build a model for predicting submissive behavior, we first performed univariate analysis using Mann-Whitney U test (for non-parametric data) or t-test (for parametric data) and chi-square, Fisher’s exact test (for categorical variables) to assess for potential predictors to be used in the multivariate analysis for groups of submissive and dominant behavior. Variables with a p value ≤ 0.05 in the univariate analyses were included in the multivariate model. Potential predictors that differed between dominant and submissive behavior (for the variable "First to reach feeder"), were analyzed by principal component analysis, which allowed optimally identifying variables that accounted for the variability within the animals and discarding variables which would not provide substantial additional information and subsequently grouping them into factors. In the next phase, we conducted a discriminant function analysis (DFA). In this study, we use step wise DFA, discriminant distance adopting the mahalanobis distance. With Spearman’s test (for non-parametric data) or Pearson's test (for parametric data), we were able to calculate the correlation between behavioral parameters and dominant–submissive obtained results (using the variable - "Time spent at feeder"). Normally distributed data and continuous variables were presented as an average ± SEM. Nonparametric data were presented as a median ± inner quartile range. Results were considered statistically significant when p < 0.05, and highly significant when p < 0.01. 3. Results 3.1. Depressive-like behavior 3.1.1. Sucrose test The MS rats had significantly lower sucrose preference compared to the control rats: MS = 74.9% ± 2.7% vs. Control = 89.8% ± 0.7%., t 4 Behavioural Brain Research 372 (2019) 112025 D. Frank, et al. Fig. 2. a-h. Anxiety-like behavior a. Time in open arms of the EPM, b. Time spent in platform of the EPM, c. Platform entries in EPM, d. Time in dark zone of the black-white box, e. Total Distance Travelled in Open Field, f. Distance Travelled in Center Zone in Open field, g. Entries into Center Zone in Open field and h. Duration in Center Zone in Open field. as sec or counts and presented as median and (25–75) percentile range. 3.2.1.2. Predator-based behaviors 3.2.1.2.1. Odor associated avoidance behavior. T-tests revealed no statistically significant differences in hide times, approach times and locomotor activity for MS and control rats. 3.2.2.2. Conflict model 3.2.2.2.1. Conflict drinking test (Vogel test). A Mann-Whitney test indicated that number of licks in the Conflict Drinking Test was significantly reduced in the MS compared to the control group (0.4 s. ± 0.3 s. vs. 2.9 s. ± 1.1 s.), U = 274, p < 0.01, r = - 0.36. (See Fig. 3c). The data are measured as sec and presented as mean ± SEM. 3.2.2. Models based on learning 3.2.2.1. Frustration (non-reward) 3.2.2.1.1. Shock-probe defensive burying test. A Mann-Whitney U test indicated that defensive burying time in the Shock-probe test was significantly greater for MS vs control rats (33.1 s. ± 8.1 s. vs. 4.5 s. ± 2.1 s, U = 114, p < 0.001, r = - 0.47. (See Fig. 3a). The data are measured as sec and presented as mean ± SEM. A Mann-Whitney test indicated that reaction to shock in the Shockprobe defensive burying test was significantly greater for MS than control rats (Mdn. = 3. Range = (3–4). vs. Mdn. = 2. Range = (1–2.5), U = 100, p < 0.001, r = - 0.48. (See Fig. 3b). The data are measured 3.3. Learning and memory 3.3.1. Barnes maze (for spatial reference memory) The Barnes maze was used as test of visual-spatial learning and memory; however, we found no significant differences in the behavior of MS vs Control rats in this assay. 5 Behavioural Brain Research 372 (2019) 112025 D. Frank, et al. control resident animals, but they displayed non-social exploration more often. 3.4.2. Dominant–submissive behavior An independent-samples t-test indicated that time spent at feeder in Dominant–Submissive behavior test for MS rats was significantly lower than for Control rats, according to the t-test 134 s. ± 2.7 s. vs. 27.1 s. ± 3.5 s., t (56) = 2.96, p < 0.01, d = -0.79 (see Fig. 4g). The data are measured as sec and presented as mean ± SEM. The number of rats who came first to the feeder in Dominant–Submissive behavior test for MS rats (4 from 28) was significantly lower than for Control rats (18 from 30) p < 0.001, according to Chi-square, Fisher’s exact test. (see Fig. 4h). According to Dominant-Submissive behavior test the level of dominance or submission reflects behavior of animals in pairs in competition for food resources. Rodents exposed to MS appeared more submissive, spent less time at the feeder and were less likely than control animals to arrive first at the trough. 3.5. Multivariable statistics and correlation analysis According to the dominant–submissive behavior test ("First rat arriving at feeder”) of the 58 rats, 36 (62.1%) were submissive and 22 (38.9%) were dominant. The characteristics of the dominant and submissive rats showing in Table 1 (Table 2). The results of the discriminant function analyses are presented in Fig. 5 and Table 3a–c. Discriminant analysis was conducted to determine which behavioral tests best discriminated between the twopattern behavior (dominant behavior vs. submissive behavior). Four variables (Attack Latency, Time Spent in Open Arms, Duration in Center, Sucrose Preference) were found to be the variables that best discriminated between dominant vs. submissive behavior. The above 4 behavioral tests were found to be able to classify rats into the dominant or submissive with an accuracy of 86.2% following validation (Wilks’ λ = 0.508, p < 0.001) (Table 3c). Coefficients whose values are less than 0.3 were not shown in the table. To define variables as predictors for Dominant-Submissive behavior from each group of variables (behavioral test), 1 predictor with the highest coefficient value (marked with an asterisk) was selected (Tables 4 and 5). Fig. 3. a-c. Anxiety-like behavior based upon learning a. Shock-probe burying time, b. shock reaction score, c. Vogel Conflict test, number of licks. 