Behavioral Effects of Early Life Maternal Trauma Witness In Rats

Abstract

Background

Earlier, we have reported that post-traumatic stress disorder (PTSD)-like behaviors developed in rats that witnessed their cage mates undergo repeated traumatic stress. More recently, we published that early life physical traumatic stress leads to later life depression-like behaviors in rats. Whether early life trauma witness causes later life PTSD-like behaviors is not known. Also unclear are sex-specific stress-induced behavioral variations in later life. The early life witness component of stress is an important aspect of stress-induced psychopathologies and must be investigated.

Objective

Here, we have examined the impact of early life repeated witnessing of traumatic events by pups from post-natal day (PND) 21- PND27, on later life behaviors at PND60, and the behavioral impact of postpartum traumatic stress in female rats.

Methods

We used a modified version of rodent social defeat model to induce postpartum stress in female rats and trauma witness stress in pups. One female Sprague-Dawley rat (intruder) was introduced into the cage of an aggressive Long–Evans male rat (resident). The encounter between the two resulted in attacks between the female rat and the Long-Evans male rat. Three exposures of social defeat (attacks) were given for 7 consecutive days. The social defeat traumatic events were witnessed by 6 pups (offspring of the intruder female rat, PND21-27), placed in six separate enclosures surrounding the cage. The objective of this experiment was three-fold: 1) to test later life behavioral effects in pups from witnessing maternal defeat, 2) to examine gender susceptibility of pups in maternal defeat witness-induced behaviors, 3) to test behavioral effect in female rats 24h after receiving the last social defeat exposure.

Results

We observed that while anxiety-like behavior assessed in open-field and elevated plus-maze tests, was not affected in male or female rats upon witnessing repeated maternal traumatic stress, depression-like behavior in forced-swim test was observed at PND60 in both male and female rats, with greater effect in male rats. No change was observed in learning and memory functions using radial arm water maze test in both male and female rats. Interestingly, socially defeated female rats (dams: mother of the pups) developed both anxiety and depression-like behavior with no change in learning-memory function when compared to control female rats.

Conclusions

Our findings suggest that early life maternal stress witness history leads to depression-like behavior in both male and female adult rats, and dams developed both anxiety and depression-like behaviors.

1. Introduction

According to American Psychological Association, ~15.5 million children in the United States witness physical or emotional abuse of a parent, most commonly that of their mother⁽¹⁾. Clearly, children raised in abusive households who witness parental abuse carry the psychological burden of helplessness, which often expresses in the form of conduct disorders or behavioral deficits⁽²⁾. Additionally, parental violence causes post-natal depression⁽³⁾, which can impair childcare ability of the mother, increasing the likelihood of child abuse and neglect. Taken together, the effects of maternal abuse are damaging not only for the mother but also for the children. While examining the link between maternal abuse and mental well being of the children as well as that of the mother are important to help devise appropriate behavioral and therapeutic interventions, conducting these studies in children with abuse or trauma history or in abused women, are difficult to carry out. Therefore, animal models are valuable in studying the behavioral consequences and the biological imprinting of trauma. Although animal models cannot accurately reveal the impact of traumatic events but are excellent tools that can provide useful insights and guide both therapeutic and management interventional strategies.

