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The influence of daily social stimulation in ameliorating PTSD-like behavioral and physiological changes in rats exposed to chronic psychosocial stress
h [electronic resource] /
by Shyam Seetharaman.
[Tampa, Fla] :
b University of South Florida,
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Thesis (M.A.)--University of South Florida, 2009.
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ABSTRACT: Individuals exposed to life-threatening trauma are at increased risk for developing post-traumatic stress disorder (PTSD). Not all people exposed to trauma, however, go on to develop PTSD. Some evidence suggests that individuals who receive social stimulation, such being involved in supportive social networks, are less likely to develop PTSD compared to those lacking social interactions. Although human research has been effective in demonstrating associations between higher levels of social stimulation and lower incidences of PTSD, there has been a lack of experimental evidence suggesting that social stimulation protects against the onset of the disorder after trauma. Here, we tested the hypothesis that providing animals with daily social stimulation (DSS) would ameliorate psychosocial stress-induced changes in behavior and physiology produced by our previously developed animal model of PTSD which generates responses comparable to patients with the disorder. The major findings of this study revealed that providing animals with DSS initiated shortly after an acute stress experience blocked the development of PTSD-like responses in adult rats exposed to chronic psychosocial stress, such as heightened anxiety, exaggerated startle, and contextual fear. These results are consistent with human research suggesting that social stimulation may confer resistance of a subset of the traumatized population to develop PTSD. This level of analysis in an animal model of PTSD underlies the importance of continuing clinical research examining social phenomena in identifying risk factors for PTSD, as well as non-pharmacological treatments (e.g. social support systems) for the disorder.
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Advisor: David Diamond, Ph.D.
t USF Electronic Theses and Dissertations.
The Influence of Daily Social Stim ulation in Ameliorating PTSD-Like Behavioral and Physiological Changes in Rats Exposed to Chronic Psychosocial Stress by Shyam Seetharaman A thesis submitted in partial fulfillment of the requirements for the degree of Master of Arts Department of Psychology College of Arts and Sciences University of South Florida Major Professor: David Diamond, Ph.D. Jennifer Bosson, Ph.D. Mark Goldman, Ph.D. Paul Spector, Ph.D. Date of Approval: May 21, 2009 Keywords: enrichment, trauma, animal, model, support Copyright 2009, Shyam Seetharaman
This work is dedicated to my dad, who has never stopped believing in me, and never let me stop believing in myself. I would also like to dedicate this to my wife, Lucky. Without her love, patience, and encouragement, this would not have been possible. 1
2 Acknowledgements I would like to acknowledge my major professor Dr David Diamond for giving me the opportunity to conduct research in hi s laboratory. His guida nce and expertise in the field has been valuable in my progression as a scientist. I would also like to thank my fellow colleagues Josh Halonen, Collin Park, and Phil Zoladz for their patience, guidance, and friendship which helped immensely in the completion of this work. Additionally, this project would not have been possible without the friendship and guidance of both Dr. Chris Bloom and Dr. Ken Carter who instilled in me the love of scientific research.