3.4. Social behavior 3.4.1. The resident-intruder paradigm Attack Latency was significantly longer for MS vs Control rats, according to the Mann-Whitney test (314 s. ± 31 s. vs. 107 s. ± 7.6 s.), U = 4, p < 0.001, r = - 0.6. (See Fig. 4a). Move Toward was significantly lower in the MS group compared to control rats, according to t-test 1.4 s. ± 0.3 s. vs. 4.8 s. ± 0.6 s, t (43.7) = 4.9, p < 0.001, d = 1.28. Because the Levene’s test indicated unequal variances (F = 6.7, p < .05), degrees of freedom were adjusted from 56 to 43.7. (see Fig. 4b). Upright Posture was significantly lower for the MS than for Control rats, according to the Mann-Whitney test (1.6 s. ± 0.5 s. vs. 3.7 s. ± 0.6 s.), U = 214, p < 0.001, r = - 0.43. (See Fig. 4c). Clinch Attack in was significantly lower for the MS than for Control rats, according to the Mann-Whitney test (1.7 s. ± 0.5 s. vs. 49 s. ± 4.5 s.), U = 1.5, p < 0.001, r = - 0.87. (See Fig. 4d). Keep Down in the Social behavior test was significantly lower for MS than for the Control rats, according to the Mann-Whitney test (4.3 s. ± 2.3 s. vs. 20.7 s. ± 4.2 s.), U = 192, p < 0.001, r = - 0.51. (See Fig. 4e). An independent-samples t-test indicated that Non-social exploration for MS was significantly greater than for Control rats, according to the ttest 307 s. ± 17.3 s. vs. 249 s. ± 7.8 s., t (43.1) = -4.33, p < 0.001, d = 1.15. Since the Levene’s test indicated unequal variances (F = 6.1, p < .05), degrees of freedom were adjusted from 56 to 43.1. (see Fig. 4f). The data are measured as sec and presented as mean ± SEM. The Resident-Intruder test is one commonly used social stress paradigm, where territoriality plays an important role. The frequency of aggressive interactions on the part of MS resident rats was lower than 4. Discussion A number of clinical studies indicate that early life stressful experiences such as child neglect and abuse may increase the risk for psychiatric disorders later in life [56]. Moreover, the stress of poverty affects brain development in children [57]. In the current study, a challenging method of daily post-natal maternal deprivation had longterm effects on measures of anxiety, depression and social behavior, while not affecting learning. Anxiety manifested itself by less time spent in the central zone of an open field, less time exploring the open arms and the central platform of the elevated plus maze, less time in the light zone of the light-dark box. In addition, MS rats showed sevenfold more defensive shock-probe burying and significantly greater reaction to shock and reduced number of licks in Vogel conflict drinking test, which indicates anxiety. The two main components of the mammalian response to early aversive conditions are the sympathetic adrenomedullary (SAM) system and HPA axis. Both are modulated by neural circuits involving areas of the prefrontal cortex, hippocampus, amygdala, hypothalamus, and brain stem nuclei. Characteristic biochemical features of these stress reactions is the presence of a dysregulated hypothalamic-pituitaryadrenal (HPA) - axis as evidenced by a hypersecretion of corticotropinreleasing factor (CRF), as well as abnormalities in the basal secretion of adrenocorticotropin hormone (ACTH) and cortisol. Increase CRF leads 6 Behavioural Brain Research 372 (2019) 112025 D. Frank, et al. Fig. 4. a-h. Social behavior assessments for rats exposed to MS compared to control rats. a–f Resident intruder test behavioral scores: a. Attack Latency, b. Move Toward, c. Upright Posture Duration, d. Clinch Attack Duration, e. Keep Down Duration, f. Non-social exploratory behavior duration. g–h. Dominant-submissive Test, g. Time at Feeder, h. First Arrival at Feeder. the hippocampal-dependent memory induced by MS might be due to heterogeneity of MS protocols and the use different animal strains. For example, Levy et al. (2003) established that Sprague-Dawley rats, which in the preweanling period were subjected to maternal deprivation, did not show impairment of spatial memory in adulthood, but they had lower scores on the social learning tasks [62]. But results of later studies [63,64] showed impairment of spatial memory in adult Wistar rats which had undergone MS on days 2–14 or 2–21 for 180 min daily. The inconsistencies among different studies on the effects of MS on different models of learning and locomotor activity might be related to differences in strain and in methodology [63,65]. In the resident-intruder test, adult males who had undergone MS in the preweanling period showed a longer latency to approach an intruder and markedly attenuated aggressive behavior in the presence of the intruder. In the competitive dominant-submissive behavioral assay, the MS rats showed more submissive behavior when facing the nonstressed control rats. Social behavior contributes not only to the individual survival of the individual animal, but also to