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Effect of chronic social defeat stress on behaviors and dopamine receptor in adult mice
Progress in Neuro-Psychopharmacology & Biological Psychiatry 66 (2016) 73–79
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Effect of chronic social defeat stress on behaviors and dopamine receptor in adult mice☆ Guang-Biao Huang a,b,⁎,1, Tong Zhao a,1, Xiao-Lei Gao a, Hong-Xing Zhang c, Yu-Ming Xu d, Hao Li c, Lu-Xian Lv a,b,⁎ a
Department of Psychiatry, The Second Affiliated Hospital of Xinxiang Medical University, Xinxiang, Henan 453002, China Henan Key Lab of Biological Psychiatry, Xinxiang Medical University, Xinxiang, Henan 453002, China Department of Psychology, Xinxiang Medical University, Xinxiang, Henan 453003, China d Department of Neurology, The First Affiliated Hospital of Zhengzhou University, Zhengzhou, Henan 450052, China b c
a r t i c l e
i n f o
Article history: Received 6 September 2015 Received in revised form 1 December 2015 Accepted 2 December 2015 Available online 3 December 2015 Keywords: Social defeat stress Susceptible Unsusceptible Behavior test Dopamine receptor
a b s t r a c t Victims of bullying often undergo depression, low self-esteem, high anxiety and post-traumatic stress disorder symptoms. The social defeat model has become widely accepted for studying experimental animal behavior changes associated with bullying; however, differences in the effects in susceptible and unsusceptible individuals have not been well studied. The present study investigated the effects of social defeat stress on behavior and the expression of dopamine receptors D1 and D2 in the brains of adult mice. Adult mice were divided into susceptible and unsusceptible groups after 10 days of social defeat stress. Behavioral tests were conducted, and protein levels in the brains were assessed by Western blotting. The results indicate that all mice undergo decreased locomotion and increased anxiety behavior. However, decreased social interaction and impaired memory performance were only observed in susceptible mice. A significantly decreased expression of D1 was observed in the prefrontal cortex and amygdala of susceptible mice only. No significant differences in D2 expression were shown between control and defeated mice in any area studied. These data indicate that depression-like behavior and cognition impairment caused by social defeat stress in susceptible mice may be related to changes in the dopamine receptor D1. © 2015 Elsevier Inc. All rights reserved.
1. Introduction Victims of bullying often undergo depression, low self-esteem (Björkqvist et al., 1982), high anxiety (Olweus, 1978) and typical posttraumatic stress disorder symptoms (Leymann, 1992). The social defeat model has become widely accepted for studying experimental animal behavior changes induced by chronic stress. This model is similar to human bullying in that the dominant animals bully the subordinate animals. Competition awareness for territory or food is used to induce animals to fight fiercely until one of them yields. As a result of the physical
☆ Animal ethics statement: This study was carried out in strict accordance with the recommendations in the Guide for the Care and Use of the Laboratory Animals of the National Health and Medical Research Council of China. The experimental protocol was approved by the Department of Psychiatry, The Second Affiliated Hospital of Xinxiang Medical University Animal Ethics Committee [SYXK(YU)2014-0005], and was endorsed by the Research and Ethics Committee of Henan Key Lab of Biological Psychiatry, China. ⁎ Corresponding authors at: Department of Psychiatry, Second Affiliated Hospital of Xinxiang Medical University, No. 388, Jianshe Middle Road, Xinxiang 453002, China. E-mail addresses: [email protected] (G.-B. Huang), [email protected] (L.-X. Lv). 1 Contributed equally.
http://dx.doi.org/10.1016/j.pnpbp.2015.12.002 0278-5846/© 2015 Elsevier Inc. All rights reserved.
and psychological stress, the loser will have a significant mood disorder. Therefore, through the use of this model, the etiology of the emotional repercussions of bullying can be assessed. It has been reported that in animal experiments, social defeat can induce anxiety (Denmark et al., 2010; Kinsey et al., 2007) and depressionlike behaviors (Schloesser et al., 2010), which are similar to those experienced by the victims of bullying. Moreover, there is evidence (Razzoli et al., 2011; Adamcio et al., 2009) that social defeat can contribute to cognition impairment. According to the report of Yu et al. (2011), mice that undergo social defeat exhibit reduced memory in T maze tests, and reduced learning and memory in water maze tests. In object recognition tests, the learning ability of defeated mice is reduced when they are affected by social pressure caused by plundering (El Hage et al., 2004). Furthermore, social defeat can alter the signaling of dopamine, which participates in the adjustment of learning, memory and emotional activities through its receptors D1 and D2. Therefore, the social defeat model provides well-defined markers for assessing the effects of defeat for different populations of mice. Despite the attributes of the social defeat model, few studies have been undertaken to investigate whether there is a difference in behavioral responses between susceptible and unsusceptible subpopulations of defeated mice. We hypothesized that the quality of depression,
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anxiety-related behaviors and cognitive impairment in mice undergoing social defeat may differ between susceptible and unsusceptible individuals. To test this hypothesis, we assessed several different parameters of behavior. Furthermore, to explore the underlying molecular mechanisms for behavioral responses after social defeat stress, we assessed the effects of social defeat in susceptible and unsusceptible mice on levels of D1 and D2. 2. Material and method 2.1. Experimental animals Seven-week-old (22–25 g) and 14-week-old (22–25 g) male C57BL/ 6J and CD1 mice (Beijing Vital River Laboratory Animal Technology Co., Ltd., China) were used in the present study. Dedicated efforts were made to minimize animal suffering and the number of animals used in accordance with the Guidelines for Animal Experiments of Xinxiang Medical University. 2.2. Administration of chronic social defeat stress Induction of social defeat stress was carried out as previously reported (Berton et al., 2006; Tsankova et al., 2006). CD-1 mice, selected on the basis of their attack latencies (shorter than 30 s on three consecutive screening tests) were used as aggressive residents. Briefly, C57BL/6J mice were exposed to a different CD1 aggressor mouse each day for 10 min for 10 days. After the social defeat sessions, the resident CD1 mouse and the intruder mouse were housed in different halves of the cage separated by a perforated Plexiglas divider to allow visual, olfactory, and auditory contact for the remainder of the 24-h period. At 24 h after the last session, all mice were housed individually. Control mice were housed in similar cages but with members of the same strain, which changed daily. A scientifically and ethically refined chronic social defeat stress protocol was used (Azzinnari et al., 2014). 