Table of Contents List of Tables iv List of Figures v Abstract vii Chapter One: Background 1 Introduction to Post-Traumatic Stress Disorder and Factors Which Influence its Etiology 1 Post Traumatic Stress Disorder 2 Animal Models 3 Social Stimulation 4 Social Stimulation and Stress 5 Social Stimulation and PTSD 6 Limitations of Social Stimulation and PTSD Studies 8 Effects of Social Stimulation on the Brain 8 Environmental Enrichment 10 Environmental Enrichment and Stress 12 Utilizing Environmental Enrichment to Study PTSD 13 Chapter Two: Experiment 14 Purpose and Hypotheses of the Present Study 14 Methods 15 Animal and Housing Conditions 15 Power Analysis: Sample Size Estimation 15 Design 16 Psychosocial Stress Procedure 16 Daily Social Stimulation Exposures 18 Behavioral Testing 19 Fear Memory 19 Elevated Plus Maze 20 Startle Response 21 Novel Object Recognition 22 Physiological Testing 23 Blood Sampling and Cardiovascular Measurements 23 Statistical Analyses 24 Experimental Design and General Analyses 24 Fear Memory 25 1 i
Elevated Plus Maze 25 Startle Response 26 Novel Object Recognition 26 Heart Rate and Blood Pressure 26 Growth Rate 27 Organ Weights 27 Results Context Test Immobility 27 Context Test Fecal Boli 28 Cue Test Immobility 29 Cue Test Fecal Boli 31 Elevated Plus Maze 32 Percent Time in Open Arms 33 Percent Time in Closed Arms 34 Movement 35 Velocity 36 Head Dips 37 Startle Response 38 90 dB Acoustic Stimuli 39 100 dB Acoustic Stimuli 39 110 dB Acoustic Stimuli 39 Novel Object Recognition 40 Ratio Time 5 Minute Test 40 Ratio Time First Minute of Test 41 Growth Rate 42 Cardiovascular Testing 43 Heart Rate 44 Systolic Blood Pressure 44 Diastolic Blood Pressure 45 Organ Weights 46 Adrenal Gland 46 Thymus Gland 48 Kidney 49 Heart 50 Discussion 53 Summary of Major Findings 53 Possible Mechanisms of Action Underlying Findings 54 Blunted Stress Response System 54 Prefrontal Co rtex: Extinction Learning and Fear Suppression 56 2 ii
Neur obiological Changes in the Prefrontal Cortex 60 Neurochemical Changes in the Prefrontal Cortex 62 Conditioned Fear and PTSD 62 Nucleus Accumbens: Decreased Anxiety, and Increased Exploratory-Like Behavior 62 Hippocampus: Recognition Memory 63 Antidepressant-Like Changes in the Brain 64 Prevention of Psychosocial Stress-Induced Changes in Organ Weight 65 Prevention of Psychosocial Stress-Induced Reduction in Growth Rate 67 Heart Rate and Blood Pressure 67 General Conclusions 68 Im plications and Clinical Relevance 69 Limitations and Future Directions 70 Concluding Remarks 73 References 75 3 iii
List of Tables Table 1. Measures, Hypotheses, and Outcomes 51 4 iv
5 List of Figures Figure 1. Effects of Psychosocial Stress and Environment on Immobility During the Contextual Fear Test 28 Figure 2. Effects of Psychosocial Stress and Environment on Fecal Boli Produced During the Contextual Fear Test 29 Figure 3. Effects of Psychosocial Stress and Environment on Immobility Before and During the Tone in the Cue Fear Test 31 Figure 4. Effects of Psychosocial Stress and Environment on Fecal Boli Produced During the Cue Fear Test 32 Figure 5. Effects of Psychosocial Stress and Environment on Percent Time Spent in the Open Arms of the Elevated Plus Maze 33 Figure 6. Effects of Psychosocial Stress and Environment on Percent Time Spent in the Closed Arms on the EPM 34 Figure 7. Effects of Psychosocial Stress and Environment on Movement on the EPM 35 Figure 8. Effects of Psychosocial Stress and Environment on Velocity on the EPM 37 Figure 9. Effects of Psychosocial Stress and Environment on Head Dips on the EPM 38 Figure 10. Effects of Psychosocial Stress and Environment on Startle Response to the 90, 100, and 110 dB Acoustic Stimuli 40 Figure 11. Effects of Psychosocial Stre ss and Environment on Novel Object Recognition During the 5 Minute Test 41 Figure 12. Effects of Psychosocial Stre ss and Environment on Novel Object Recognition During the First Minute of the Test 42 v
Figure 13. Effects of Psychosocial Stre ss and Environment on Growth Rate 43 Figure 14. Effects of Psychosocial Stress a nd Environment on Heart Rate 44 Figure 15. Effects of Psychosocial Stress and Environment on Systolic Blood Pressure 45 Figure 16. Effects of Psychosocial Stress and Environment on Diastolic Blood Pressure 46 Figure 17. Effects of Psychosocial Stress and Environment on Adrenal Gland Weight 47 Figure 18. Effects of Psychosocial Stress and Environment on Thymus Gland Weight 48 Figure 19. Effects of Psychosocial Stress and Environment on Kidney Weight 49 Figure 20. Effects of Psychosocial Stre ss and Environment on Heart Weight 50 6 vi