improving the chances of survival of the to activation of the autonomic nervous system, elevation of peripheral catecholamines, and increased heart rate and blood pressure, which is characteristic of the state of fear and anxiety. In this way early life stress mediates neurodevelopmental changes through alterations to the CRF and the HPA axis, which relate to the pathophysiology of mood and anxiety disorders [58]. Altered HPA function can be associated with dysregulation in noradrenergic (NA) and serotonergic (5-HT) systems [59]. Changes to monoamines are considered to be relevant for the regulatory developmental ontogeny of anxiety at critical time windows. Hypoactivity or hyperactivity of the 5-HT system causes anxiety and mood disorders [60]. We found no difference in ability of rats of both groups to learn and remember the location of a target zone in the Barnes maze, which indicates the lack of influence of MS on spatial learning and memory. The Barnes maze is similar to the Morris water maze test, but does not utilize a strong aversive stimulus (stress induced by swimming). Behavioral tasks involving high levels of stress can influence the animal's performance on the task [61], so the Barnes maze is ideal for eliminating stress-induced confounds. Contradictory data on deficits in 7 D. Frank, et al. Table 1 Comparison of the characteristics of the Dominant and Submissive Groups. Variables with a p value less than 0.05 were identified as potential predictors for dominant–submissive behavior. Variables Assessment anxiety-like behavior Mean and SEM for parametric data / median and range for non-parametric data Models based upon spontaneous responses (unconditioned) Exploratory behaviors Elevated plus maze test Open field test Predator-based behaviors 8 Models based upon learned (conditioned) Frustration (non-reward) Conflict model The social behavior assessments Assessment of cognitive and memory impairments Shock-robe defensive burying test Vogel test The Residentintruder Paradigm Barnes maze Sucrose Preference test Statistics reporting* t(27.6) = 3.7 d = 1.22 t(56) = 2 d = 0.57 t(56) = 2.8 d = 0.7 Dominant n = 22 Submissive n = 36 Time spent in open arms 66.2 ± 6.9 sec. 31.4 ± 4 sec. p < 0.001 Platform entries 20.6 ± 0.9 17.4 ± 0.9 p < 0.05 Time spent in Platform 112 ± 6.4 sec. 83.3 ± 8.1 sec. p < 0.01 Total Traveled Distance Traveled Distance in Center In Center Frequency 7376 ± 300 cm. 2175 ± 250 cm. 23.2 ± 1.8 6932 ± 292 cm. 1609 ± 253 cm. 17.6 ± 1.9 n.s. n.s. p < 0.05 Duration in Center 78.2 ± 6.9 sec. 54.4 ± 6.6 sec. p < 0.05 Time Spent in Dark Zone The time spent in the compartment containing of the odorant stimulus The number of crossing between compartments Reaction to shock Defensive burying time 243 ± 4.9 sec. 72.6 ± 9.7 sec. 246 ± 6.4 sec. 78.8 ± 8.9 sec. n.s. n.s. 3.64 ± 0.51 sec. 4.2 ± 0.34 sec. n.s. 2 (1-2) 12.79 ± 5.46 sec. 3 (2-4) 18.86 ± 6.14 sec. p < 0.001 p < 0.001 U = 105, r = 0.49 U = 115, r = 0.49 Number of licks 3.64 ± 1.5 0.5 ± 0.27 p < 0.01 U = 274, r = 0.36 Attack latency Move toward 91 ± 6.5 sec. 5.14 ± 0.8 sec. 229 ± 24 sec. 1.97 ± 0.3 sec. p < 0.001 p < 0.01 Upright posture Clinch attack Keep down Non-social explore Number of errors before finding the escape box Successful finding escape box Time to Spend in sector I Time to Spend in sector II Time to Spend in sector III Time to Spend in sector IV Total Time to Spend in arena Sucrose preference 3.23 ± 0.81 sec. 44.6 ± 6.7 sec. 24.2 ± 5 sec. 266 ± 12 sec. 3.7 ± 0.5 2.36 ± 0.49 sec. 14.9 ± 3.7 sec. 5.8 ± 2.4 sec. 292 ± 12 sec. 4 ± 0.8 p < 0.01 p < 0.001 p < 0.001 n.s. n.s. U = 4, r = 0.6 t(27.6) = 3.6 d = 1.06 U = 215, r = 0.43 U = 408, r = 0.87 U = 192, r = 0.51 22 (100%) 26 ± 4.6 sec. 4.8 ± 2.2 sec. 23 ± 6 sec. 3 ± 1.5 sec. 53 ± 7.8 sec. 89 ± 1.3 % 34 (94.4%) 33 ± 7.3 sec. 5.3 ± 2.2 sec. 28 ± 7 sec. 1.8 ± 0.8 sec. 68 ± 13.3 sec. 80.7 ± 1.1 % n.s. n.s. n.s. n.s. n.s. n.s. p < 0.001 *In T-tests Mean and SEM, degrees of freedom, statistics t and Cohen’s d are presented. *In Mann-Whitney Test. The median and range, statistics U, and effect size, r = Z / √N are reported. *In Fisher’s exact test (for categorical variables “Successful finding escape box in Barnes maze test”) The Chi-square (χ2) and degrees of freedom are reported. t(56) = 2 d = 0.58 t(56) = 2.4 d = 0.66 t(56) = 4.8 d = 1.3 Behavioural Brain Research 372 (2019) 112025 Assessment depressive-like behavior Light-dark test Odor associated avoidance behavior p‑value (twotailed) Behavioural Brain Research 372 (2019) 112025 D. Frank, et al. Table 2 The results of the principal component analysis are presented in Table 2 (Rotated Component Matrix: Varimax with Kaiser Normalization). The following tests best describe the predictors within the study group. Assessment anxiety-like behavior Models based upon spontaneous responses (unconditioned) Exploratory behaviors Behavioral tests Variables 1 2 Elevated plus maze test Time spent in open arms Platform entries Time spent in Platform In Center Frequency Duration in Center Reaction to shock Defensive burying time Number of licks .700* −.326 Attack latency Move toward Upright posture Clinch attack Keep down Sucrose preference −.613* Open field test Models based upon learned (conditioned) Frustration (non-reward) Conflict model The social behavior assessments Assessment depressive-like behavior Shock-probe defensive burying test Vogel test The Residentintruder Paradigm Sucrose Preference test 3 4 5 .440 .735 .807 .837 .338 .863* −.479 −.381 −.551 −.359 .863 −.393 −.412 .799 −.312 .831 .401 .310* .626 .347 .701 .550 .425 .308 whole community and species, therefore submissive behavior could potentially impair individual or group survival. In communities, each animal occupies a definite position in the hierarchy, performs certain functions and also establishes between other animals the procedure for using various "benefits", in the first place, food. Price et al. [66] suggested that dominance and subordination were equivalent to mania and depression, respectively. Many studies have examined the similarity between submissive behavior in animals and depression in humans [67]. Subordinate animals, similarly to depressed humans, show increased defensive behavior, weight loss and major alterations in sleep, eating and active behaviors. Animal models based on dominant–submissive behavior may be more valid indicators of the neuralemotional systems that are disturbed in depression and mania than models based on changes in locomotors activity [40]. In summary, we have shown that submissive animals in the dominant-submissive models demonstrated subordinate behavior in the resident–intruder test and increased anxiety even though cognitive behaviors were not impaired. Other studies have reported increased anxiety and depression associated with increased aggressiveness in rats subjected to a 3 h/day protocol of MS [42] or a reduction in some measures of aggression such as boxing and avoidance of attacks, using evasions [68]. Another factor that can mediate the effect of MS is the reaction of the dam following reunion with the entire litter. MS stress affects maternal behavior, when the nursing dam is deprived of all her pups for a long period [69]. Three-hour daily separation was found to increase maternal arched back nursing in rats [17] and increased time in the nest in several mouse strains [14], but reduced maternal licking of pups on PND 6, but not on PND 3 or 9 [11]. Isolating the dam from the litter has been found to induce more intense maternal behavior [68] and increase corticosterone levels [7]. Other forms of early life stress which do not separate the dam from the pups also affect maternal and pup behavior. Exposing the dam and litter to an intruder male enhanced both nursing and pup retrieval as a function of the pups’ age and reduced the expression of oxytocin and prolactin transcripts, which was correlated to altered maternal behavior [70]. Restricting nesting material [71] lead to entropy and disorganized maternal care, without reducing the total amount of time that the dam spent with the pup [71–73]. The adult offspring had reduced weight, higher levels of plasma corticosterone, and impaired memory in both spatial and non-spatial learning assays. Fig. 5. Histogram of values of discriminant function Dominant-Submissive behavior for the predictors: Attack latency (Resident-Intruder), Time spent in open arms (EPM), Duration in Center (Open field), Sucrose preference. 9 Behavioural Brain Research 372 (2019) 112025 D. Frank, et al. Table 3 The results of the Canonical Discriminant Function Coefficients. a. The results of the Canonical Discriminant Function Coefficients d = 7.420 + 0.006*” Attack latency” -0.018* “Time spent in open arms” + 0.022* “Duration in Center” – 0.105* “Sucrose preference” Canonical Discriminant Function Coefficients Function 1 Attack latency Time spent in open arms Duration in Center Sucrose preference (Constant) 0.006 −0.018 0.022 −0.105 7.420 Classification Resultsa Original Predicted Group Membership Count Submissive behavior Dominant behavior Submissive behavior Dominant behavior % a Submissive behavior Dominant behavior Total 30 2 83.3 9.1 6 20 16.7 90.9 36 22 100.0 100.0 86.2% of original grouped cases correctly classified. c The results of the discriminant function analyses; Wilks' Lambda. Wilks' Lambda Test of Function(s) Wilks' Lambda Chi-square df Sig. 1 0.508 24.382 4 0.00007 Table 4 Comparing between Dominant–submissive behavior and anxiety-like behavior, depressive-like behavior social behavior, cognitive and memory impairments. Behavioral tests and their variables Dominant–submissive behavior: First rat comes to feeder R Assessment anxiety-like behavior Models based upon spontaneous responses (unconditioned) Exploratory behaviors Elevated plus maze test Open field test Predator-based behaviors Models based upon learned (conditioned) Frustration (non-reward) Conflict model The social behavior assessments Assessment of cognitive and memory impairments Assessment depressive-like behavior Light-dark test Odor associated avoidance behavior Shock-robe defensive burying test Vogel test The Residentintruder Paradigm Barnes maze Sucrose Preference test 10 Time spent in open arms Platform entries Time spent in Platform Total Traveled Distance Traveled Distance in Center In Center Frequency Duration in Center Time Spent in Dark Zone The time spent in the compartment containing of the odorant stimulus The number of crossing between compartments Reaction to shock Defensive burying time Rp = 0.29 P value (twotailed) n.s. n.s. p < 0.05 n.s. n.s. n.s. n.s. n.s. n.s. n.s. Rs=-0.43 p < 0.01 n.s. Number of licks Rs = 0.45 p < 0.01 Attack latency Move toward Upright posture Clinch attack Keep down Non-social explore Number of errors before finding the escape box Successful finding escape box Time to Spend in sector I Time to Spend in sector II Time to Spend in sector III Time to Spend in sector IV Total Time to Spend in arena Sucrose preference Rs=-0.39 p < n.s. n.s. p < p < p < n.s. Rs=-0.33 Rs=-0.35 Rp=-0.29 Rp = 0.43 0.05 0.05 0.01 0.05 n.s. n.s. n.s. n.s. n.s. n.s. p < 0.01 D. Frank, et al. Table 5 Comparing between social behavior, depressive-like behavior and anxiety-like behavior in study groups. The social behavior Assessments: The Resident-intruder Paradigm Assessment anxiety-like behavior Elevated plus maze test Time spent in open arms Platform entries Time spent in Platform Open field test Total Traveled Distance 11 Traveled Distance in Center In Center Frequency Duration in Center Light-dark test Time Spent in Dark Zone Odor associated avoidance behavior The time spent in the compartment containing of the odorant stimulus The number of crossing between compartments Reaction to shock Shock-robe defensive burying test Defensive burying time Number of licks Sucrose Preference test Sucrose Preference Attack latency Move toward Upright posture Clinch attack Keep down Non-social explore Rs=-0.64; p < 0.001 p - n.s. p - n.s. p - n.s. p - n.s. p - n.s. Rs = 0.60; p < 0.001 Rs = 0.62; p < 0.001 Rs = 0.68; p < 0.001 Rs = 0.67; p < 0.001 Rs = 0.73; p < 0.001 Rs = 0.73; p < 0.001 Rs = 0.71; p < 0.001 Rs=-0.43; p < 0.001 p - n.s. Rs = 0.60; p < 0.001 Rs = 0.40; p < 0.01 Rs = 0.43; p < 0.001 Rs = 0.30; p < 0.05 Rs = 0.44; p < 0.001 Rs = 0.47; p < 0.001 Rs = 0.45; p < 0.001 Rs=-0.29; p < 0.05 p - n.s. p - n.s. Rs=-0.50; p < 0.001 Rs=-0.51; p < 0.001 Rs=-0.61; p < 0.001 p - n.s. Rp = 0.39; p < 0.01 Rp = 0.40; p < 0.01 Rp = 0.34; p < 0.05 Rp = 0.33; p < 0.05 Rp = 0.32; p < 0.05 Rp = 0.36; p < 0.01 Rp = 0.37; p < 0.01 p - n.s. Rp=-0.35; p < 0.01 Rp=-0.51; p < 0.001 Rp=-0.38; p < 0.01 Rp=-0.36; p < 0.01 Rp=-0.37; p < 0.01 Rp=-0.35; p < 0.01 Rp = 0.38; p < 0.01 p - n.s. Rp = 0.49; p < 0.001 Rp = 0.59; p < 0.001 Rp = 0.65; p < 0.001 Rp = 0.59; p < 0.001 Rp = 0.61; p < 0.001 Rp = 0.60; p < 0.001 Rp = 0.62; p < 0.001 Rp=-0.28; p < 0.05 p - n.s. p - n.s. p - n.s. p - n.s. p - n.s. p - n.s. p - n.s. p - n.s. Rs = 0.41; p < 0.05 p - n.s. p - n.s. p - n.s. p - n.s. Rs=-0.46; p < 0.001 p - n.s. p - n.s. Rs=-0.33; p < 0.05 p - n.s. Rs=-0.56; p < 0.001 Rs=-0.41; p < 0.01 Rs = 0.30; p < 0.05 Rp = 0.52; p < 0.001 Rs = 0.43; p < 0.001 Rs=-0.36; p < 0.05 Rs=-0.42; p < 0.001 Rs=-0.26; p < 0.05 Rs = 0.76; p < 0.001 Rs=-0.45; p < 0.01 p - n.s. Rs=-0.51; p < 0.001 Rs=-0.50; p < 0.001 Rs = 0.43; p < 0.001 Rs = 0.41; p < 0.01 Rs = 0.32; p < 0.05 Rs = 0.36; p < 0.01 Rs = 0.39; p < 0.01 Rs = 0.35; p < 0.01 p - n.s. p - n.s. Rs=-0.41; p < 0.001 Rs = 0.60; p < 0.001 Rs = 0.44; p < 0.01 p - n.s. Rp=-0.39; p < 0.01 Behavioural Brain Research 372 (2019) 112025 Assessment depressive-like behavior Vogel test Assessment depressive-like behavior Sucrose preference test Behavioural Brain Research 372 (2019) 112025 D. Frank, et al. Anhedonia in adults following even mild early life stress is a robust phenomenon [25,74] and has been linked to aberrant circuitry of the reward and stress systems. Anhedonia following early stress has been reversed by preventing corticotropin releasing hormone (CRH) activity in the amygdala [74]. Neonatal isolation impaired partnership formation in monogamous prairie voles [75]. Hence, MS is a model that can have long-term consequences on reward and stress regulatory systems. [6] Y.D. Gilles, E.K. Polston, Effects of social deprivation on social and depressive-like behaviors and the numbers of oxytocin expressing neurons in rats, Behav. Brain Res. 328 (2017) 28–38, https://doi.org/10.1016/j.bbr.2017.03.036. [7] S. Lundberg, M. Martinsson, I. Nylander, E. Roman, Altered corticosterone levels and social play behavior after prolonged maternal separation in adolescent male but not female Wistar rats, Horm. Behav. 87 (2017) 137–144, https://doi.org/10.1016/ j.yhbeh.2016.11.016. [8] H. Ji, W. Su, R. Zhou, J. Feng, Y. Lin, Y. Zhang, X. Wang, X. Chen, J. Li, Intranasal oxytocin administration improves depression-like behaviors in adult rats that experienced neonatal maternal deprivation, Behav. Pharmacol. 27 (2016) 689–696, https://doi.org/10.1097/FBP.0000000000000248. [9] C. Heim, P.M. Plotsky, C.B. Nemeroff, Importance of studying the contributions of early adverse experience to neurobiological findings in depression, Neuropsychopharmacology 29 (2004) 641. [10] N.K. Moog, S. Entringer, J.M. Rasmussen, M. Styner, J.H. Gilmore, N. Kathmann, C.M. Heim, P.D. Wadhwa, C. Buss, Intergenerational effect of maternal exposure to childhood maltreatment on newborn brain anatomy, Biol. Psychiatry 83 (2018) 120–127, https://doi.org/10.1016/j.biopsych.2017.07.009. [11] V.V. Reshetnikov, A.V. Kovner, A.A. Lepeshko, K.S. Pavlov, L.N. Grinkevich, N.P. Bondar, Stress early in life leads to cognitive impairments, reduced numbers of CA3 neurons and altered maternal behavior in adult female mice, Genes Brain Behav. (2018) e12541. [12] D.G. Gee, L.J. Gabard-Durnam, J. Flannery, B. Goff, K.L. Humphreys, E.H. Telzer, T.A. Hare, S.Y. Bookheimer, N. Tottenham, Early developmental emergence of human amygdala–prefrontal connectivity after maternal deprivation, Proc. Natl. Acad. Sci. (2013) 201307893. [13] B. Goff, D.G. Gee, E.H. Telzer, K.L. Humphreys, L. Gabard-Durnam, J. Flannery, N. Tottenham, Reduced nucleus accumbens reactivity and adolescent depression following early-life stress, Neuroscience 249 (2013) 129–138, https://doi.org/10. 