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Delayed recovery of core body temperature from repeated social defeat may be indicative of stress vulnerability
Arjunan Gnanendran Sponsor: Seema Bhatnagar, Ph.D. Department: Anesthesiology and Critical Care Perelman School of Medicine, University of Pennsylvania ABSTRACT
Exposure to stress has been shown to lead to certain psychopathological disorders such as depression and anxiety, including posttraumatic stress disorder. However, it is unclear why only certain individuals develop stress-related diseases whereas other individuals remain resilient. The answer may be linked to the type of strategy used to cope with stress. Passive coping has been linked with the development of stress-induced disorders. Our goal is to determine the biomarkers of resilience using a rodent model of the stress of social defeat. In this experiment, we used fully implantable telemetry to measure body temperate prior to, during, and after repeated social stress events, using a rodent resident/intruder paradigm of social defeat. For seven consecutive days, experimental intruder rats underwent a 30-minute social defeat. We found that the behavioral coping differences to the stressor between LL and SL rats are associated with different core body temperature responses. Specifically, following social defeat, SL animals expressed a prolonged elevated body temperature hours after the stress event. This is in line with numerous studies that have shown that repeated social defeat induces chronic hyperthermia in rats. However, our data suggests that this chronic hyperthermia is not present in all individuals following repeated stress events, but that it is prevalent mostly in animals that exhibit passive coping behavior (SL). Since this chronic hyperthermia has also been associated with depressive-type behavior in rats, a body temperature response characterized by a prolonged recovery to baseline following stress may be a biomarker of stress vulnerability. 1. INTRODUCTION One of the most studied paradigms of social stress is the resident/ intruder test developed by Miczek (1). In this paradigm, the intruder (experimental) rats are placed into the home cage territory of an unfamiliar resident previously screened for high aggression. A typical encounter results in the intruder showing defeat, signaled by the intruder assuming a supine position for approximately 3 seconds. In this homogenous population of intruders, two subpopulations emerge, each exhibiting different coping behavior (2). These two subpopulations have been classified by their latency time to defeat. The first group, termed long latency animals (LL), resists defeat for more than 300 seconds and exhibit more proactive coping behavior. Short latency (SL) animals usually resist defeat for less than 300 seconds and typically show passive behavior toward the resident. Since most of the stress humans encounter is of a social nature, this animal model of defeat can provide valuable information regarding the neural mechanisms that govern responses to stress in the human brain (3). Repetitive social stress in rat and mice models has been shown to remodel the dendrites of the hippocampus and alter the neuroendocrine response to stress by the hypothalamic-pituitaryadrenocortical (HPA) axis (3,4). These changes in the brain and neuroendocrinology can have lasting effects on the individual, and studying their mechanisms may explain why certain individuals are vulnerable to developing psychopathological disorders. Exposure to social stressors has been shown to induce hyperthermia in rats (5,6). The elevation in core body temperature is seen during the social stress event itself, rising rapidly at its onset and gradually returning to baseline within several hours. In experiments of repeated social stress, the elevations in temperature may be seen long after the acute stress events occur (5). A study by Tsuji showed that the effects of repeated social stress induces chronic hyperthermia in rats, along with depressivetype behavior. Following a four week social stress experiment, showed and increase in baseline body temperature of about 0.20.3C. Furthermore, 8 days following the last social stressor, hyperthermia and depression-like behavior were still observed in a forced-swim test (5). A study by Koolhaas showed that the effects on body temperature depended on the rats counter-aggressiveness during the defeat (7). They saw that intruders that counterattacked the resident more often were least affected by the stress, in that their daily temperature amplitude did not shrink as much as more passive intruders (7). In this experiment, we determined the effects of a repeated social defeat stress on body temperature and assessed whether there were differences in the homeostatic response between the two subpopulations LL and SL. By focusing on these two subpopulations, we seek to further understand the physiological changes that occur in animals that exhibit proactive vs. passive coping and further explore the link to stress resilience vs. vulnerability.