2.3. Social avoidance testing On day 11 after chronic social defeat stress, social avoidance testing was performed to distinguish the “susceptible group” from the “unsusceptible group”. Testing was performed in self-made chambers (42 cm × 42 cm × 42 cm). Before the tests, the mice to be tested were placed in the test room for 1 h. To delimit the testing area, a woven metal box (10 cm × 4.5 cm) was put in the interactive area. Mice inside and outside of the metal box could see, hear and smell each other, but could not touch each other. The 8 cm area around the box was regarded as the interaction area. The test had two phases of 2.5 min each. In the first phase, the mice to be tested were placed at the far-end of the interactive area. The box in the interactive area had no CD1 mice. The movements of the mice were recorded. In the second phase, CD1 mice were placed in the box within the interactive area and then the mice to be tested were placed at the same position. The interactions between the test mice and the CD1 mice were observed. A video tracking system was used throughout the test process to record and analyze the interaction, and the interaction rate was calculated (interaction rate = 100 × length of stay in interaction area in the second phase / length of stay in interaction area in the first phase). Mice with interaction rate ≥ 100 were determined to be the “unsusceptible group” and mice with interaction rate b 100 were determined to be the “susceptible group”. Behavioral testing and Western blot analysis were performed consecutively after social avoidance testing (on day 12) as described below. 2.4. Open field testing An automated recording of the locomotor activity was conducted in an open acrylic box (30 × 40 × 50 cm) using a video tracking system with SMART software (Panlab, Barcelona, Spain). Mice were allowed
to habituate to the testing room for 30 min. Then, mice were placed into the testing apparatus, and their activity (distance moved) was measured for 30 min. 2.5. Light/dark preference determination An apparatus was constructed that consists of a rectangular acrylic box (46 × 27 × 30 cm), divided into one small (18 × 27 cm) and one large (27 × 27 cm) area, with a door-like opening (7.5 × 7.5 cm) in the center of the separation. Each animal was individually placed in the center of the bright compartment (facing away from the door), and the following parameters were measured for 5 min: latency of the initial movement from the light to dark area (latency of transition), total number of transitions between the light and dark areas, and total time spent in the light area. 2.6. Social interaction testing The social interaction apparatus consisted of a transparent acrylic box (30 × 40 × 50 cm) without a top. Mice were habituated to the testing box for 10 min on the testing day. Then, each mouse was paired with an unfamiliar mouse of the same genetic background and similar weight. The following behaviors were recorded for 10 min under the dimly lit condition (40 lx): social sniffing, anogenital sniffing, social grooming, following, climbing or mounting, crawling under or over, aggressive or fighting behavior, and aggressive chasing. 2.7. New object recognition testing The apparatus for this task consisted of a black open-field box (30 × 40 × 50 cm) located in a sound-attenuated room and illuminated with a 40-W bulb. Before the test, mice were habituated (10 min per day) in the box for 3 days. On day 4, two identical objects, such as a Duplo Lego toy or golf ball, were placed approximately 10 cm from each corner, and each mouse was allowed to explore the box for 10 min (acquisition trial). The time that the mice spent exploring each object was recorded. Retention trials were carried out at 1- and 24-h intervals following the acquisition trial. During the retention trials, each mouse was placed back in the same box with one of the familiar objects used during the acquisition and a novel object. The mouse was then allowed to explore freely for 10 min, and the time spent exploring each object was recorded. The recognition index (RI) and discrimination index (DI), defined in terms of the amount of time spent exploring a novel object (TN) and the total time spent exploring familiar (TF) and novel (TN) objects in the retention trial, i.e., TN / (TN + TF) and (TN − TF) / (TN + TF), respectively, were used to measure cognitive functioning. 2.8. Preparation of brain tissue After the social avoidance test was completed, mice were terminated by decapitation under ether anesthesia. The prefrontal cortex, amygdala, and CA1 and dentate gyrus regions of the dorsal hippocampus were punched out bilaterally using a 1.0-mm Harris Uni-Core micropunch (Electron Microscopy Sciences, Hatfield, PA, USA). 2.9. Immune protein blotting Tissue samples were homogenized in 20 mM ice-cold Tris–HCl (pH 7.4) containing 1% protease (P2714; Sigma-Aldrich Korea, Ltd., Yongin, Kyunggi-Do, Korea) and phosphatase inhibitor (P2850; SigmaAldrich Korea, Ltd., Yongin, Kyunggi-Do, Korea). The homogenates were centrifuged for 15 min at 14,000 rpm at 4 °C, and the resulting supernatant fractions were used for Western blot analyses. The protein samples (20 μg/lane) were separated and transferred to a hydrophobic polyvinylidene difluoride membrane. The membranes were blocked
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and incubated overnight at 4 °C with monoclonal mouse anti-D1R (diluted 1:10,000; Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA), or polyclonal rabbit anti-D2R (diluted 1:1000; Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA) in 5% non-fat milk. On the next day, the membranes were washed with PBS-T, and the primary antibody was detected using goat anti-mouse horse-radish peroxidase (HRP)-linked immunoglobulin G (IgG) (1:10,000, Santa Cruz Biotechnology Inc., Santa Cruz, California, USA) for D1, or goat anti-rabbit HRP-linked IgG (Vector Laboratories, Burlingame, CA, USA) for D2 (1:5000) in PBS (60 min, 25 °C). The blots were developed using an enhanced chemiluminescence reagent (RPN2232; GE Healthcare, Inc., Piscataway, NJ, USA), visualized with a LAS-3000 Plus lumino-imaging analyzer (Fuji Photo Film Company, Kanagawa, Japan) and quantified using Multi Gauge software v3.0 (Fujifilm, Tokyo, Japan).
2.10. Data analysis Data were analyzed with SPSS version 12.0. Differences among the three groups were analyzed using one-way ANOVA. Post hoc individual comparisons were made using Tukey's honestly significantly different test. In all cases, differences were considered statistically significant at P b 0.05. Results are presented as means ± standard error of the mean (SEM).
3. Results 3.1. Administration of chronic social defeat stress and grouping of susceptible, unsusceptible and control mice To determine whether the impact of social defeat stress differs for susceptible and unsusceptible mice, C57BL/6 mice (n = 113) were subject to social defeat stress for 10 days through daily interactions with aggressive CD1 mice. Control mice were housed in similar cages but with members of the same strain, which changed daily. All C57BL/6 mice that were subjected to social stress showed signs of failure (surrender posture, lack of movement, fleeing, lying down or quitting) after being attacked by CD1 mice, and 6.2% (n = 7) died during the attacks. Mice with injuries that could affect further testing were not included in the behavioral experiments. The mice that underwent social defeat stress were subject to a final social avoidance test to classify them as “susceptible” or “unsusceptible”. The mice were placed into a box with CD1 mice but were separated from direct contact with a metal mesh. The interactions between the test mice and the CD1 mice were observed, and test mice with higher interaction rates (≥ 100) were designated the “unsusceptible group”, while mice with lower interaction rates (b100) were designated the “susceptible group”.