1016/j.neuroscience.2012.12.010. [14] R.A. Millstein, A. Holmes, Effects of repeated maternal separation on anxiety-and depression-related phenotypes in different mouse strains, Neurosci. Biobehav. Rev. 31 (2007) 3–17. [15] R. Gardner, Mechanisms in manic-depressive disorder: an evolutionary model, Arch. Gen. Psychiatry 39 (1982) 1436–1441. [16] R.D. Romeo, A. Mueller, H.M. Sisti, S. Ogawa, B.S. McEwen, W.G. Brake, Anxiety and fear behaviors in adult male and female C57BL/6 mice are modulated by maternal separation, Horm. Behav. 43 (2003) 561–567. [17] J. Maniam, M.J. Morris, Long-term postpartum anxiety and depression-like behavior in mother rats subjected to maternal separation are ameliorated by palatable high fat diet, Behav. Brain Res. 208 (2010) 72–79. [18] J.H. van Heerden, V. Russell, A. Korff, D.J. Stein, N. Illing, Evaluating the behavioural consequences of early maternal separation in adult C57BL/6 mice; the importance of time, Behav. Brain Res. 207 (2010) 332–342. [19] D.B. Parfitt, J.K. Levin, K.P. Saltstein, A.S. Klayman, L.M. Greer, D.L. Helmreich, Differential early rearing environments can accentuate or attenuate the responses to stress in male C57BL/6 mice, Brain Res. 1016 (2004) 111–118. [20] F.R. D’Amato, S. Cabib, R. Ventura, C. Orsini, Long-term effects of postnatal manipulation on emotionality are prevented by maternal anxiolytic treatment in mice, Dev. Psychobiol. 32 (1998) 225–234. [21] S.M. O’Mahony, N.P. Hyland, T.G. Dinan, J.F. Cryan, Maternal separation as a model of brain–gut axis dysfunction, Psychopharmacology (Berl.) 214 (2011) 71–88. [22] K. Björkqvist, Social defeat as a stressor in humans, Physiol. Behav. 73 (2001) 435–442. [23] I.D. Chase, K. Seitz, Self-structuring properties of dominance hierarchies a new perspective, Adv. Genet. 75 (2011) 51. [24] R.J. Blanchard, K.J. Flannelly, D.C. Blanchard, Life-span studies of dominance and aggression in established colonies of laboratory rats, Physiol. Behav. 43 (1988) 1–7. [25] D. Riga, J.T. Theijs, T.J. de Vries, A.B. Smit, S. Spijker, Social defeat-induced anhedonia: effects on operant sucrose-seeking behavior, Front. Behav. Neurosci. 9 (2015) 195, https://doi.org/10.3389/fnbeh.2015.00195. [26] V. Krishnan, E.J. Nestler, Animal models of depression: molecular perspectives, Curr. Top. Behav. Neurosci. 7 (2011) 121, https://doi.org/10.1007/7854_2010_ 108. [27] M. Broom, A. Koenig, C. Borries, Variation in dominance hierarchies among groupliving animals: modeling stability and the likelihood of coalitions, Behav. Ecol. 20 (2009) 844–855, https://doi.org/10.1093/beheco/arp069. [28] E. Malatynska, R. Goldenberg, L. Shuck, A. Haque, P. Zamecki, G. Crites, N. Schindler, R.J. Knapp, Reduction of submissive behavior in rats: a test for antidepressant drug activity, Pharmacology 64 (2002) 8–17, https://doi.org/10. 1159/000056145. [29] E. Malatyńska, W. Kostowski, The effect of antidepressant drugs on dominance behavior in rats competing for food, Pol. J. Pharmacol. Pharm. 36 (1984) 531–540. [30] M. Lund, Social mechanisms and social structure in rats and mice, Ecol. Bull. (1975) 255–260. [31] O. Kaidanovich-Beilin, T. Lipina, I. Vukobradovic, J. Roder, J.R. Woodgett, Assessment of social interaction behaviors, J. Visualized Exp.: JoVE (2011), https:// doi.org/10.3791/2473. [32] G. Karvat, T. Kimchi, Systematic autistic-like behavioral phenotyping of 4 mouse strains using a novel wheel-running assay, Behav. Brain Res. 233 (2012) 405–414, https://doi.org/10.1016/j.bbr.2012.05.028. [33] E.B. Defensor, B.L. Pearson, R.L.H. Pobbe, V.J. Bolivar, R.J. Blanchard, D.C. Blanchard, A novel social proximity test suggests patterns of social avoidance and gaze aversion-like behavior in BTBR T+ tf/J mice, Behav. Brain Res. 217 (2011) 302–308, https://doi.org/10.1016/j.bbr.2010.10.033. 5. Conclusions The main finding of this study was that the early stress caused by maternal deprivation of pups leads to submissive behavior in the population of rats in adulthood. Analysis of a battery of tests showed that submissive behavior could be predicted from four standard behavioural tests: two measures based on the EPM test, anhedonia, as measured by sweet preference, and aggression, as measured by the latency to attack in the Resident-Intruder test. Based on results of multivariate statistical analysis, we conclude that the dominant submissive behavior correlates with depressive, anxiety and attack behavior and can be predicted with an accuracy of 86.2% (the dominant behavior with an accuracy of 90.9%, submissive with an accuracy of 83.3%). Since in our sample there were no rats with cognitive and memory impairments (according to Barnes maze), the social behavior deficits cannot be attributed to cognitive factors. This study adds to the literature on the long-term effects of MS stress, by emphasizing how anxiety and depressive behavior predict predominantly submissive social behavior. We suggest that future studies investigate the submissive trait as a predictor of reactions to subsequent stressors. 6. Declaration of competing interest None. Acknowledgments The authors gratefully acknowledge Dr. S. Swees resident in general surgery department, Dr. A. Alkhazov, resident in orthopedic surgery department and Dr. I. Sief, resident in the Division of anesthesiology and critical care, Soroka University Medical Center, Ben-Gurion University of the Negev, for their help in analyzing of video records of the social organization test. Special thanks to the Y. Bykova, Applicant of the Dnipropetrovs’k regional Regional Institute of Public Administration of National Academy of Public Administration, Office of the President of Ukraine, for her contribution to training and practical assistance in building models of behavior under stressful situations. This research was partly supported by the ISRAEL SCIENCE FOUNDATION (grant No. 1490/15) awarded to Matthew Boyko and Alexander Zlotnik. References [1] S.S. Janetsian-Fritz, N.M. Timme, M.M. Timm, A.M. McCane, A.J. Baucum Ii, B.F. O’Donnell, C.C. Lapish, Maternal deprivation induces alterations in cognitive and cortical function in adulthood, Transl. Psychiatry 8 (2018), https://doi.org/10. 