Recently, we reported that rats witnessing traumatic events (social defeat of a cage-mate) exhibited severe behavioral deficits resembling post-traumatic stress disorder (PTSD)-like behaviors^(4,5). More recently, we published that early life stress in pups caused later life behavioral deficits in rats⁽⁶⁾. In the present study, by combining the two approaches, we examined later life effect of early life maternal witness stress in rats. Briefly, a female Sprague-Dawley rat (dam: mother) was introduced into the cage of a Long Evans (LE) rat. The female rat (dam) underwent aggressive attacks from the LE rat. Natural litters at PND21 of the female rat were placed in separate chambers surrounding the cage witnessing these attacks. Pups were exposed to daily witness of repeated attacks between their mother and the male LE for 7 consecutive days, from PND21–PND27. Four and a half weeks later, when the pups became 60 days old (considered adults), behavioral outcomes of witnessing maternal stress were examined. It is important to clarify if memories of repeated witness experience of any fearful and/or traumatic event in early life are damaging for later behaviors, or is maternal stress witness central to modulation of later life behaviors? Several groups of rats were included to control for factors of fear unrelated to maternal stress witness, such as that of isolation from dams or induction of fearful stimuli from sighting of larger rats. One group of pups were made to repeatedly witness only the male aggressor rat in order to evaluate if the sighting of a large male rat is enough to induce behavioral impairments in pups. Another group of pups were made to witness a larger female rat for the same purpose. Third group of pups were allowed witness of a dam alone in isolation, in order to examine if maternal separation witness alone can cause deficits in pups. Fourth group of pups witnessed a new female rat undergo social defeat. This female rat was not a dam, unrelated to pups. The purpose of inclusion of this group was to determine if witness of social defeat exposures of any female rat induces traumatic memories in pups which might elicit later life behavioral deficits. Finally, age-matched pups that did not witness maternal social defeat were also included as controls. Behavioral and cognitive analysis was also conducted in the dams after conclusion of social defeat procedure. Behavioral data of dams was compared with two other set of females. The purpose of this comparison was to examine if post-partum social defeat induced behavioral deficits in dams are related to post-partum trauma. One group of a non-lactating female rats did not undergo social defeat attacks, while another group of non-lactating female rats underwent social defeat attacks.

2. Methods

2.1 Animal Housing and Care

Upon arrival at the animal facility, rats were housed on a 12-h light/dark cycle in a climate-controlled room with food and water provided ad libitum. Experiments with rats were conducted in accordance with the NIH guidelines using protocols approved from the University of Houston Animal Care and Use Committee. Sprague-Dawley pups arrived on post-natal day (PND) 14 along with their dams. They were placed with the dams for 4 days for acclimatization. Before PND 18, pups and dams were housed together to allow bonding. Then age-matched pups of the same gender were reared in littermate groups. Female Sprague-Dawley rats (225–250 g) were used as controls or intruders, and male Long-Evans (LE) retired breeders (400–500 g) served as resident aggressors (Charles River, Wilmington, MA).

2.2. Animal Model

The animal model used in this study is a modified version of our previously published trauma witness model⁽⁴⁾ which is based on the social defeat model of stress⁽⁷⁾. One female Sprague-Dawley rat (intruder) was introduced into the cage of an aggressive Long–Evans male rat (resident). During the encounter, the male Long-Evans rat attacked the female Sprague-Dawley rat. Three exposures of social defeat (attacks) were given for 7 consecutive days. Typically in social defeat paradigm the resident dominates the intruder and fully defeats it with the intruder assuming a supine position. Here, the resident attacks were met with more frequent defensive postures by the intruder, than typically observed in the social defeat paradigm. This can be attributed as protective attempts by the dam to shield the pups. The social defeat traumatic events were witnessed by 6 pups (offspring of the intruder female rat, PND21-27), which were placed in six separate enclosures surrounding the cage. As indicated in figure 1A, the apparatus was designed to include a central enclosure of social defeat, with six separate small witness enclosures. Pups were natural litters of the dam and were exposed to daily witnessing of repeated social defeat (attacks) of their mother by an unfamiliar aggressive LE male rat for 45 minutes (3 intervals, 10 minutes of social defeat followed by 5 minutes of rest), for 7 consecutive days. Male and female pups were separately placed and their behavioral parameters recorded separately. As indicated in the experimental design (Figure 1B), after conclusion of social defeat witness, pups were left undisturbed for a month in their home cages and their behavior assessment was conducted at PND60 (considered adult). As another segment of this study, 24 hour after conclusion of the social defeat procedures, dams were examined for behavioral and cognitive functions (Figure 1C) and results compared with another set of female rats not subjected to social defeat but exposed to control exposures as previously published⁽⁴⁾. Control exposures include subjecting the female rats to a novel cage without the presence of a male LE rat but in the presence of pups, which are placed in transparent separate enclosures for 10 min each exposure. This way the pups can see the female rat separated from them but since the male LE rat is not present, no social defeat takes place.