Co-sponsor: Phillip Rea, Ph.D. Department: Biology, University of Pennsylvania

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2. MATERIALS & METHODS Animals Intruders were male Sprague-Dawley rats (Charles River) weighing 225-250g on arrival were used as the experimental animals. Residents were retired male Long Evans breeders (Charles River). Rats were individually housed with a 12-h light, 12-h dark cycle (lights on at 0700 h) in a climate-controlled room with ad libitum food and water. Studies were approved by the Childrens Hospital of Philadelphia Institutional Animal Care and Use Committee and conformed to the National Institutes of Health Guide for the Use of Laboratory Animals. Animals were given 5 days of acclimation prior to any procedures. All defeats took place between 1030h and 1100h. Telemetry Data was recorded from hardware and software purchased from Data Sciences International (St. Paul, MN). DSI PhysioTel F40EET transmitters were implanted into the peritoneal cavity of each rat anesthetized with Isoflurane. The animals were given 4 days to recover from the surgery before any experimental procedures began. Animals were housed individually and were placed atop a wireless receiver. Temperature and locomotor activity (parameter values) were continuously recorded every 30 seconds throughout the experiment. These recordings were then averaged into 10-minute bins and graphed and analyzed using Prism software. We have focused on the 9:30 (1 hour prior to defeat) to 5:30 time period. Social Defeat Stress During each episode of social stress, a rat was placed into the home cage territory of an unfamiliar Long-Evans resident previously screened for high aggression. A typical agonistic encounter resulted in intruder subordination or defeat, signaled by the

intruder assuming a supine position for approximately 3 sec. After defeat, a wire mesh enclosure was placed in the cage to prevent physical contact between the resident and intruder but allowing visual, auditory, and olfactory contact for the remainder of the 30min defeat session. Latency to assume a submissive posture was recorded and averaged over the seven daily defeat exposures. If an intruder resisted defeat for 15 min, rats were separated with the wire partition for the remainder of the session. Controls were placed behind a wire partition in a novel cage for 30 min daily. Rats were returned to their home cage after each session. Experimental Design Residents were randomly assigned to either a social defeat or control group. Defeat animals were exposed to a 30-min social defeat, while control animals underwent novel cage exposure, for 7 consecutive days. Following the 7-day defeat period, on day 8, residents were placed in a resident cage where the resident was not present in an anticipation test. After 30 minutes had passed, rats were returned to their home cages. Prior to sac, the defeat and control groups underwent a novel stressor, a 30- minute restraint, followed by a 30-minute recovery. All defeats and novel cage placements took place between 1030h and 1100h. (See figure below) Statistical Analysis For analysis of the body temperature response to the repeated social defeat and anticipation test on days 1-8, two way [Stress Group Day] repeated measures ANOVAS were conducted on each time point (averaged 10-minuted bin) from 9:30 AM to 5:30 PM on days -1,1,5,7, and 8. All data was analyzed using PRISM 5.0c. P 0.05 was considered statistically significant for all analyses.

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3. RESULTS: Temperature Response to Repeated Social Defeat

FIGURE 1: Body temperature is shown in rats exposed to acute social defeat or novel cage placement (CTRL n=5) for 30 minutes for seven consecutive days. Based on average latency times to defeat over the course of the 7-day defeat stress period, two groups, designated long latency (LL; n=11) and short latency (SL; n=4) were defined. Temperatures are shown during a one-hour baseline period (9:30 10:30 AM), 30-minute social defeat or novel cage period (10:30 11:00 AM), a 30-minute recovery period (11:00 11:30 AM), and a prolonged recovery period (11:30 AM 5:30 PM) on days 1,5, and 7 of social defeat. *Both SL & LL rats significantly higher than CTRL rats at the time points indicated (P 0.05) +Only LL rats significantly higher than CTRL rats at the time points indicated (P 0.05) ++Only SL rats significantly higher than CTRL rats at the time points indicated (P 0.05) **SL rats significantly higher than CTRL & LL rats at the time points indicated (P 0.05) *** SL rats significantly higher than LL (but not CTRL) rats at the time points indicated (P 0.05)

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FIGURE 2: Body temperature (Mean & SEM) is shown for LL, SL and control rats exposed to either social defeat or novel cage placement. Data is from days: -2, -1, 1, 4, 5, 7, and 8 (Anticipation Test). Starting on day four, we begin to see an increase in the SLs body temperature following defeat, with a slower recovery to a baseline temperature. During the anticipation test, where stress group rats were placed in a empty resident cage, we see an temperature response very similar to an actual defeat.

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4. DISCUSSION The present study supports others that show repeated social defeat increases rats body temperature long after the stress events have concluded. However, our analysis shows that all individuals of the population do not show the same temperature response both during and after the stress event. As the experiment progressed, the baseline temperature of the LL animals tracked the control group closely, while the SL animals temperature remained elevated hours after defeat. The chronic hyperthermia induced by the social defeat is much more pronounced in the SL group, with many time points significantly higher (P 0.05) than control in the recovery period, than the LL group. For example, on day 5 of defeat, SL rats were significantly above control at 20 10-minute time periods while the LL group was only significantly higher in 1 10-minute period. Furthermore, at three of those time points, the SL group was higher than both the LL and CTRL groups. Splitting the population in the two subpopulations based on latency time to defeat, as detailed by Wood et al. (2010), has illuminated this trend, which would have been hidden in an average of the entire stress group. SL rats showed an elevated temperature during defeat as well as a delayed temperature recovery to baseline following defeat. This trend developed over the course of the experiment, becoming more pronounced after days 4 and 5 of defeat. This is interesting because it is thought that the two subpopulations begin to split in latency time, with the LL animals taking longer to defeat each successive day, after day 4. Thus, the temperature differences we observe may be linked to other changes occurring to the neuroendocrine response, particularity in the HPA axis.