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3.2. Effects of social defeat stress on locomotion To determine whether susceptible and unsusceptible mice are affected in their locomotion activity, which is reflective of anxiety, we performed opening tests, which track the movement of mice in an open surrounding. We measured the duration that the mice from susceptible, unsusceptible and control groups stayed in the center and the spatial movement distance among mice that moved. There was a significant difference among the three groups (F [2, 57] = 12.088, P b 0.001) (Fig. 1). Compared to the control group, both the time that the mice of the susceptible group stayed in the center (P b 0.001) and the movement distance (P = 0.003) were significantly reduced. A similar reduction in the time (P b 0.001) and movement distance (P = 0.009) was observed for the unsusceptible group as compared to the control group. However, the susceptible and unsusceptible groups did not differ from each other in either the time or distance (P N 0.05). These results suggest that social defeat stress affects anxiety behavior for both susceptible and unsusceptible mice, and that the extent of the effect does not differ significantly for the two groups. 3.3. Effects of social defeat stress on light/dark preference To verify the findings of the locomotion test, we examined the light/ dark response of susceptible and unsusceptible mice. Mice were placed into a lit compartment of a box which had free access to a dark compartment and the time until initial movement of the mice from bright to dark areas and the total time that mice spent in the bright area were assessed over a 5 min test span. There was a significant difference among the susceptible, unsusceptible and control groups with regard to both the latency of remaining in the bright area (F [2, 57] = 40.551, P b 0.001) and duration of hiding (F [2, 57] = 7.709, P = 0.001) (Fig. 2). Compared to the control group, the latency period (P b 0.001) and the overall time spend in the bright compartment (P = 0.016) were reduced for mice from the susceptible group. Similar findings were obtained for the unsusceptible group as compared to the control group (P = 0.002 and P = 0.034, respectively). However, there were no significant differences between the susceptible and unsusceptible groups in these measurements (P N 0.05). These results suggest that social defeat stress affects light/dark preference for both susceptible and unsusceptible mice and that the extent of the effect does not differ significantly for the two groups. Because light/dark preference serves as an additional measure of anxiety, these results support the determination that both susceptible and unsusceptible mice are prone to anxietyrelated behavior. 3.4. Effects of social defeat stress on social interaction To determine whether susceptible and unsusceptible mice differ in their social interactions after undergoing social defeat stress, we
Fig. 1. Effects of social defeat stress on locomotion: opening tests were performed to assess locomotion. a. Time spent in the central zone; b. distance moved. ** P b 0.01, compared to the control group (n = 18–20 per group).
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Fig. 2. Effects of social defeat stress on light/dark preference: movement between a light and dark area was assessed for 5 min after placement in a lit area. a. Time until initial movement from the light to dark area (latency of transition), b. total time spent in lit area. ** P b 0.01, compared to the control group (n = 18–20 per group).
exposed mice to unfamiliar mice of the same genetic background and similar weight and examined their behavior for 10 min. There was a significant difference (F [2, 57] = 113.078, P b 0.001] with regard to the total time of social sniffing of mice in the susceptible, unsusceptible and control groups (Fig. 3A). The social sniffing time in the susceptible group was significantly reduced in comparison to both the control group (P b 0.001) and the unsusceptible group (P b 0.001). However, the sniffing time of the unsusceptible group was not significantly reduced in comparison to the control groups (P N 0.05). Furthermore, the following time (F [2, 57] = 0.329, P N 0.05) and crawling or climbing down time (F [2, 57] = 0.14, P N 0.05) was not significantly different among the three groups. Additionally, the number of times that the mice engaged in social sniffing (F [2, 57] = 1.662, P N 0.05), following (F [2, 57] = 0.575, P N 0.05) or crawling or climbing down (F [2, 57] = 0.299, P N 0.05) was not significantly different among these three groups (Fig. 3B). These results suggest that susceptible mice show reduced social sniffing time, but that no differences can be observed in the other social parameters that were tested. 3.5. Effects of social defeat stress on new object recognition To determine whether susceptible and unsusceptible mice differ in cognitive ability, we conditioned mice to one object and then exposed mice to new objects after they had 1 or 24 h of rest. After 1 h, a significant difference among the susceptible, unsusceptible and control groups was observed with regard to recognition index (F [2, 57] = 340.795, P b 0.001) and resolution index (F [2, 57] = 340.795, P b 0.001) (Fig. 4). The susceptible group had significantly reduced recognition and resolution indices compared to both the control group and unsusceptible group (P b 0.001); however, the values for the unsusceptible group were not significantly different from those for the control group (P N 0.05). Similar results were observed after 24 h with regard to both the recognition index (F [2, 57] = 367.517, P b 0.001) and the
resolution index (F [2, 57] = 367.517, P b 0.001). These results suggest that susceptible mice have reduced cognitive ability. 3.6. Effects of social defeat stress on the expression of D1 and D2 in the prefrontal cortex and amygdale The receptors D1 and D2 facilitate dopamine signaling, which is associated with mood changes. To examine whether social defeat stress causes reduction in the levels of D1 and D2 and whether the effect differs for susceptible and unsusceptible mice, we performed Western blotting. A significant difference (F [2, 56] = 70.664, P b 0.001) was observed among the susceptible, unsusceptible and control groups with regard to the expression level of D1 in the prefrontal cortex area (Fig. 5). Compared to both the unsusceptible group (P b 0.001) and the control group (P b 0.001), D1 expression was significantly reduced in the susceptible group, and there were no significant differences between the unsusceptible and control groups (P N 0.05). Similar results were observed for D1 in the amygdala region (P = 0.001, susceptible versus unsusceptible group; P = 0.002, susceptible versus control; P = 0.069, unsusceptible versus control). However, no statistical differences in D1 expression were observed in the hippocampus. In contrast to the findings for D1, there were no significant differences among the three groups of mice in the D2 expression level in prefrontal cortex (F [2, 56] = 0.64, P N 0.05), amygdala (F [2, 56] = 0.4, P N 0.05), or hippocampus (F [2, 56] = 0.59, P N 0.05) (Fig. 6). Collectively, these results suggest that susceptible mice undergo physiological changes that lead to the reduction of D1, but not D2 levels, within the prefrontal cortex and amygdala. 4. Discussion Few studies have reported the effect of social defeat stress on dopamine receptors in the brain. In this study, we aimed to explore the effect
Fig. 3. Effects of social defeat stress on social interaction: social interaction with unfamiliar mice of the same genetic background and similar weight were examined over 10 min. a. Total time spent in active social behaviors, b. total number of social contacts. * P b 0.05, compared to the control group. † P b 0.05, compared to the unsusceptible group (n = 18–20 per group).