Fig 1.

A schematic representation of the maternal trauma witness apparatus comprising of a central enclosure where social defeat events occur and surrounding enclosures from where the social defeat events are witnessed by the pups (A), and the experimental design depicting the experimental protocol (B). Group designations are as follows: Pups at PND21 witnessed social defeat of dams (M-TW: maternal trauma witness), pups at PND21 witnessed only LE (LE-W: witness Long Evans rat only), pups at PND21 witnessed dams alone (W-D), pups at PND21 witnessed social defeat of a new female rat which is not a dam (F-TW: female trauma witness), pups at PND21 witnessed female rat only without social defeat (F-W), dams that underwent social defeat SD-D (social defeat-dams) and control females undergoing control exposures (CON). In addition to this, the experimental design depicting the experimental protocol to access the effects of maternal abuse on lactating female in the presence of her pups is showed in (C).

Group designations and controls

(1) pups at PND21 witnessed social defeat of dams (M-TW: maternal trauma witness), (2) pups at PND21 witnessed only LE (LE-W: witness Long Evans rat only) (3) pups at PND21 witnessed dams alone (W-D), (4) pups at PND21 witnessed social defeat of a new female rat which is not a dam (F-TW: female trauma witness), (5) pups at PND21 witnessed female rat only without social defeat (F-W). Thus, several controls were included in the study. We used age-matched pups that did not witness maternal social defeat. Inclusion of different control groups is important to control for factors of fear unrelated to maternal stress witness, such as that of isolation from dams or induction of fearful stimuli from sighting of larger rats. Pups were placed into the chambers surrounding the resident cage of their mother in the absence of the aggressive male LE rat. This control group involved the natural litters of the dam witnessing their mother in her resident cage for 45 minutes, for 7 consecutive days (W-D). An additional control group was included to confirm that the changes observed in adult rats were a result of witnessing maternal social defeat, and not a consequence of fear from the sight of the aggressive LE rat. Pups were placed into the chamber surrounding the resident cage of LE rat. This time the dam was not present throughout the procedure (7 days) (LE-W). Dams that underwent social defeat are referred as SD-D (social defeat-dams) while control females undergoing control exposures: (1) Ctrl Dam: the dams that did not undergo social defeat (attacks), (2) Ctrl Female: the non-lactating female rats that did not undergo social defeat (attacks), (3) SD-Female: the non-lactating female rats that underwent social defeat (attacks).

2.3 General Body Parameters

Body weight, food and water intake were recorded from the beginning of social defeat procedure to the end of behavioral assessments. Trauma witnessing paradigm lasted 7 days, depression- and anxiety-like behavior tests were conducted 24 h later, maintaining a gao of 24 rest in between tests, as published^(4,6). Cognitive functions including short-term and long-term memory tests using radial arm water maze (RAWM) paradigm were performed as published^(4,6). Rats were sacrificed after the conclusion of behavior tests.

2.4 Behavior Analysis

The order of behavior tests conducted was the following; 1) open-field test, 2) elevated-plus maze 3) short-term memory test, 4) long-term memory test, 5) forced swim test. In general, anxiety-like behavior tests are conducted in the order such that the least stressful test is conducted first, followed by the most stressful one. There was a gap of 24h between each test. In our experience, twenty four hour rest period has proven to be sufficient to remove memory of previous stress. We have published that if this order of anxiety behavior tests is maintained allowing a gap of twenty four hour between each test, the behavioral performance of rats is not affected^(4,6).

Anxiety-like behavior tests

First, open-field test was conducted followed by elevated-plus maze (EPM) test as previously published^(4,6).