and SL animals have very similar temperature responses but as the experiment progresses, SL animals peak temperature during defeat remains elevated while LL temperature decreases. This along with the aforementioned prolonged recovery to a baseline temperature may be evidence of poor habituation to the repeated social defeat. Although more analysis must be conducted to find definitive evidence, these findings are exciting, since they may provide a noninvasive, readily measured biomarker of stress vulnerability, easily translatable to humans. Further analysis in this experiment will look at the changes to sleep architecture caused by the repeated social defeat. Changes to circadian rhythms will also be analyzed, since they are an important part of HPA regulation. Stress is regulated by a complex interconnected response, and so the differences shown in the temperature response must be seen as just one endpoint in the broader physiological changes found in stress vulnerable individuals. Hopefully, this data can help elucidate the changes caused by chronic stress in the underlying neural circuitry. 5. ACKNOWLEDGEMENTS: I thank Dr. Seema Bhatnagar and Elizabeth Ver Hoeve for taking the time to teach me and help steer my analysis during the course of this experiment. 6. AUTHOR CONTRIBUTIONS: Project conception and planning provided by S. Bhatnagar. Experiments were conducted by E. Ver Hoeve and A.G. Set up of hardware, software and data collection management by A.G. Data analyses preformed by A.G.
References

HPA hypo-responsiveness, due to excessive negative feedback caused by stress, may negatively affect the normal homeostatic response, leading to depressive disorders (6). In normal functioning, the HPA axis is activated when the hypothalamus releases a combination of corticotropin releasing hormone (CRH), vasopressin, and oxytocin, which act on the anterior pituitary gland (6). The anterior pituitary, in turn, releases adrenocorticotropic hormone (ACTH), which triggers the adrenal cortex to stimulate the secretion of glucocorticoids (6). Abnormality in this pathway has been seen in a number of psychopathological disorders, and it is thought that poor habituation to stress may be one cause of HPA dysfunction. Although we still must analyze the plasma of our animals to determine if they have levels of hormones indicative poor habituation, for example high levels of ACTH, we may see evidence supporting this in the temperature response. Habituation refers to the decline in magnitude of the stress response with repeated exposure to a stress stimulus. For example, peak ACTH levels should be highest on day one of a social defeat experiment, and decrease over the course of the experiment. Looking at our temperature data, we see that SL animals do not seem to follow this general trend. On day one, LL

1. Miczek KA. A new test for aggression in rats without aversive stimulation: Differential effects of d-amphetamine and cocaine. Psychopharmacology 1979;60(3):253-9. 2. Wood SK, Walker HE, Valentino RJ, Bhatnagar S. Individual differences in reactivity to social stress predict susceptibility and resilience to a depressive phenotype: Role of corticotropin-releasing factor. Endocrinology 2010 April 01;151(4):1795-805. 3. Buwalda B, Kole MHP, Veenema AH, Huininga M, de Boer SF, Korte SM, Koolhaas JM. Long-term effects of social stress on brain and behavior: A focus on hippocampal functioning. Neuroscience & Biobehavioral Reviews 2005 2;29(1):83-97. 4. Bowens N, Heydendael W, Bhatnagar S, Jacobson L. Lack of elevations in glucocorticoids correlates with dysphoria-like behavior after repeated social defeat. Physiol Behav 2011 2/28;105(4):958-65. 5. Hayashida S, Oka T, Mera T, Tsuji S. Repeated social defeat stress induces chronic hyperthermia in rats. Physiol Behav 2010 8/4;101(1):124-31. 6. Bhatnagar weight S, and Vining food C, Iyer with Boer V, Kinni V. Changes in hypothalamic-pituitary-adrenal body 7. intake A, De function, body temperature, social JM. stress Long-

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exposure in rats. J Neuroendocrinol 2006;18(1):13-24. Meerlo P, Sgoifo Koolhaas lasting consequences of a social conflict in rats: Behavior during the interaction predicts subsequent changes in daily rhythms of heart rate, temperature, and activity. Behavioral Neuroscience;Behavioral Neuroscience 1999;113(6):1283-90.

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