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Fig. 4. Effects of social defeat stress on new object recognition: new object recognition was assessed as a measure of cognition. a. Recognition index TN / (TN + TF), b. discrimination index (TN − TF) / (TN + TF). *** P b 0.001, compared to the control group. ††† P b 0.001, compared to the unsusceptible group (n = 18–20 per group).
of social defeat stress on behavior and dopamine receptors in the brains of mice. To more clearly distinguish the effects of social defeat, we subdivided the defeated mice into susceptible and unsusceptible populations. We observed that the defeated mice of both the susceptible and the unsusceptible group performed poorly in locomotion and light/dark preference testing. However, the effects of social defeat stress were only detectable for the susceptible group when examined using tests for social interaction and new object recognition response. Furthermore, the susceptible mice showed a reduced level of expression of the D1 receptor in the prefrontal cortex and amygdala. These results suggest that the susceptible mice undergoing social defeat stress had depression-like behavior and cognition impairment, while the response for the unsusceptible mice was less severe. The defeated mice from both the susceptible and unsusceptible groups were found to have anxiety-like behavior in the opening (locomotion) experiment and the dark compartment experiment. These findings for mice subjected to social defeat stress are consistent with the results reported by Kinsey et al. (2007) and Razzoli et al. (2010), though susceptible and unsusceptible mice were not subdivided in the latter studies. Krishnan et al. (2007) separated the susceptible group from unsusceptible mice and observed that mice in both the susceptible
Fig. 5. Effects of social defeat on D1 levels in the prefrontal cortex (PFC), amygdala (Amyg), and hippocampus (HIP). Top: A representative Western blot is shown. β-Actin was tested as a loading control. Bottom: Values represent the mean ± SEM of the relative band densities from three independent experiments as determined by densitometry and were standardized to 1.0 in the control. ** P b 0.01, compared to the control group. †† P b 0.01, compared to the unsusceptible group (n = 18–19 per group).
and unsusceptible groups showed a diminished response to light exposure; however, there was no significant difference in spontaneous activities of the mice among the susceptible, unsusceptible and control groups, which may be because they used a different study method from those in this study. We recorded the spontaneous activity of the mice in an open space for 30 min on the 12th day, but Krishnan et al. recorded spontaneous activity of the mice for 5 min on the 11th day. Interestingly, the mice from the susceptible groups had a reduced level of social interaction as assessed by social sniffing time, but not by other parameters of testing (following, crawling, climbing). To our knowledge, this is the first study to examine the effect of social defeat stress on social interaction. Our findings suggest that social defeat stress may cause social withdrawal behavior, which is similar to human depression. Consistent with our findings, it has been reported that after social defeat stress, sucrose preference and enjoyment-like reactions are reduced for mice in the susceptible group (Krishnan et al., 2007) and the defeated mice (Schloesser et al., 2010; Razzoli et al., 2010; Yu et al., 2011). Therefore, these findings support the use of the chronic social defeat stress model as an animal model of depression and anxiety disorders. We also determined using new object recognition experiments that cognition memory of the mice in the susceptible group is reduced.
Fig. 6. Effects of social defeat on D2 levels in the prefrontal cortex (PFC), amygdala (Amyg), and hippocampus (HIP). Top: A representative Western blot is shown. β-Actin was tested as a loading control. Bottom: Values represent the mean ± SEM of the relative band densities from three independent experiments as determined by densitometry and were standardized to 1.0 in the control (n = 18–19 per group).
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A limited number of studies have explored cognition memory of social stress defeat through new object recognition experiments. It has been reported that in new object recognition experiments, the learning ability of mice is reduced if they are exposed to predators, in which case they forget the information they learned (El Hage et al., 2004). Therefore, it is likely that social defeat stress causes subtle cognition impairment to mice of the susceptible group, though more spontaneous behavior test experiments are needed to further verify these findings. Our results demonstrate that the dopamine D1 receptor in the prefrontal cortex and amygdala are also only reduced in the mice of the susceptible group. The density of dopamine receptors is known to become reduced upon increase in dopamine release. Therefore, our results suggest that susceptible mice have increased dopamine release caused by social defeat stress. Several studies support the idea that dopamine levels (Louilot et al., 1986; Puglisi-Allegra et al., 1990) and the release of endogenous dopamine (Tidey and Miczek, 1996) increases somewhat under social defeat stress, but these studies did not divide the mice into susceptible and unsusceptible groups. Cao et al. (2010) divided the socially defeated mice into susceptible and unsusceptible groups and observed increased dopamine levels and release in the ventral tegmental area only in mice of the susceptible group. The dopamine signaling pathway serves as an important neurotransmitter in the brain. A large number of reports demonstrate that unbalanced dopamine can lead to the generation of depression (Braestrup et al., 1975; Lambert et al., 2000). Recently, the dopamine system has been shown to play an important role in the mechanism of some antidepressants (Bymaster et al., 2003; Pania and Gessab, 2002). Furthermore, methylphenidate, a dopamine medication, can affect the spontaneous activity of mice and cause anxiety-like behavior (Homsi et al., 2001). It has been well established that the expression level of dopamine in the hippocampus is associated with depression. There are also reports indicating that the expression level of dopamine in the prefrontal cortex participates in the formation of the working memory (Goldman-Rakic, 1996) and further influences reasoning and judgment. Depression and bipolar diseases are exacerbated when the working memory is damaged (Goldman-Rakic, 1994; Meyer-Lindenberg et al., 2005). Therefore, it can be inferred that the reduction in D1 levels in our study provides a basic mechanism for cognitive impairment, as observed in the new object recognition experiment. This reduction also could account partially for the anxiety-like and depression-like behavior of the mice in the susceptible group. Though a reduction in D1 was observed in our study, no significant differences in the levels of the D2 receptor were observed in three encephalic regions. These results are consistent with the results of several previous studies. In social isolation experiments, the dopamine D2 receptor had no significant change in the amygdala and nucleus accumbens of rats (Malone et al., 2008). Furthermore, no significant changes were observed in adolescent rats in the expression of the dopamine D2 receptor in the prefrontal cortex and nucleus (Burke et al., 2011). However, some studies suggest that the levels of the D2 receptor fluctuate depending on the stimuli. The density of the dopamine D2 receptor is reduced in the prefrontal cortex and nucleus when rats are passively exposed to attack (Suzuki et al., 2010) or subjected to the smell of a cat (Wright et al., 2008). In the amygdala expression of genes important in dopamine function were de-regulated including down-regulated Drd2 and Darpp-32 after chronic social defeat stress (Azzinnari et al., 2014); however the density of the dopamine D2 receptor is significantly increased when rats are repeatedly exposed to visible caves (Lucas et al., 2004). One possible explanation for why we observed a reduction in D1, but not D2 is that the internalization rate of D1 and D2 receptors after stress are different, with D2 internalization being a slower process. Internalization of D1 has been observed in numerous in vitro and in vivo experiments (Ng et al., 1995). Furthermore, the intensity of social defeat stress may not be enough to change the expression of D2, or the density of D2 may be relatively low in each area of the brain.