Open field (OF) test

The open field task was carried out in 60×40 cm open field surrounded by 50 cm high walled Plexiglas chambers in standard room lighting conditions. The rats were placed at the center of the compartment and left free to explore the arena for 15 min. Activity was quantitated using a computer-operated Opto-Varimex Micro Activity Meter v2.00 system (Optomax, Columbus Instruments; OH) that utilizes sensors containing 8 infrared light emitting diodes and 8 phototransistors that emit and detect modulated infrared light beams. Sensors were positioned to form two-dimensional cages each with rearing monitoring. Movement was detected by beam breaks and data from three test chambers was recorded simultaneously, one rat per chamber, collected in 3 min intervals over a 15-min test session. The program tabulates activity counts, zone entries, zone times, center time and the periphery time, distance traveled and rearing for every cage in the system. For center time analysis, an approximately 25 cm × 25 cm square in center of the open field arena was defined as the center zone for data analysis. The total time spent in the center of the arena and rearing will be calculated for each group as described in our publications^(4,6).

Elevated plus-maze (EPM)

A standard rat elevated plus-maze with 43 cm arms extending from a 10 cm central area 90 cm above the floor was used (Med Associates Inc., St. Albans, VT). The rat’s movements were tracked visually by an observer that was blinded to treatment. Each session lasted 5 min and was started by placing the rat in the central area facing the open arms of the maze. Shorter the time spent in open arms, the higher is anxiety and vice versa. The maze exploits the conflict between the innate fear that rodents have of open placers versus their desire to explore novel environments. The amount of time the rat spent in the open arms was determined^(4,6).

2.5 Depression-like behavior test

Forced Swim Test (FST)

The FST is a test used for measuring depression-like behavior in rodents⁽⁸⁾. This test was used with some modifications in our study including lack of the pretest phase period^(9,10). Rats were individually placed in a water tank of 24cm × 50cm, filled with water (25°C) for 5-min. At some point after being placed in the water the rat assumes an immobile posture, marked by motionless floating and cessation of struggling. The total time spent immobile was recorded^(4,6). Higher immobile time is an indication of depression-like behavior.

2.6 Memory Function Test

Radial Arm Water Maze (RAWM)

The RAWM procedures were done as published^(4,6). Each rat was randomly assigned a goal arm, which contains a hidden black platform near the end of the arm. The rats were released at an arm other than the goal arm, and the rat must swim and locate the platform, which was submerged about 1 cm under the water. The rats were allowed a maximum time of 1 minute for each learning trial or memory test. An error was counted when the rat’s entire body entered more than halfway into an arm other than the goal arm. An error was also counted if the rat’s entire body entered more than half of the goal arm but failed to approach the platform. The number of errors range from 1 to a maximum of 7 as the rat can swim into 7 arms within 1 minute. If the rat failed to locate the platform within 1 minute, the rat was manually guided to the platform and was scored with 7 errors. Once the rat reached the platform, the rat was allowed 15 seconds sitting time on the platform before the next trial began. The rats were subjected to the first set of six learning trials (trials # 1–6) followed by a 5-min rest period and then a second set of six learning trials (trials # 7–12). Short-term memory was assessed by repeating the 6 trials a third time 30-min after the end of 12^th trial. Long term memory was assessed by returning the rats to their home cages and then repeating the 6 trials a fourth time 24 h after the end of the 12^th trial.

2.7 Data Analysis

Data are expressed as mean +SEM. Significance was determined by one-way ANOVA applying Tukey’s post-hoc test (GraphPad Software, Inc. San Diego, CA). A value of P< 0.05 was considered significant. The experimental design is summarized in Fig. 1B and C.

3. Results

3.1 General body parameters

Daily food and water intake and body weight gain starting from social defeat witness protocol to the day of sacrifice were measured in all pups. No significant changes were observed in total weight gain, food intake or water intake in the M-TW when compared to the control groups (Table. 1)

Table 1. Examination of general body parameters.

A and B daily food intake, C and D daily water intake, E and F daily body weight gain in rats. Group designations: pups at PND21 witnessed social defeat of dams (M-TW: maternal trauma witness), pups at PND21 witnessed only LE (LE-W: witness Long Evans rat only), pups at PND21 witnessed dams alone (W-D), pups at PND21 witnessed social defeat of a new female rat which is not a dam (F-TW: female trauma witness), pups at PND21 witnessed female rat only without social defeat (F-W).