The Influence of Daily Social Stimulation in Ameliorating PTSD-Like Behavioral and Physiological Changes in rats Expos ed to Chronic Psychosocial Stress Shyam Seetharaman ABSTRACT Individuals exposed to life-thr eatening trauma are at incr eased risk for developing post-traumatic stress disorder (PTSD). Not a ll people exposed to trauma, however, go on to develop PTSD. Some evidence suggests that individuals who receive social stimulation, such being involve d in supportive social networks are less likely to develop PTSD compared to those lack ing social interact ions. Although human research has been effective in demonstrating associations between higher levels of so cial stimulation and lower incidences of PTSD, there has been a la ck of experimental evidence suggesting that social stimulation protects against the onset of the disorder after trauma. Here, we tested the hypothesis that providing animals with daily social stimulation (DSS) would ameliorate psychosocial stress-induced changes in behavior a nd physiology produced by our previously developed animal model of PTSD which generates responses comparable to patients with the disorder. The major fi ndings of this study re vealed that providing animals with DSS initiated shortly after an acute stress experience blocked the 7 vii
8 development of PTSD-like responses in adult rats exposed to chroni c psychosocial stress, such as heightened anxiety, exaggerated star tle, and contextual fear. These results are consistent with human research suggesting that social stimulation ma y confer resistance of a subset of the traumatized population to de velop PTSD. This level of analysis in an animal model of PTSD underl ies the importance of con tinuing clinical research examining social phenomena in identifyi ng risk factors for PTSD, as well as nonpharmacological treatments (e.g. social support systems) for the disorder. viii
Chapter One Background Introduction to Post Traumatic Stress Disorder and Factors which In fluence its Etiology Individuals exposed to horrifi c, life-threatening trauma are at increased risk for developing post-traumatic stress disorder (PTSD) It is a debilitating disorder which can produce symptoms such as hypervigilance, heightened anxiety, and intrusive memories (Elzinga & Bremner, 2002; Stam, 2007a). Although trauma exposur e increases the risk of developing PTSD, not all traumatized individuals go onto develop PTSD. Findings suggest that only about 25% of those exposed to trauma develop PTSD (Yehuda, 2001). Evidence has suggested that environmental fact ors, such as social support, education and cognitive stimulation are asso ciated with decreased rate s of PTSD among victims of trauma. For instance, post-com bat social support was associat ed with decreased rates of PTSD among Vietnam veterans (King, King, Fairbank, Keane & Adams, 1998). Although findings in social and clinical research have been effective in demonstrating relationships between environmen tal factors, such as social support, and lower rates of PTSD (King et al, 1998), ther e is a lack of experiment al evidence suggesting that environmental factors are effective in pr otecting against PTSD. Experimentally manipulating environmental factors in an anim al model, therefore, may be useful in 1
2 facilitating conclusions based on causality. This aim of this study was to manipulate social and environmental factors in an animal model of PTSD previously developed by our group (Zoladz, Fleshner & Diamond, 2008) through the use of daily social stimulation (DSS) to examine its influence on the development of symptoms in rats analogous to those seen in patients with PTSD In this study, I tested the hypothesis that providing animals with social and environmen tal enrichment will ameliorate PTSD-like behavioral and physiological ch anges of rats exposed to ch ronic psychosocial stress. Findings of this study may provide experimental insight into the influence of social phenomena on the development of PTSD-like symptoms. Post Traumatic Stress Disorder Individuals exposed to horrifi c, life-threatening trauma such as rape, combat, or natural disasters, are at increased risk to develop PTSD. These individuals respond to traumatic experiences with intense feelings of helplessness, panic, fear, anxiety, and horror which can persist for many years afte r trauma exposure (American Psychiatric Association 1994). PTSD is a debilitating di sorder involving symptoms of increased anxiety, exaggerated startle, hyperarousal and cognitive impairments (Elzinga & Bremner, 2002; Stam, 2007a). These sympto ms may be further exacerbated by intrusive flashback memories of the original trauma which are not simply reminders but, rather, are reported as feeling like actual re-experiences of the trauma (Reynolds & Brewin, 1999). As a result, PTSD patients often make great e fforts to avoid situations or stimuli which remind them of the trau matic experience.