5. Conclusion In conclusion, this study shows that chronic social defeat stress can lead to a reduction in spontaneous movement and an increase in anxiety-like behavior of all the defeated mice. However, we only observed reduced social interaction and cognition impairment in susceptible mice. Furthermore, we observed a significant reduction of D1 in the prefrontal cortex and amygdala only in mice of the susceptible group. These data indicate that depression-like behavior and cognition impairment caused by social defeat stress may be related to changes in the dopamine D1 receptor, indicating that selective reduction of dopamine receptors in the prefrontal cortex and amygdala can be used as a marker to distinguish susceptible animals. Acknowledgements Funding for this study was provided by the National Natural Science Foundation of China (No. 81401112). References Adamcio, B., Havemann-Reinecke, U., Ehrenreich, H., 2009. 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Progress in Neuro-Psychopharmacology & Biological Psychiatry journal homepage: www.elsevier.com/locate/pnp
Effect of chronic social defeat stress on behaviors and dopamine receptor in adult mice☆ Guang-Biao Huang a,b,⁎,1, Tong Zhao a,1, Xiao-Lei Gao a, Hong-Xing Zhang c, Yu-Ming Xu d, Hao Li c, Lu-Xian Lv a,b,⁎ a
Department of Psychiatry, The Second Affiliated Hospital of Xinxiang Medical University, Xinxiang, Henan 453002, China Henan Key Lab of Biological Psychiatry, Xinxiang Medical University, Xinxiang, Henan 453002, China Department of Psychology, Xinxiang Medical University, Xinxiang, Henan 453003, China d Department of Neurology, The First Affiliated Hospital of Zhengzhou University, Zhengzhou, Henan 450052, China b c
a r t i c l e
i n f o
Article history: Received 6 September 2015 Received in revised form 1 December 2015 Accepted 2 December 2015 Available online 3 December 2015 Keywords: Social defeat stress Susceptible Unsusceptible Behavior test Dopamine receptor
a b s t r a c t Victims of bullying often undergo depression, low self-esteem, high anxiety and post-traumatic stress disorder symptoms. The social defeat model has become widely accepted for studying experimental animal behavior changes associated with bullying; however, differences in the effects in susceptible and unsusceptible individuals have not been well studied. The present study investigated the effects of social defeat stress on behavior and the expression of dopamine receptors D1 and D2 in the brains of adult mice. Adult mice were divided into susceptible and unsusceptible groups after 10 days of social defeat stress. Behavioral tests were conducted, and protein levels in the brains were assessed by Western blotting. The results indicate that all mice undergo decreased locomotion and increased anxiety behavior. However, decreased social interaction and impaired memory performance were only observed in susceptible mice. A significantly decreased expression of D1 was observed in the prefrontal cortex and amygdala of susceptible mice only. No significant differences in D2 expression were shown between control and defeated mice in any area studied. These data indicate that depression-like behavior and cognition impairment caused by social defeat stress in susceptible mice may be related to changes in the dopamine receptor D1. © 2015 Elsevier Inc. All rights reserved.
1. Introduction Victims of bullying often undergo depression, low self-esteem (Björkqvist et al., 1982), high anxiety (Olweus, 1978) and typical posttraumatic stress disorder symptoms (Leymann, 1992). The social defeat model has become widely accepted for studying experimental animal behavior changes induced by chronic stress. This model is similar to human bullying in that the dominant animals bully the subordinate animals. Competition awareness for territory or food is used to induce animals to fight fiercely until one of them yields. As a result of the physical
☆ Animal ethics statement: This study was carried out in strict accordance with the recommendations in the Guide for the Care and Use of the Laboratory Animals of the National Health and Medical Research Council of China. The experimental protocol was approved by the Department of Psychiatry, The Second Affiliated Hospital of Xinxiang Medical University Animal Ethics Committee [SYXK(YU)2014-0005], and was endorsed by the Research and Ethics Committee of Henan Key Lab of Biological Psychiatry, China. ⁎ Corresponding authors at: Department of Psychiatry, Second Affiliated Hospital of Xinxiang Medical University, No. 388, Jianshe Middle Road, Xinxiang 453002, China. E-mail addresses: [email protected] (G.-B. Huang), [email protected] (L.-X. Lv). 1 Contributed equally.
http://dx.doi.org/10.1016/j.pnpbp.2015.12.002 0278-5846/© 2015 Elsevier Inc. All rights reserved.
and psychological stress, the loser will have a significant mood disorder. Therefore, through the use of this model, the etiology of the emotional repercussions of bullying can be assessed. It has been reported that in animal experiments, social defeat can induce anxiety (Denmark et al., 2010; Kinsey et al., 2007) and depressionlike behaviors (Schloesser et al., 2010), which are similar to those experienced by the victims of bullying. Moreover, there is evidence (Razzoli et al., 2011; Adamcio et al., 2009) that social defeat can contribute to cognition impairment. According to the report of Yu et al. (2011), mice that undergo social defeat exhibit reduced memory in T maze tests, and reduced learning and memory in water maze tests. In object recognition tests, the learning ability of defeated mice is reduced when they are affected by social pressure caused by plundering (El Hage et al., 2004). Furthermore, social defeat can alter the signaling of dopamine, which participates in the adjustment of learning, memory and emotional activities through its receptors D1 and D2. Therefore, the social defeat model provides well-defined markers for assessing the effects of defeat for different populations of mice. Despite the attributes of the social defeat model, few studies have been undertaken to investigate whether there is a difference in behavioral responses between susceptible and unsusceptible subpopulations of defeated mice. We hypothesized that the quality of depression,
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anxiety-related behaviors and cognitive impairment in mice undergoing social defeat may differ between susceptible and unsusceptible individuals. To test this hypothesis, we assessed several different parameters of behavior. Furthermore, to explore the underlying molecular mechanisms for behavioral responses after social defeat stress, we assessed the effects of social defeat in susceptible and unsusceptible mice on levels of D1 and D2. 2. Material and method 2.1. Experimental animals Seven-week-old (22–25 g) and 14-week-old (22–25 g) male C57BL/ 6J and CD1 mice (Beijing Vital River Laboratory Animal Technology Co., Ltd., China) were used in the present study. Dedicated efforts were made to minimize animal suffering and the number of animals used in accordance with the Guidelines for Animal Experiments of Xinxiang Medical University. 2.2. Administration of chronic social defeat stress Induction of social defeat stress was carried out as previously reported (Berton et al., 2006; Tsankova et al., 2006). CD-1 mice, selected on the basis of their attack latencies (shorter than 30 s on three consecutive screening tests) were used as aggressive residents. Briefly, C57BL/6J mice were exposed to a different CD1 aggressor mouse each day for 10 min for 10 days. After the social defeat sessions, the resident CD1 mouse and the intruder mouse were housed in different halves of the cage separated by a perforated Plexiglas divider to allow visual, olfactory, and auditory contact for the remainder of the 24-h period. At 24 h after the last session, all mice were housed individually. Control mice were housed in similar cages but with members of the same strain, which changed daily. A scientifically and ethically refined chronic social defeat stress protocol was used (Azzinnari et al., 2014). 