	Male Pups					Female Pups
Food Intake (gms/day)	23.65±0	23.65±1.1	22.53±1.6	23.61±2.0	22.58±1.1	18.26±0.0	16.80±0.37	15.69±1.88	15.33±1.28	17.19±0.84
Water Intake (ml/day)	31.89±0	31.29±0.37	30.70±1.88	32.48±1.28	38.56±0.84	21.25±0.0	26.31±1.44	20.81±2.13	22.00±1.33	26.32±2.60
Weight Gain (gm/day)	7.23±0.18	4.58±0.202	6.87±0.136	6.24±0.20	6.80±0.32	3.61±0.78	3.36±0.27	3.20±0.19	2.99±0.06	3.59±0.26

3.2 Analysis of Anxiety-like behavior of rats

In the elevated-plus maze test (EPM), male M-TW rats spent 178.4 ± 36.77 seconds in the open arms of the EPM apparatus when compared to LE-W (185.3 ± 21.01 seconds), W-D (132.6 ± 36.82 seconds), F-TW (108.3 ± 36.03 seconds) and F-W (137.0 ± 33.22 seconds) rats (Fig. 2A). The data suggests no significant change in time spent in the open-arms of the EPM apparatus. Similarly, female M-TW rats spent 141.0 ± 60.62 seconds in the open arms of the EPM apparatus when compared to LE-W (150.0 ± 14.14 seconds), W-D (133.4 ± 39.54 seconds), F-TW (117.4 ± 22.16 seconds) and F-W (108.3 ± 34.38 seconds) rats (Fig. 2B). Increased time spent in the closed arms during a 5-min session is indicative of high anxiety-like behavior⁽⁶⁾. No change was observed in anxiety-like behavior of rats that witnessed the attacks between their mother and a Long-Evans rat in this test.

Fig 2. Examination of anxiety-like behavior.

Total time spent in the open arms in the elevated plus maze (EPM) test (A, B, and E) as well as the ratio of time spent in the center of the open arena and the time spent in the periphery of the open arena in the open field test (C, D, and F) was used to measure anxiety-like behavior. Group designations: pups at PND21 witnessed social defeat of dams (M-TW: maternal trauma witness, n=18), pups at PND21 witnessed only LE (LE-W: witness Long Evans rat only, n=18), pups at PND21 witnessed dams alone (W-D, n=18), pups at PND21 witnessed social defeat of a new female rat which is not a dam (F-TW: female trauma witness, n=18), pups at PND21 witnessed female rat only without social defeat (F-W, n=18), dams that underwent social defeat SD-D (social defeat-dams, n=5) and control females undergoing control exposures: (1) Ctrl Dam: the dams that did not underdo social defeat (attacks), (2) Ctrl Female: the non-lactating female rats that did not undergo social defeat (attacks), (3) SD-Female: the non-lactating female rats that underwent social defeat (attacks). (*) indicates significantly different at, P<0.05. Bars represent means ± SEM, n =18 rats/group for the pups, n=3–5 rats/group for the adult female rats.

In the open field test (OF), the ratio of the time spent in the open arena and the time spent in the peripheral arena (Central/Peripheral time) was calculated for each rat. The Central/Peripheral time is 0.7911 ± 0.1204 for M-TW when compared to LE-W (0.6707 ± 0.06319), W-D (0.6436 ± 0.1298), F-TW (0.8401 ± 0.1420) and F-W (0.7413 ± 0.09485) rats (Fig. 2C). Similarly, female M-TW rats had 0.7178 ± 0.1572 Central/Peripheral in the OF apparatus when compared to LE-W (0.6314 ± 0.06346), W-D (0.6098 ± 0.1955), F-TW (0.6542 ± 0.1666) and F-W (0.6460 ± 0.1456) rats (Fig. 2D). Decreased time spent in the open arena of the OF apparatus during a 15-min session is indicative of high anxiety-like behavior^(4,6). No change was observed in anxiety-like behavior of rats that witnessed the attacks between their mother and a Long-Evans rat in the OF test. All groups covered similar distance, suggesting no change in activity in between groups (Fig. 2E,F). In summary, in two distinct tests assessing anxiety-like behavior, both male and female M-TW did not exhibit anxiety-like behavior when compared to control groups.