3 There are various biological outcomes that are prevalent among those diagnosed with PTSD. Relative to control participants, PT SD patients have significant elevations in cortisol (CORT; cortisol in humans and cort icosterone in rodents), a main physiological marker of stress, in response to laboratory stressors (Bre mner, Vythilingam, Vermetten, Adil & Khan, 2003), and reminders of trau matic experiences (Elzinga, Schmahl, Vermetten, van Dyck & Bremner, 2003). A lthough inconsistent, findings have also indicated lower baseline CORT levels am ong those with PTSD compared to control participants (Yehuda, Golie r & Kaufman, 2005). PTSD patients also demonstrate significantly elevated levels of cardiovas cular reactivity (i.e. heart rate and blood pressure) when exposed to acute laboratory stressors (Orr, Lasko, Shalev, & Pitman, 1995), and reminders of traumatic experien ces (McFall, Murburg, Ko, & Veith, 1990). Research also suggests that PTSD patients e xhibit significant increase s in baseline heart rate and blood pressure levels comp ared to controls (Pole, 2007). Animal Models. Preclinical researchers have utilized different paradigms to model PTSD in rodents. Such paradigms have included the use of electric shock, underwater trauma, predator exposure, or predator-related cues such as odor (Stam, 2007b). Our group has developed an animal mo del of PTSD that generates PTSD-like responses in rats based on multiple physiol ogical and behavioral tests (Zoladz et al, 2008). It utilizes cat exposure an ethologically relevant st ressor, shown to elicit intense defensive, fear-related behaviors in rats (Blanchard, Blanchard, Rodgers & Weiss, 1990; Blanchard, Yang, I Li, Gervacio & Blanchar d, 2001; Blanchard, Canteras, Markham, Pentkowski & Blanchard, 2005). This model, which incorporates two cat exposures in
4 conjunction with daily social instability, produces robust behavioral changes such as exaggerated startle responses, heightened anxiety, cognitive impairments, as well as physiological changes consiste nt with symptoms analogous to those exhibited by people with PTSD (Zoladz et al, 2008). Developing an understanding of so cial factors, which may mitigate PTSD severity and development, will be beneficial in understanding individuals susceptibility to develop PTSD, and may contribute to the body of clinical research focused on treating the disorder. Social Stimulation Accumulating evidence suggests that PTSD risk and recovery are associated with factors such as social stimulation, such as social support, which refers to individuals social network size and comple xity (Charuvastra & Cloitr e, 2008). Positive social interactions with spouses, family members, or a community may provide individuals with support in the form of attachment, love, or advice which may promote better health (Cohen & Wills, 1985; Uchino, Cacciopo & Kiecolt -Glaser, 1996). There is evidence indicating that social inter actions may be beneficial to those who have experienced intense and, possibly, traumatic events. Speci fically, studies have found an association between social support and an attenua tion of detrimental effects of stress on psychological or physical well-being in vic tims of sexual assault (Kimerling & Calhoun, 1994), and childhood abuse (Runtz, 1997). High leve ls of social support have also been related to lower cardiovascular reactivity to la boratory stressors compared to low-social support controls (Lepore, Allen & Evans, 1993 ; Pruitt & Zoellner, 2008)
5 Social Stimulation & Stress Stress can generate general feelings of helplessness (Cohen & Wills, 1985), increasing the likelihood of individuals turning to maladaptiv e coping strategies such as alcohol abuse (Brady & Sonne, 1999). Social support can increase feelings within individuals that others will provide necessary resources for coping with stressful situations and, in turn, reduce feelings of helplessness and uncontrollability, compared to those who lack support (Cohen & Wills, 1985). In this fashion, social support may, in theory, buffer against the onset of pathol ogical disorders by intervening between a stressor and stress reaction by attenuating the perceived th reat of the experience and promoting healthier behaviors (Cohen & W ills, 1985). A more recent report, however, suggested that social support was associated with actual decreases in the occurrences of stressors, as opposed to being mobilized duri ng stressful situations in a meta-analysis examining workplace stress (Viswesvaran, Sanchez & Fisher, 1999). These findings imply that social support may simply redu ce the likelihood of st ressful experiences, rather than act to buffer against the detrimen tal effects of stress on health. Conflicting correlation-based findings evident in human research reinforces the need for more experimental evidence which may provide insi ght into the causal effects of social stimulation on responses to stress and its im pact on health. Nonetheless, there is evidence indicating that individuals may be able to draw on their own social networks for