2.3. Social avoidance testing On day 11 after chronic social defeat stress, social avoidance testing was performed to distinguish the “susceptible group” from the “unsusceptible group”. Testing was performed in self-made chambers (42 cm × 42 cm × 42 cm). Before the tests, the mice to be tested were placed in the test room for 1 h. To delimit the testing area, a woven metal box (10 cm × 4.5 cm) was put in the interactive area. Mice inside and outside of the metal box could see, hear and smell each other, but could not touch each other. The 8 cm area around the box was regarded as the interaction area. The test had two phases of 2.5 min each. In the first phase, the mice to be tested were placed at the far-end of the interactive area. The box in the interactive area had no CD1 mice. The movements of the mice were recorded. In the second phase, CD1 mice were placed in the box within the interactive area and then the mice to be tested were placed at the same position. The interactions between the test mice and the CD1 mice were observed. A video tracking system was used throughout the test process to record and analyze the interaction, and the interaction rate was calculated (interaction rate = 100 × length of stay in interaction area in the second phase / length of stay in interaction area in the first phase). Mice with interaction rate ≥ 100 were determined to be the “unsusceptible group” and mice with interaction rate b 100 were determined to be the “susceptible group”. Behavioral testing and Western blot analysis were performed consecutively after social avoidance testing (on day 12) as described below. 2.4. Open field testing An automated recording of the locomotor activity was conducted in an open acrylic box (30 × 40 × 50 cm) using a video tracking system with SMART software (Panlab, Barcelona, Spain). Mice were allowed
to habituate to the testing room for 30 min. Then, mice were placed into the testing apparatus, and their activity (distance moved) was measured for 30 min. 2.5. Light/dark preference determination An apparatus was constructed that consists of a rectangular acrylic box (46 × 27 × 30 cm), divided into one small (18 × 27 cm) and one large (27 × 27 cm) area, with a door-like opening (7.5 × 7.5 cm) in the center of the separation. Each animal was individually placed in the center of the bright compartment (facing away from the door), and the following parameters were measured for 5 min: latency of the initial movement from the light to dark area (latency of transition), total number of transitions between the light and dark areas, and total time spent in the light area. 2.6. Social interaction testing The social interaction apparatus consisted of a transparent acrylic box (30 × 40 × 50 cm) without a top. Mice were habituated to the testing box for 10 min on the testing day. Then, each mouse was paired with an unfamiliar mouse of the same genetic background and similar weight. The following behaviors were recorded for 10 min under the dimly lit condition (40 lx): social sniffing, anogenital sniffing, social grooming, following, climbing or mounting, crawling under or over, aggressive or fighting behavior, and aggressive chasing. 2.7. New object recognition testing The apparatus for this task consisted of a black open-field box (30 × 40 × 50 cm) located in a sound-attenuated room and illuminated with a 40-W bulb. Before the test, mice were habituated (10 min per day) in the box for 3 days. On day 4, two identical objects, such as a Duplo Lego toy or golf ball, were placed approximately 10 cm from each corner, and each mouse was allowed to explore the box for 10 min (acquisition trial). The time that the mice spent exploring each object was recorded. Retention trials were carried out at 1- and 24-h intervals following the acquisition trial. During the retention trials, each mouse was placed back in the same box with one of the familiar objects used during the acquisition and a novel object. The mouse was then allowed to explore freely for 10 min, and the time spent exploring each object was recorded. The recognition index (RI) and discrimination index (DI), defined in terms of the amount of time spent exploring a novel object (TN) and the total time spent exploring familiar (TF) and novel (TN) objects in the retention trial, i.e., TN / (TN + TF) and (TN − TF) / (TN + TF), respectively, were used to measure cognitive functioning. 2.8. Preparation of brain tissue After the social avoidance test was completed, mice were terminated by decapitation under ether anesthesia. The prefrontal cortex, amygdala, and CA1 and dentate gyrus regions of the dorsal hippocampus were punched out bilaterally using a 1.0-mm Harris Uni-Core micropunch (Electron Microscopy Sciences, Hatfield, PA, USA). 2.9. Immune protein blotting Tissue samples were homogenized in 20 mM ice-cold Tris–HCl (pH 7.4) containing 1% protease (P2714; Sigma-Aldrich Korea, Ltd., Yongin, Kyunggi-Do, Korea) and phosphatase inhibitor (P2850; SigmaAldrich Korea, Ltd., Yongin, Kyunggi-Do, Korea). The homogenates were centrifuged for 15 min at 14,000 rpm at 4 °C, and the resulting supernatant fractions were used for Western blot analyses. The protein samples (20 μg/lane) were separated and transferred to a hydrophobic polyvinylidene difluoride membrane. The membranes were blocked
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and incubated overnight at 4 °C with monoclonal mouse anti-D1R (diluted 1:10,000; Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA), or polyclonal rabbit anti-D2R (diluted 1:1000; Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA) in 5% non-fat milk. On the next day, the membranes were washed with PBS-T, and the primary antibody was detected using goat anti-mouse horse-radish peroxidase (HRP)-linked immunoglobulin G (IgG) (1:10,000, Santa Cruz Biotechnology Inc., Santa Cruz, California, USA) for D1, or goat anti-rabbit HRP-linked IgG (Vector Laboratories, Burlingame, CA, USA) for D2 (1:5000) in PBS (60 min, 25 °C). The blots were developed using an enhanced chemiluminescence reagent (RPN2232; GE Healthcare, Inc., Piscataway, NJ, USA), visualized with a LAS-3000 Plus lumino-imaging analyzer (Fuji Photo Film Company, Kanagawa, Japan) and quantified using Multi Gauge software v3.0 (Fujifilm, Tokyo, Japan).
2.10. Data analysis Data were analyzed with SPSS version 12.0. Differences among the three groups were analyzed using one-way ANOVA. Post hoc individual comparisons were made using Tukey's honestly significantly different test. In all cases, differences were considered statistically significant at P b 0.05. Results are presented as means ± standard error of the mean (SEM).
3. Results 3.1. Administration of chronic social defeat stress and grouping of susceptible, unsusceptible and control mice To determine whether the impact of social defeat stress differs for susceptible and unsusceptible mice, C57BL/6 mice (n = 113) were subject to social defeat stress for 10 days through daily interactions with aggressive CD1 mice. Control mice were housed in similar cages but with members of the same strain, which changed daily. All C57BL/6 mice that were subjected to social stress showed signs of failure (surrender posture, lack of movement, fleeing, lying down or quitting) after being attacked by CD1 mice, and 6.2% (n = 7) died during the attacks. Mice with injuries that could affect further testing were not included in the behavioral experiments. The mice that underwent social defeat stress were subject to a final social avoidance test to classify them as “susceptible” or “unsusceptible”. The mice were placed into a box with CD1 mice but were separated from direct contact with a metal mesh. The interactions between the test mice and the CD1 mice were observed, and test mice with higher interaction rates (≥ 100) were designated the “unsusceptible group”, while mice with lower interaction rates (b100) were designated the “susceptible group”.