Anxiety-like behavior tests were also conducted in the dams following 7-days of social defeat in the presence of pups in the surrounding safe enclosures. In EPM test, the SD-D rats exhibited significant anxiety-like behavior compared to their age matched controls, indicated by decrease in time spent in the open arms by SD-D rats (SD-D: 89.00±10.15 seconds, Ctrl Dam: 193.3±5.508 seconds, Ctrl Female: 167.3 ± 29.67 seconds, SD-Female: 161.0 ± 19.08 seconds, P<0.05) (Fig. 2G). Similarly, SD-D rats exhibited significantly less Central/Peripheral time in the OF apparatus when compared to control rats (Ctrl Dam: 0.7508 ± 0.07739, Ctrl Female: 0.8695 ± 0.1215, SD-Female: 0.7113 ± 0.03973, SD-D: 0.5332 ± 0.07261, P<0.05) (Fig. 2H). In summary, SD-D rats exhibited anxiety-like behavior in both anxiety tests when compared to control rats.

3.3 Analysis of depression-like behavior of rats

In the forced swim test (FST), male M-TW rats spent 146.0±36.28 seconds being immobile, a sign of increased depression like behavior⁽⁶⁾, when compared to LE-W (17.00 ± 6.083 seconds), W-D (60.44±40.61 seconds), F-TW (44.30 ± 27.83 seconds) and F-W (93.30 ± 72.69 seconds) rats (Fig. 3A). Similarly, female M-TW rats spent 83.43±40.82 seconds being immobile, a sign of increased depression like behavior⁽⁶⁾, when compared to LE-W (16.50 ± 17.68 seconds), W-D (29.50 ± 19.58 seconds), F-TW (47.60 ± 33.71 seconds) and F-W (27.17 ± 19.81 seconds) rats (Fig. 3B). Male pups spent 52% time immobile in FST in response to maternal trauma witness, while female rats spent 35% time immobile in response to trauma witness, suggesting that male pups exhibited approximately 2 fold greater depression-like behavior than female pups. There was no significant difference between F-W and F-TW, while M-TW spent significantly longer time of immobility as compared to W-D in both male and female rats.

Fig 3. Examination of depression-like behavior.

Total immobility time in the Forced swim test (FST) was used to examine depression-like behavior. Group designations: pups at PND21 witnessed social defeat of dams (M-TW: maternal trauma witness, n=18), pups at PND21 witnessed only LE (LE-W: witness Long Evans rat only, n=18), pups at PND21 witnessed dams alone (W-D, n=18), pups at PND21 witnessed social defeat of a new female rat which is not a dam (F-TW: female trauma witness, n=18), pups at PND21 witnessed female rat only without social defeat (F-W, n=18), dams that underwent social defeat SD-D (social defeat-dams) and control females undergoing control exposures: (1) Ctrl Dam: the dams that did not underdo social defeat (attacks), (2) Ctrl Female: the non-lactating female rats that did not undergo social defeat (attacks), (3) SD-Female: the non-lactating female rats that underwent social defeat (attacks). (*) indicates significantly different at, P<0.05, Bars represent means ± SEM, n =18 rats/group for the pups, n=3–5 rats/group for the adult female rats.

FST was also conducted in the dams which underwent 7-days of social defeat in the presence of pups in the safe enclosures. SD-D rats spent 133.7 ± 15.95 seconds being immobile when compared to Ctrl Dam (91.33 ± 4.041 seconds), Ctrl Female (13.00 ± 8.718 seconds), and SD-Female (28.00 ± 21.93 seconds) rats (Fig. 3C), a sign of increased depression like behavior⁽⁶⁾. In summary, both male and female M-TW rats as well as SD-D rats exhibited depression-like behavior when compared to respective control groups.