6 support after traumatic stress (Ullman, 1999), and may confer resistance of a subset of the traumatized population to develop PTSD. Social Stimulation & PTSD Research has shown that only about 25% of individuals exposed to horrific, lifethreatening trauma develop PTSD (Yehuda 2001). Studies have shown a significant relationship between social support received after trauma exposure and PTSD severity, development, and, in some cases, remission of the disorder. Specifically, analyses of responses from Vietnam veterans revealed a significant correla tion between social support received at homecoming and lower ra tes of PTSD (King et al, 1998). Other evidence indicates higher incidences of PTSD in those not receiving social support among a sample of Jews and Arabs exposed to repeated acts of terrorism in Israel (Hobfall, Canetti-Nisim, Johnson, Palmieri Varley & Galea, 2008) compared to individuals with support from others. In addition, Vietnam veterans diagnosed with PTSD reported significantly lower levels of social network sizes and positive social interactions in the first 1-3 months follo wing military discharge compared to those combat veterans not diagnosed with PT SD (Keane, Scott, Chavoya, Lamparski & Fairbank, 1985). The association between postcombat social support and lower rates of PTSD was also illustrated in studies exam ining former prisoners of the Korean War (Sutker, Winstead, Galina, & Allain, 1991), World War II (Engdahl, Dikel, Eberly & Blank, 1997; Gold, Engdahl, Eberly, Blake, Pa ge & Frueh, 2000), and veterans of the
7 Persian Gulf War (Ozer, Best, Lipsey & Weiss, 2003). In a 14 year longitudinal study, Vietnam veterans diagnosed with PTSD with higher levels of community involvement were significantly more likely to show a remission of symptoms over time compared to patients who withdrew from society (Koenen, Stellman, Stellman & Sommer, 1998, 2003). Interestingly, this relationship between social interaction and symptom remission implies that social support may contribute to PTSD recovery even after clinical diagnosis. Social support received after traumatic even ts, such as interpersonal violence (Astin, Lawrence & Foy, 1993; Ozer et al, 2003; Perrin, Van Hasselt, Basilio & Hersen 1996), accidents (Perry, Difede, Musgni & Frances, 199 2), natural disasters (Ozer et al, 2003), and sexual abuse (Kimerling & Calhoun, 1994; Ozer et al, 2003) were also significantly correlated with lower rates of PTSD. These findings indica te that post-trauma social support may play a critical role in prot ecting against the deve lopment of PTSD. Although limited, there is some evidence in dicating that social support received during ongoing trauma may be effective in lowering risk for PTSD development. One such study conducted on Israeli-Lebanon war ve terans showed a sign ificantly negative correlation between rates of PTSD diagnosis and cohesiveness within military units during combat (Solomon, Mikulincer & Hobfall, 1987). The authors posited that soldiers who did not form positive social bonds with fellow combatants were more likely to have increased feelings of uncont rollability and helplessness a nd, in turn, develop PTSD, compared to those experiencing supportive relationships within their military units (Solomon et al, 1987). This finding suggests that social support may protect against the onset of PTSD when it is received by indivi duals during situations, such as combat, in
8 which both acute trauma (e.g. w itnessing the mutilati on of bodies) (King et al, 1998), and chronic social stress (Solomon et al, 1987) may be experienced. Limitations of Social St imulation & PTSD Studies Human studies have indicated a strong relationship between social stimulation and lo wer rates of PTSD. Although human research has demonstrated correlations between higher levels of social stimulation and lower rates of PTSD, it has lacked expe rimental evidence suggesting that social factors are effective in protecting against PT SD. In addition, there may also be various sources of error in studies focused on combat veterans stemming from their retrospective nature. In studies examining social phenomen a, for instance, combat veterans are asked to recall events, such as levels of social support they received dur ing combat, or after returning home. Veterans w ho, in some cases, have deve loped a debilitating disease such as PTSD, may not be able to, for