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3.2. Effects of social defeat stress on locomotion To determine whether susceptible and unsusceptible mice are affected in their locomotion activity, which is reflective of anxiety, we performed opening tests, which track the movement of mice in an open surrounding. We measured the duration that the mice from susceptible, unsusceptible and control groups stayed in the center and the spatial movement distance among mice that moved. There was a significant difference among the three groups (F [2, 57] = 12.088, P b 0.001) (Fig. 1). Compared to the control group, both the time that the mice of the susceptible group stayed in the center (P b 0.001) and the movement distance (P = 0.003) were significantly reduced. A similar reduction in the time (P b 0.001) and movement distance (P = 0.009) was observed for the unsusceptible group as compared to the control group. However, the susceptible and unsusceptible groups did not differ from each other in either the time or distance (P N 0.05). These results suggest that social defeat stress affects anxiety behavior for both susceptible and unsusceptible mice, and that the extent of the effect does not differ significantly for the two groups. 3.3. Effects of social defeat stress on light/dark preference To verify the findings of the locomotion test, we examined the light/ dark response of susceptible and unsusceptible mice. Mice were placed into a lit compartment of a box which had free access to a dark compartment and the time until initial movement of the mice from bright to dark areas and the total time that mice spent in the bright area were assessed over a 5 min test span. There was a significant difference among the susceptible, unsusceptible and control groups with regard to both the latency of remaining in the bright area (F [2, 57] = 40.551, P b 0.001) and duration of hiding (F [2, 57] = 7.709, P = 0.001) (Fig. 2). Compared to the control group, the latency period (P b 0.001) and the overall time spend in the bright compartment (P = 0.016) were reduced for mice from the susceptible group. Similar findings were obtained for the unsusceptible group as compared to the control group (P = 0.002 and P = 0.034, respectively). However, there were no significant differences between the susceptible and unsusceptible groups in these measurements (P N 0.05). These results suggest that social defeat stress affects light/dark preference for both susceptible and unsusceptible mice and that the extent of the effect does not differ significantly for the two groups. Because light/dark preference serves as an additional measure of anxiety, these results support the determination that both susceptible and unsusceptible mice are prone to anxietyrelated behavior. 3.4. Effects of social defeat stress on social interaction To determine whether susceptible and unsusceptible mice differ in their social interactions after undergoing social defeat stress, we
Fig. 1. Effects of social defeat stress on locomotion: opening tests were performed to assess locomotion. a. Time spent in the central zone; b. distance moved. ** P b 0.01, compared to the control group (n = 18–20 per group).
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Fig. 2. Effects of social defeat stress on light/dark preference: movement between a light and dark area was assessed for 5 min after placement in a lit area. a. Time until initial movement from the light to dark area (latency of transition), b. total time spent in lit area. ** P b 0.01, compared to the control group (n = 18–20 per group).
exposed mice to unfamiliar mice of the same genetic background and similar weight and examined their behavior for 10 min. There was a significant difference (F [2, 57] = 113.078, P b 0.001] with regard to the total time of social sniffing of mice in the susceptible, unsusceptible and control groups (Fig. 3A). The social sniffing time in the susceptible group was significantly reduced in comparison to both the control group (P b 0.001) and the unsusceptible group (P b 0.001). However, the sniffing time of the unsusceptible group was not significantly reduced in comparison to the control groups (P N 0.05). Furthermore, the following time (F [2, 57] = 0.329, P N 0.05) and crawling or climbing down time (F [2, 57] = 0.14, P N 0.05) was not significantly different among the three groups. Additionally, the number of times that the mice engaged in social sniffing (F [2, 57] = 1.662, P N 0.05), following (F [2, 57] = 0.575, P N 0.05) or crawling or climbing down (F [2, 57] = 0.299, P N 0.05) was not significantly different among these three groups (Fig. 3B). These results suggest that susceptible mice show reduced social sniffing time, but that no differences can be observed in the other social parameters that were tested. 3.5. Effects of social defeat stress on new object recognition To determine whether susceptible and unsusceptible mice differ in cognitive ability, we conditioned mice to one object and then exposed mice to new objects after they had 1 or 24 h of rest. After 1 h, a significant difference among the susceptible, unsusceptible and control groups was observed with regard to recognition index (F [2, 57] = 340.795, P b 0.001) and resolution index (F [2, 57] = 340.795, P b 0.001) (Fig. 4). The susceptible group had significantly reduced recognition and resolution indices compared to both the control group and unsusceptible group (P b 0.001); however, the values for the unsusceptible group were not significantly different from those for the control group (P N 0.05). Similar results were observed after 24 h with regard to both the recognition index (F [2, 57] = 367.517, P b 0.001) and the
resolution index (F [2, 57] = 367.517, P b 0.001). These results suggest that susceptible mice have reduced cognitive ability. 3.6. Effects of social defeat stress on the expression of D1 and D2 in the prefrontal cortex and amygdale The receptors D1 and D2 facilitate dopamine signaling, which is associated with mood changes. To examine whether social defeat stress causes reduction in the levels of D1 and D2 and whether the effect differs for susceptible and unsusceptible mice, we performed Western blotting. A significant difference (F [2, 56] = 70.664, P b 0.001) was observed among the susceptible, unsusceptible and control groups with regard to the expression level of D1 in the prefrontal cortex area (Fig. 5). Compared to both the unsusceptible group (P b 0.001) and the control group (P b 0.001), D1 expression was significantly reduced in the susceptible group, and there were no significant differences between the unsusceptible and control groups (P N 0.05). Similar results were observed for D1 in the amygdala region (P = 0.001, susceptible versus unsusceptible group; P = 0.002, susceptible versus control; P = 0.069, unsusceptible versus control). However, no statistical differences in D1 expression were observed in the hippocampus. In contrast to the findings for D1, there were no significant differences among the three groups of mice in the D2 expression level in prefrontal cortex (F [2, 56] = 0.64, P N 0.05), amygdala (F [2, 56] = 0.4, P N 0.05), or hippocampus (F [2, 56] = 0.59, P N 0.05) (Fig. 6). Collectively, these results suggest that susceptible mice undergo physiological changes that lead to the reduction of D1, but not D2 levels, within the prefrontal cortex and amygdala. 4. Discussion Few studies have reported the effect of social defeat stress on dopamine receptors in the brain. In this study, we aimed to explore the effect
Fig. 3. Effects of social defeat stress on social interaction: social interaction with unfamiliar mice of the same genetic background and similar weight were examined over 10 min. a. Total time spent in active social behaviors, b. total number of social contacts. * P b 0.05, compared to the control group. † P b 0.05, compared to the unsusceptible group (n = 18–20 per group).