1038/s41398-018-0119-5 71-15. [2] E. Uribe, E. Sánchez-Mendoza, N. Nieves, G. Merchor, Neonatal administration of memantine enhances social cognition in adult rats subjected to early maternal deprivation, Exp. Neurobiol. 25 (2016) 328–332, https://doi.org/10.5607/en.2016. 25.6.328. [3] M. Banqueri, M. Méndez, J.L. Arias, Spatial memory-related brain activity in normally reared and different maternal separation models in rats, Physiol. Behav. 181 (2017) 80–85, https://doi.org/10.1016/j.physbeh.2017.09.007. [4] J. Menezes, B. Neves, M. Souza, P.B. Mello-Carpes, Green tea protects against memory deficits related to maternal deprivation, Physiol. Behav. 182 (2017) 121–127. [5] A. Diamantopoulou, T. Kalpachidou, G. Aspiotis, I. Gampierakis, F. Stylianopoulou, A. Stamatakis, An early experience of mild adversity involving temporary denial of maternal contact affects the serotonergic system of adult male rats and leads to a depressive-like phenotype and inability to adapt to a chronic social stress, Physiol. Behav. 184 (2018) 46–54, https://doi.org/10.1016/j.physbeh.2017.11.004. 12 Behavioural Brain Research 372 (2019) 112025 D. Frank, et al. [34] J.J. Nadler, S.S. Moy, G. Dold, N. Simmons, A. Perez, N.B. Young, R.P. Barbaro, J. Piven, T.R. Magnuson, J.N. Crawley, Automated apparatus for quantitation of social approach behaviors in mice, Genes Brain Behav. 3 (2004) 303–314, https:// doi.org/10.1111/j.1601-183X.2004.00071.x. [35] T. Strekalova, R. Spanagel, D. Bartsch, F.A. Henn, P. Gass, Stress-induced anhedonia in mice is associated with deficits in forced swimming and exploration, Neuropsychopharmacology 29 (2004) 2007. [36] A. Burokas, J. Gutiérrez‐Cuesta, E. Martín‐García, R. Maldonado, Operant model of frustrated expected reward in mice, Addict. Biol. 17 (2012) 770–782. [37] M.M. Holmes, L. Niel, J.J. Anyan, A.T. Griffith, D.A. Monks, N.G. Forger, Effects of Bax gene deletion on social behaviors and neural response to olfactory cues in mice, Eur. J. Neurosci. 34 (2011) 1492–1499. [38] N.N. Kudryavtseva, Use of the "partition" test in behavioral and pharmacological experiments, Neurosci. Behav. Physiol. 33 (2003) 461 doi: 1023411217051. [39] D.F. Avgustinovich, O.V. Gorbach, N.N. Kudryavtseva, Comparative analysis of anxiety-like behavior in partition and plus-maze tests after agonistic interactions in mice, Physiol. Behav. 61 (1997) 37–43, https://doi.org/10.1016/S0031-9384(96) 00303-4. [40] E. Malatynska, R.J. Knapp, Dominant–submissive behavior as models of mania and depression, Neurosci. Biobehav. Rev. 29 (2005) 715–737, https://doi.org/10.1016/ j.neubiorev.2005.03.014. [41] A. Pinhasov, J. Crooke, D. Rosenthal, D. Brenneman, E. Malatynska, Reduction of Submissive Behavior Model for antidepressant drug activity testing: study using a video-tracking system, Behav. Pharmacol. 16 (2005) 657–664, https://doi.org/10. 1097/00008877-200512000-00009. [42] Alexa H. Veenema, Inga D. Neumann, Maternal separation enhances offensive playfighting, basal corticosterone and hypothalamic vasopressin mRNA expression in juvenile male rats, Psychoneuroendocrinology 34 (2008) 463–467, https://doi.org/ 10.1016/j.psyneuen.2008.10.017. [43] M. Lippmann, A. Bress, C.B. Nemeroff, P.M. Plotsky, L.M. Monteggia, Long-term behavioural and molecular alterations associated with maternal separation in rats, Eur. J. Neurosci. 25 (2007) 3091, https://doi.org/10.1111/j.1460-9568.2007. 05522.x. [44] Jaclyn M. Spivey, Eimeira Padilla, Jason D. Shumake, F. Gonzalez-Lima, Effects of maternal separation, early handling, and gonadal sex on regional metabolic capacity of the preweanling rat brain, Brain Res. 1367 (2010) 198–206, https://doi.org/ 10.1016/j.brainres.2010.10.038. [45] M. Boyko, R. Kutz, B. Gruenbaum, H. Cohen, N. Kozlovsky, S. Gruenbaum, Y. Shapira, A. Zlotnik, The influence of aging on poststroke depression using a rat model via middle cerebral artery occlusion, Cogn. Affect. Behav. Neurosci. 13 (2013) 847–859, https://doi.org/10.3758/s13415-013-0177-3. [46] M. Boyko, A.N. Azab, R. Kuts, B.F. Gruenbaum, S.E. Gruenbaum, I. Melamed, E. Brotfain, Y. Shapira, E. Cesnulis, A. Zlotnik, The neuro-behavioral profile in rats after subarachnoid hemorrhage, Brain Res. 1491 (2013) 109–116. [47] M. Boyko, R. Kutz, J. Grinshpun, V. Zvenigorodsky, B.F. Gruenbaum, S.E. Gruenbaum, E. Brotfain, Y. Shapira, A. Zlotnik, Establishment of an animal model of depression contagion, Behav. Brain Res. 281 (2015) 358–363, https://doi. org/10.1016/j.bbr.2014.12.017. [48] M. Bourin, M. Hascoët, The mouse light/dark box test, Eur. J. Pharmacol. 463 (2003) 55–65. [49] D.V. Garg, V.J. Dhar, A. Sharma, R. Dutt, Experimental model for antianxiety activity: a review, Pharmacology 1 (2011) 394–404. [50] G.B. Fox, L. Fan, R.A. LeVasseur, A.I. Faden, Effect of traumatic brain injury on mouse spatial and nonspatial learning in the Barnes circular maze, J. Neurotrauma 15 (1998) 1037–1046, https://doi.org/10.1089/neu.1998.15.1037. [51] C.A. Barnes, Memory deficits associated with senescence: a neurophysiological and behavioral study in the rat, J. Comp. Physiol. Psychol. 