inst ance, accurately recall the level of social support they received after re turning home from combat many years prior to assessment by researchers or clinicians. As a result, their responses during a ssessment sessions may not reflect actual experiences and, theref ore, may lead investigators to flawed conclusions. Nonetheless, there is evidence suggesting that social stimulation may exert a profound influence on the brain and, in tu rn, influence the impact of stress on the development of PTSD. Effects of Social Stimulation on the Brain There is accumulating research in the social neuroscience field which has attempted to identify the possible neural unde rpinnings of evidence s uggesting that social
9 stimulation buffers against the detrimental effe cts of stress, and is associated with lower rates of PTSD. Patients with PTSD, in some cases, are withdrawn in nature and fail to seek out others for support (Norris & Kaniasty, 1996). There is some evidence suggesting that this failure to seek support is the result of emotional numbing, which is related to deficits in brain reward circuits. One study revealed that, relative to normal controls, participants diagnosed with PTSD exhibited signifi cantly smaller activation in areas related to reward seeking, such as th e nucleus accumbens in response to social reward stimuli (pictures of a ttractive or pleasant faces). The investigators, utilizing fMRI, also found that PTSD patients exhibited signific ant less activation in th e prefrontal cortex (PFC) compared to healthy controls perf orming the task (Elman, Frederick, Ariely, Dunlap & Rodolico, 2005). This study sugge sts that PTSD may produce deficits in reward circuitry and those who, for instance, are provided with, or seek social stimulation may be less likely to develop such deficits. Additionally, it indicates that PTSD patients may exhibit inhibited executive control ba sed on impaired PFC functioning. Other research has examined specific neurochemi cal changes which may underlie the possible protective nature of social st imulation against PTSD. Speci fically, some investigations have examined the neuropeptides oxytocin (OT) and vasopressin (AVP), which have been identified as essential chemical medi ators of social stimulation, pair bonding, mating, (Charuvastra & Cloitre, 2008; DeVries, Glasper & Detillon, 2003) and may relate the effects of social stimulation on PTSD-rela ted circuitry. Animal studies have shown that a large number of AVP and OT recepto rs are located in the amygdala, which governs emotional responses. Stimulating OT receptors in the amygdala may, in turn, inhibit its
10 activity under situations wh ich may provoke, for example, fear, or anxiety (LeDoux, 2000). Some work has indicated, in fact, that exogenous administration of OT to female rats decreased anxiety-like behaviors. In one human study, male participants administered OT or placebo intranasally were assessed for amygda la activity utilizing fMRI after viewing pictures of threatening, or not threatening fa ces. Findings revealed that OT significantly suppressed amygdala ac tivation relative to the placebo in the threatening face condition (Kirsch, Esslinger, Chen, Mier, Lis et al, 2005). Another study, which specifically examined PTSD pa tients, found that administering OT to Vietnam veterans with the disorder resu lted in a significant decrease in their physiological responses to combat imagery relative to placebo (Pitman, Orr & Lasko, 1993). These findings suggest that enha nced social stimulation may elevate neuropeptides, such as OT, thereby suppres sing the activation of brain areas governing fear and anxiety-related res ponses, and, in turn, physiologica l responses to reminders of the patients trauma. Social stimulati on, therefore, maybe an important nonpharmacological candidate of treating symptoms associated with PTSD. Environmental Enrichment The modern concept of EE is based on early anecdotal evidence by Donald Hebb who, in 1947, noted that rats he kept as domestic pets exhibited superior mazeperformance compared to cohorts housed in sm all cages in his laboratory. He speculated that these animals may have ha d larger brain sizes, as a re sult of increased opportunities to explore the relatively dive rse environment of his home, which contributed to their enhanced performance. Pioneering experiment al work demonstrated that EE, involving