3.4 Analysis of learning-memory function in rats

Short-term memory (STM) and long-term memory (LTM) function tests did not show any significant changes among different groups in the RAWM apparatus. All groups including M-TW, LE-W, W-D, F-TW and F-W made comparable errors (Fig. 4A–D). Errors made in the STM: male M-TW (0.2222 ± 0.4410), male LE-W (0.3333 ± 0.5774), male W-D (0.7500 ± 0.7071), male F-TW (0.6000 ± 0.6992) and male F-W (0.4444 ± 0.7265); female M-TW (1.000 ± 1.581), female LE-W (0.0 ± 0.0), female W-D (0.7500 ± 1.035), female F-TW (3.375 ± 3.068) and female F-W (0.6250 ± 1.188). Errors made in the LTM: male M-TW (1.333 ± 2.345), male LE-W (3.000 ± 3.464), male W-D (4.444 ± 3.005), male F-TW (0.7000 ± 1.059) and male F-W (2.200 ± 2.700); female M-TW (2.778 ± 3.270), female LE-W (7.000 ± 0.0), female W-D (1.556 ± 2.404), female F-TW (2.875 ± 2.696) and female F-W (2.375 ± 2.615). SD-D and the control groups also made comparable errors in both tests (Fig. 4E and F). Errors made in the STM: SD-D (1.667 ± 2.887), Ctrl Dam (1.667 ± 1.155), Ctrl Female (0.3333 ± 0.5774), and SD-Female (2.000 ± 1.732). Errors made in the LTM: SD-D (3.667 ± 3.512), Ctrl Dam (4.667 ± 2.082), Ctrl Female (1.333 ± 1.155), and SD-Female (0.6667 ± 0.5774).

Fig 4. Examination of memory function.

Number of errors made in the short-term memory (STM) test (A) and long-term memory (LTM) test (B) examined learning and memory function using radial arm water maze (RAWM) test. Group designations: pups at PND21 witnessed social defeat of dams (M-TW: maternal trauma witness, n=18), pups at PND21 witnessed only LE (LE-W: witness Long Evans rat only, n=18), pups at PND21 witnessed dams alone (W-D, n=18), pups at PND21 witnessed social defeat of a new female rat which is not a dam (F-TW: female trauma witness, n=18), pups at PND21 witnessed female rat only without social defeat (F-W, n=18), dams that underwent social defeat SD-D (social defeat-dams) and control females undergoing control exposures: (1) Ctrl Dam: the dams that did not underdo social defeat (attacks), (2) Ctrl Female: the non-lactating female rats that did not undergo social defeat (attacks), (3) SD-Female: the non-lactating female rats that underwent social defeat (attacks). (*) indicates significantly different at, P<0.05, Bars represent means ± SEM, n =18 pups/group, n=3–5 rats/group for the adult female rats.

4. Discussion

Our previously published work has shown that rats witnessing traumatic events of social defeat of a cage-mate exhibited behavioral deficits resembling PTSD-like behaviors^(4,5). Studies from other groups also have supported this observation^(11–14). More recently, we have published that early life stress in pups caused later life behavioral deficits in adult rats⁽⁶⁾. These studies have prompted us to ask some relevant questions: First, can early life witness stress cause one or several behavioral and/or cognitive deficits in later life? Second, can maternal witness and general stress witness induce similar behavioral and/or cognitive deficits in later life? Third, are there sex-specific differences in behavioral consequences of maternal witness stress? Finally, what is the behavioral and cognitive impact on the dams after undergoing social defeat in the presence of their pups? To address these questions, we combined the two approaches used in our previous studies^(4,6), and examined later life effect of early life maternal witness stress in rats. Anxiety-like and depression-like behavior as well as learning and memory functions were analyzed in PND60 rats. These measures also were conducted in the dams.