93 (1979) 74–104, https:// doi.org/10.1037/h0077579. [52] I. Belcheva, S. Belcheva, V.D. Petkov, V.V. Petkov, C. Hadjiivanova, Behavorial responses to the 5-HT1A receptor antagonist NAN190 injected into rat CA1 hippocampal area, Gen. Pharmacol. 28 (1997) 435–441, https://doi.org/10.1016/ S0306-3623(96)00185-1. [53] C.O. Bondi, G. Barrera, M.D.S. Lapiz, T. Bedard, A. Mahan, D.A. Morilak, Noradrenergic facilitation of shock-probe defensive burying in lateral septum of rats, and modulation by chronic treatment with desipramine, Prog. Neuropsychopharmacol. Biol. Psychiatry 31 (2007) 482–495, https://doi.org/10. 1016/j.pnpbp.2006.11.015. [54] E. Malatynska, A. Pinhasov, J.J. Crooke, V.L. Smith-Swintosky, D.E. Brenneman, Reduction of dominant or submissive behaviors as models for antimanic or [55] [56] [57] [58] [59] [60] [61] [62] [63] [64] [65] [66] [67] [68] [69] [70] [71] [72] [73] [74] [75] 13 antidepressant drug testing: technical considerations, J. Neurosci. Methods 165 (2007) 175–182, https://doi.org/10.1016/j.jneumeth.2007.05.035. J. Koolhaas, C.M. Coppens, S.F. Boer, B. Buwalda, P. Meerlo, P.J.A. Timmermans, The resident-intruder paradigm: a standardized test for aggression, violence and social stress, J. Visualized Exp.: JoVE (2013), https://doi.org/10.3791/4367. L. Marais, S.J. van Rensburg, J.M. van Zyl, D.J. Stein, W.M.U. Daniels, Maternal separation of rat pups increases the risk of developing depressive-like behavior after subsequent chronic stress by altering corticosterone and neurotrophin levels in the hippocampus, Neurosci. Res. 61 (2008) 106–112, https://doi.org/10.1016/j. neures.2008.01.011. N.E. Holz, R. Boecker, E. Hohm, K. Zohsel, A.F. Buchmann, D. Blomeyer, C. JennenSteinmetz, S. Baumeister, S. Hohmann, I. Wolf, The long-term impact of early life poverty on orbitofrontal cortex volume in adulthood: results from a prospective study over 25 years, Neuropsychopharmacology 40 (2015) 996. S.A. Syed, C.B. Nemeroff, Early life stress, mood, and anxiety disorders, Chronic Stress 1 (2017) 2470547017694461. K.J. Ressler, C.B. Nemeroff, Role of serotonergic and noradrenergic systems in the pathophysiology of depression and anxiety disorders, Depress. Anxiety 12 (2000) 2–19 doi: AID-DA2&3.0.CO;2-4. Matteo M. Pusceddu, Philip Kelly, Nurbazilah Ariffin, John F. Cryan, Gerard Clarke, Timothy G. Dinan, n-3 PUFAs have beneficial effects on anxiety and cognition in female rats: effects of early life stress, Psychoneuroendocrinology 58 (2015) 79–90, https://doi.org/10.1016/j.psyneuen.2015.04.015. C. Hölscher, Stress impairs performance in spatial water maze learning tasks, Behav. Brain Res. 100 (1999) 225–235, https://doi.org/10.1016/S0166-4328(98) 00134-X. F. Lévy, A.I. Melo, B.G. Galef, M. Madden, A.S. Fleming, Complete maternal deprivation affects social, but not spatial, learning in adult rats, Dev. Psychobiol. 43 (2003) 177–191, https://doi.org/10.1002/dev.10131. B.árbara Aisa, Rosa Tordera, Berta Lasheras, Joaquín Del Río, Maria J. Ramírez, Cognitive impairment associated to HPA axis hyperactivity after maternal separation in rats, Psychoneuroendocrinology 32 (2007) 256–266, https://doi.org/10. 1016/j.psyneuen.2006.12.013. Couto FSd, V.L. Batalha, J.S. Valadas, J. Data-Franca, J.A. Ribeiro, L.V. Lopes, Escitalopram improves memory deficits induced by maternal separation in the rat, Eur. J. Pharmacol. 695 (2012) 71–75, https://doi.org/10.1016/j.ejphar.2012.08. 020. C. Estanislau, S. Morato, Prenatal stress produces more behavioral alterations than maternal separation in the elevated plus-maze and in the elevated T-maze, Behav. Brain Res. 163 (2005) 70–77, https://doi.org/10.1016/j.bbr.2005.04.003. J. Price, L. Sloman, R. Gardner Jr., P. Gilbert, P. Rohde, The social competition hypothesis of depression, Br. J. Psychiatry 164 (1994) 309–315, https://doi.org/10. 1192/bjp.164.3.309. E. Nesher, I. Koman, M. Gross, T. Tikhonov, M. Bairachnaya, M. Salmon-Divon, Y. Levin, G. Gerlitz, I. Michaelevski, G. Yadid, A. Pinhasov, Synapsin IIb as a functional marker of submissive behavior, Sci. Rep. 5 (2015) 10287, https://doi. org/10.1038/srep10287. B. Zimmerberg, K.A. Sageser, Comparison of two rodent models of maternal separation on juvenile social behavior, Front. Psychiatry 2 (2011), https://doi.org/ 10.3389/fpsyt.2011.00039. R.C. Newberry, J.C. Swanson, Implications of breaking mother–young social bonds, Appl. Anim. Behav. Sci. 110 (2008) 3–23. C.A. Murgatroyd, B.C. Nephew, Effects of early life social stress on maternal behavior and neuroendocrinology, Psychoneuroendocrinology 38 (2013) 219–228. J. Molet, K. Heins, X. Zhuo, Y.T. Mei, L. Regev, T.Z. Baram, H. Stern, Fragmentation and high entropy of neonatal experience predict adolescent emotional outcome, Transl. Psychiatry 6 (2016) e702. E.E. Gilles, L. Schultz, T.Z. Baram, Abnormal corticosterone regulation in an immature rat model of continuous chronic stress, Pediatr. Neurol. 15 (1996) 114–119, https://doi.org/10.1016/0887-8994(96)00153-1. C.J. Rice, C.A. Sandman, M.R. Lenjavi, T.Z. Baram, A novel mouse model for acute and long-lasting consequences of early life stress, Endocrinology 149 (2008) 4892–4900. J.L. Bolton, J. Molet, A. Ivy, T.Z. Baram, New insights into early-life stress and behavioral outcomes, Curr. Opin. Behav. Sci. 14 (2017) 133–139. C.E. Barrett, S.E. Arambula, L.J. Young, The oxytocin system promotes resilience to the effects of neonatal isolation on adult social attachment in female prairie voles, Transl. Psychiatry 5 (2015) e606.