11 exposing groups of animals to complex environments, was indeed effective in significantly increasing neuroanatomical change s in the brain relative to a few animals housed together in smaller, less complex standard cages (Rosen zweig & Bennett, 1984, 1996). Specifically, they found that when r odents were provided with EE, involving placing groups of animals into a large appa ratus containing blocks running wheels, and increased opportunities for soci al interactions compared to standard laboratory housing conditions, they had significantly larger ove rall cortical weights (Bennett, Rosenzweig, Diamond, Moromito & Hebert, 1974; Diamond, Krech & Rosenzweig, 1964; Rosenzweig, Love & Bennett, 1968; Di amond, 2001; Welch, Brown, Welch & Lin, 1974), increased dendritic branching ( Diamond et al, 1964; Diamond, 2001), and increased numbers of synapses (Mollgaar d, Diamond, Bennett & Rosenzweig, 1971;West & Greenough, 1972) in the cerebral cortex, sugge stive of enhanced cognitive abilities compared to animals housed under standard conditions. More recent studies established that EE significantly enhanced hippocampal synaptic plasticity, (Abel & Nguyen, 2001; Artola, Frijtag, Fermont, Gispen & Schram a, 2006; Duffy, Craddock, Ickes et al, 2000; Faherty, Kerley & Smeyne, 2003; Fernandez-Tr uel, Gimenez-Llort, Escorihuela, Gil & Aguilar, 2002; Leggio, Mandolesi, Federico, Spirito & Ricci, 2005; Olsson et al, 1994; Segovia et al, 2008; Sharp, Mc Naughton & Barnes, 1985), neur otrophic factors relating to plasticity (Ickes, Pham, Sanders, Alb eck & Mohammed, 2000; Olsson, Mohammed, Donaldson, Henrikkson & Seckl, 1994; Segovi a, Arco, de Blas, Garrido & Mora, 2008), neurogenesis (Brown, Cooper-Kuhn, Kempermann, Van Praag, Kempermann, & Gage, 2000; Bruel-Jungerman, Laroche & Rampon, 2005; Kempermann, Kuhn & Gage, 1997;
12 Kempermann, Gast & Gage, 2002; Nilsson, Perfilieva, Johansson, Orwar & Eriksson, 1999 ), and reduced hippocampal cell death (Young, Lawlor, Leone, Dragunow & During, 1999) compared to controls, all neurobi ological changes related to increases in cognitive performance. Other work indicat es that EE improves behavioral performance on cognitive tasks (Lee, Hsu & Ma, 2003; Leggi o et al, 2005; Nilsson et al, 1999; Meshi, Drew, Saxe, Ansorge & David, 2006; Schrij ver, Bahr, Weiss & Wurbel, 2002), and reduces anxiety-like behaviors (Brenes, Rodriguez & Fornaguera, 2008; BruelJungerman et al, 2005; Chapillon, Mannech e, Belzung & Caston, 1999; Fernandez-Truel et al, 1997; Friske & Gammie, 2005; Klein, Lambert, Durr, Schaefer & Waring, 1994; Meshi et al, 2006; Teather, Magnusson, Chow & Wurtman, 2002; Widman & Rosellini, 1990; Zimmerman, Stauffacher, Langhans & Wurbel, 2001) compared to standard housing. Experimental evidence also suggests that the beneficial effects of EE on the brain and behavior may play a role in protec ting against the detrimental effects of stress on the brain, behavior, and physiology of organisms. Environmental Enrichment & Stress Although limited, findings suggest that EE is effective in reversing the adverse effects of stress on the brain and behavior of r odents. EE, for instance, has been shown to block increases in behavioral measures of f ear and anxiety in rode nts exposed to shock (Benaroya-Milshtein, Hollander, Apter, Kukulan sky, Raz & Will, 2004). In addition, rats exposed to early-post natal maternal separation (Bredy, Humpartzoomian, Cain & Meaney, 2003) and low maternal care (Y ang, Hou, Liu, Zhang, Zhou, Xu & Li, 2007)
13 exhibited memory impairments which were bl ocked by EE. Exposing animals to EE also protected against stress-induced reductions of synaptic plasticity (Yang et al, 2007), impaired behavioral performance on spatial memory tasks (Larsson, Winblad, & Mohammed, 2002; Wright & Conrad, 2008), and in creased anxiety-like behaviors of rats exposed to predator odor (Roy, Belzung, Delarue & Chapillon, 2001). Utilizing EE to Study PTSD EE may exert its protective efficacy by mitigating stress-induced increases in physiological markers of stress. This hypot hesis has been supported with evidence showing that EE blocked the effect of maternal separation stress on elevated corticosterone (CORT), a main physiological ma rker of stress, levels when rat pups were subjected to acute restraint stress as adol escents (Francis, Diorio, Plotksy & Meaney, 2002), adults (Morley-Fletcher, Rea, Maccari & Laviola, 2003), and when adult mice were exposed to predator odor (Benaroya-Mil shtein et al, 2003; Roy et al, 2001). These findings suggest that EE may render the st ress response system of the brain more adaptive and efficient by reducing hormone leve ls released in response to stress and, in turn, facilitate recovery after exposure to stressful situations (Fox, Merali & Harrison, 2008). As a result, studying the effects of envi ronmental manipulations on the etiology of PTSD-like symptoms may provi de insight into identifying mechanisms responsible for the development of PTSD, and reinforcing th e importance of social and environmental treatments for the disorder.