The results of this study suggest that witnessing early life maternal stress by pups caused depression-like behavior in later life at PND60 (adults) and this observation was evident in both male and female rats suggesting no sex-specific variation in this behavior. Interestingly, neither male nor female rats exhibited anxiety-like behavior following early life maternal witness stress. These observations were made more interesting by the fact that depression-like behavior was observed only in the group of PND60 rats that witnessed social defeat/attacks of the dams but not when they witnessed social defeat of another female rat (not the dam) or from sighting of the LE rat or from simply seeing the dam in an exposed anxiogenic environment. This suggests the specificity of the phenotype as it developed from witnessing of maternal social defeat only.

Furthermore, our postulation is that social defeat and/or traumatic stress increases alertness and causes hyperactivity in young rats as a result of which they are able to pass movement or motion based tests such as anxiety-like behavior tests which record rat’s movement. This postulation is based upon our observations in open-field test where we have noted that while young rats cover equal distance amongst groups, but, these rats covered approximately 25%–40% more distance than adult Sprague-Dawley rats⁽⁴⁾. And, this may be the reason why there was no change noted in STM and LTM tests. Due to increased alertness and hyperactivity the rats are able to locate the platform quickly without making errors. The phenotype emerges in FST where despair-like behavior is indicated when a rat is provided a no-escape scenario. Interestingly, despite the hyperactivity of TW rats, their immobility pattern suggestive of despair-like behavior in FST is compelling.

Some studies have reported sex-dependent effect of stress on a variety of behaviors. Both pre-clinical^{(15,16,17,18)} and clinical⁽¹⁹⁾ studies have reported different reactions to stress between males and females. In general rodent studies report resilience in female rats in response to chronic stress with female rats exhibiting resilience to cognitive impairments⁽²⁰⁾ and behavioral deficits^(21,22). However, behavioral test dependent variations also exist with some studies suggesting presence of stress-induced behavioral deficits in both males and female animals⁽²³⁾ while others suggesting the deficits only in females⁽²⁴⁾ or in males⁽²⁵⁾. Studies using both female and male rats showed that stressed female showed depression-like behavior in social interaction test but not accompanied by other exploratory behaviors, while stressed male rats did not show changes in social interaction test, but increased freezing behavior⁽²⁶⁾. Thus the type, length and manner of stressor used, strain and age of rodents might explain the variation in results.

Furthermore, dams exhibited increased stress reactivity as both anxiety and depression-like behavior phenotype was evident while learning-memory function was not impaired. Hyper reactivity to stress can be attributed to excessive stressful stimuli^(27,11)_, as social defeat stress is most likely intensified by the presence of the pups. The dams were threatened not only by the LE rats but also feared attacks on the pups by the LE, a behavior well documented in rodents⁽²⁸⁾. The lactating female rats showed aggressive behavior. This can be attributed as a response to both fearful and defensive behaviors. The protective behavior of the female rats serves as a defensive mechanism against the potentially dangerous male LE rat. Previous studies have shown that maternal aggression may be linked with the anxiety and depression-like behavior⁽²⁹⁾. Brain neuropeptides oxytocin, which is a maternal neuropeptide, also plays an important role in regulating anxiety-like behavior⁽³⁰⁾. Our postulation is that hyper-responsivity of the stress system, over activity of the fear circuitry, combined with the disruption of the emotional connectivity leads to the development of anxiety and depression-like behavior in the lactating female rats.

5. Conclusion

Our results indicate that early life trauma witness experiences are directly linked with development of later life depression-like phenotype. And, there seems to be no gender difference in susceptibility to developing depression-versus anxiety-like behavior later at PND60, however, the extent of depression-like behavior was more in male rats than in female rats. This study stems from our curiosity of trying to understand the long-term impact of witnessing maternal abuse. While the intent is rooted in a social issue but we understand that drawing close parallels between animal research and social issues is unreasonable, yet, our observations can be considered as translationally relevant and informative.

Highlights.

• Early life maternal witness stress causes depressive phenotype in later life in rats

• Early life maternal witness stress does not cause anxiety-like behavior in later life in rats

• There is no sex-based selectivity in terms of the behavioral impairments in rats

• Post-partum maternal stress causes behavioral impairments in the dams