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Fig. 4. Effects of social defeat stress on new object recognition: new object recognition was assessed as a measure of cognition. a. Recognition index TN / (TN + TF), b. discrimination index (TN − TF) / (TN + TF). *** P b 0.001, compared to the control group. ††† P b 0.001, compared to the unsusceptible group (n = 18–20 per group).
of social defeat stress on behavior and dopamine receptors in the brains of mice. To more clearly distinguish the effects of social defeat, we subdivided the defeated mice into susceptible and unsusceptible populations. We observed that the defeated mice of both the susceptible and the unsusceptible group performed poorly in locomotion and light/dark preference testing. However, the effects of social defeat stress were only detectable for the susceptible group when examined using tests for social interaction and new object recognition response. Furthermore, the susceptible mice showed a reduced level of expression of the D1 receptor in the prefrontal cortex and amygdala. These results suggest that the susceptible mice undergoing social defeat stress had depression-like behavior and cognition impairment, while the response for the unsusceptible mice was less severe. The defeated mice from both the susceptible and unsusceptible groups were found to have anxiety-like behavior in the opening (locomotion) experiment and the dark compartment experiment. These findings for mice subjected to social defeat stress are consistent with the results reported by Kinsey et al. (2007) and Razzoli et al. (2010), though susceptible and unsusceptible mice were not subdivided in the latter studies. Krishnan et al. (2007) separated the susceptible group from unsusceptible mice and observed that mice in both the susceptible
Fig. 5. Effects of social defeat on D1 levels in the prefrontal cortex (PFC), amygdala (Amyg), and hippocampus (HIP). Top: A representative Western blot is shown. β-Actin was tested as a loading control. Bottom: Values represent the mean ± SEM of the relative band densities from three independent experiments as determined by densitometry and were standardized to 1.0 in the control. ** P b 0.01, compared to the control group. †† P b 0.01, compared to the unsusceptible group (n = 18–19 per group).
and unsusceptible groups showed a diminished response to light exposure; however, there was no significant difference in spontaneous activities of the mice among the susceptible, unsusceptible and control groups, which may be because they used a different study method from those in this study. We recorded the spontaneous activity of the mice in an open space for 30 min on the 12th day, but Krishnan et al. recorded spontaneous activity of the mice for 5 min on the 11th day. Interestingly, the mice from the susceptible groups had a reduced level of social interaction as assessed by social sniffing time, but not by other parameters of testing (following, crawling, climbing). To our knowledge, this is the first study to examine the effect of social defeat stress on social interaction. Our findings suggest that social defeat stress may cause social withdrawal behavior, which is similar to human depression. Consistent with our findings, it has been reported that after social defeat stress, sucrose preference and enjoyment-like reactions are reduced for mice in the susceptible group (Krishnan et al., 2007) and the defeated mice (Schloesser et al., 2010; Razzoli et al., 2010; Yu et al., 2011). Therefore, these findings support the use of the chronic social defeat stress model as an animal model of depression and anxiety disorders. We also determined using new object recognition experiments that cognition memory of the mice in the susceptible group is reduced.
Fig. 6. Effects of social defeat on D2 levels in the prefrontal cortex (PFC), amygdala (Amyg), and hippocampus (HIP). Top: A representative Western blot is shown. β-Actin was tested as a loading control. Bottom: Values represent the mean ± SEM of the relative band densities from three independent experiments as determined by densitometry and were standardized to 1.0 in the control (n = 18–19 per group).
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A limited number of studies have explored cognition memory of social stress defeat through new object recognition experiments. It has been reported that in new object recognition experiments, the learning ability of mice is reduced if they are exposed to predators, in which case they forget the information they learned (El Hage et al., 2004). Therefore, it is likely that social defeat stress causes subtle cognition impairment to mice of the susceptible group, though more spontaneous behavior test experiments are needed to further verify these findings. Our results demonstrate that the dopamine D1 receptor in the prefrontal cortex and amygdala are also only reduced in the mice of the susceptible group. The density of dopamine receptors is known to become reduced upon increase in dopamine release. Therefore, our results suggest that susceptible mice have increased dopamine release caused by social defeat stress. Several studies support the idea that dopamine levels (Louilot et al., 1986; Puglisi-Allegra et al., 1990) and the release of endogenous dopamine (Tidey and Miczek, 1996) increases somewhat under social defeat stress, but these studies did not divide the mice into susceptible and unsusceptible groups. Cao et al. (2010) divided the socially defeated mice into susceptible and unsusceptible groups and observed increased dopamine levels and release in the ventral tegmental area only in mice of the susceptible group. The dopamine signaling pathway serves as an important neurotransmitter in the brain. A large number of reports demonstrate that unbalanced dopamine can lead to the generation of depression (Braestrup et al., 1975; Lambert et al., 2000). Recently, the dopamine system has been shown to play an important role in the mechanism of some antidepressants (Bymaster et al., 2003; Pania and Gessab, 2002). Furthermore, methylphenidate, a dopamine medication, can affect the spontaneous activity of mice and cause anxiety-like behavior (Homsi et al., 2001). It has been well established that the expression level of dopamine in the hippocampus is associated with depression. There are also reports indicating that the expression level of dopamine in the prefrontal cortex participates in the formation of the working memory (Goldman-Rakic, 1996) and further influences reasoning and judgment. Depression and bipolar diseases are exacerbated when the working memory is damaged (Goldman-Rakic, 1994; Meyer-Lindenberg et al., 2005). Therefore, it can be inferred that the reduction in D1 levels in our study provides a basic mechanism for cognitive impairment, as observed in the new object recognition experiment. This reduction also could account partially for the anxiety-like and depression-like behavior of the mice in the susceptible group. Though a reduction in D1 was observed in our study, no significant differences in the levels of the D2 receptor were observed in three encephalic regions. These results are consistent with the results of several previous studies. In social isolation experiments, the dopamine D2 receptor had no significant change in the amygdala and nucleus accumbens of rats (Malone et al., 2008). Furthermore, no significant changes were observed in adolescent rats in the expression of the dopamine D2 receptor in the prefrontal cortex and nucleus (Burke et al., 2011). However, some studies suggest that the levels of the D2 receptor fluctuate depending on the stimuli. The density of the dopamine D2 receptor is reduced in the prefrontal cortex and nucleus when rats are passively exposed to attack (Suzuki et al., 2010) or subjected to the smell of a cat (Wright et al., 2008). In the amygdala expression of genes important in dopamine function were de-regulated including down-regulated Drd2 and Darpp-32 after chronic social defeat stress (Azzinnari et al., 2014); however the density of the dopamine D2 receptor is significantly increased when rats are repeatedly exposed to visible caves (Lucas et al., 2004). One possible explanation for why we observed a reduction in D1, but not D2 is that the internalization rate of D1 and D2 receptors after stress are different, with D2 internalization being a slower process. Internalization of D1 has been observed in numerous in vitro and in vivo experiments (Ng et al., 1995). Furthermore, the intensity of social defeat stress may not be enough to change the expression of D2, or the density of D2 may be relatively low in each area of the brain.
5. Conclusion In conclusion, this study shows that chronic social defeat stress can lead to a reduction in spontaneous movement and an increase in anxiety-like behavior of all the defeated mice. However, we only observed reduced social interaction and cognition impairment in susceptible mice. Furthermore, we observed a significant reduction of D1 in the prefrontal cortex and amygdala only in mice of the susceptible group. These data indicate that depression-like behavior and cognition impairment caused by social defeat stress may be related to changes in the dopamine D1 receptor, indicating that selective reduction of dopamine receptors in the prefrontal cortex and amygdala can be used as a marker to distinguish susceptible animals. Acknowledgements Funding for this study was provided by the National Natural Science Foundation of China (No. 81401112). References Adamcio, B., Havemann-Reinecke, U., Ehrenreich, H., 2009. 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