14 Chapter 2: Experiment Purpose and Hypotheses of Present Study Although extensive research has revealed EE to produce robust changes in the brain and behavior of organisms, the majority of studies have incl uded running wheels in the EE apparatuses. This can result in possibly confound findi ngs in that exercise alone has been shown to exert profound changes related to the learning and memory of animals, such as neurogenesis, long-term potentiation, neurotrophic growth factors and performance on spatial memory tasks (Neep er, Gomez-Pinilla, Choi & Cotman, 1996; van Praag, Kempermann & Gage 1999; van Pr aag, Christie, Sejnowski & Gage, 1999; van Praag, 2008). This experiment addressed the possible confounding influence of exercise on findings in the EE literature by eliminating direct sources of exercise (i.e. running wheels) in the enrichment apparatus. Although it is possible that animals in the current apparatus were more active relative to those housed under standard conditions, they were not provided with a source of dir ect exercise. In doing so, this study focused on addressing the specific influence of daily so cial stimulation (DSS) on behavioral and physiological responses of rats exposed to the chronic psychosocial stress regimen. The present study was also designed to build upon the limited literature examining the interaction between chroni c stress and enrichment in adult rats. Importantly, in contrast to the majority of st udies in this area, this experiment examined
15 the influence of brief, daily stimulation impl emented in an animal model of PTSD which assesses a variety of symptoms associated with the disorder. A dditionally, this study addressed the impact of social complexity on stress effects on behavior and physiology by placing groups of animals together, in cont rast to some EE studies which utilized smaller group sizes (Marashi et al, 2003; Bennett, McRae, Levy & Frick, 2006). This work investigated the possible prof ound influence which non-pharmacological experience-related manipulations can exert on the behavior and physiology of organisms, and how observed changes may relate to th eir brain function. Th is study tested the hypothesis that 1) rats subj ected to our laboratorys pr eviously developed chronic psychosocial stress regimen (Zoladz et al, 2008 ) would demonstrate robust increases in behavioral and physiological ch anges analogous to those seen in patients clinically diagnosed with PTSD, and 2) that providing ra ts with DSS starting th e day after an acute stress experience would signi ficantly ameliorate these PTSD-like changes relative to animals kept exclusively under standard housing conditions. Methods Animals and Housing Conditions Adult male Sprague-Dawley rats (225-250g) arrived from Charles River laboratories and were housed in pairs on a 12:12 h light-dark schedule (lights on at 0700h) in Plexiglass cages (46 x 25 x 21 cm) with free access to food (Harlan Teklad Global 18% Protein Rodent Diet ; Harlan Laboratories; Indianapolis, IN) and water. Rats
16 were given one week to acclimate to the an imal housing room before any experimental manipulations took place. Power Analysis: Sample Size Estimation Given an accepted level of power (1 = .80), a relatively large effect size ( =.60), and an estimated 60 degrees of fr eedom for the mean square error, the appropriate sample size per group can be estimated as follows: n = / for 1 = .80 and 60 df, = 1.65 n = (1.65) / (.60) n = 2.7225 / .36 n = 7.5625 Previous work in our laboratory has shown sample sizes of 8 to be sufficient in reasonably interpreting data by re ducing the probability of Type I and II errors (Zoladz et al, 2008). A sample size of 10 per group, therefore, was thought to be more than sufficient for this study. Design This study will be designed to examine th e effects of chronic psychosocial stress and SES on rat behavior and physiology utilizing a 2x2 factorial design with psychosocial stress (psychosoc ial stress, no psychosocial st ress) and environment (home cage, SES) as the between subjects factors.
17 Psychosocial Stress Procedure Animals underwent stress manipulations si milar to those employed previously by our group which have produced robust enhancem ents of fear and a nxiety-like behaviors, memory impairments, as well as several physiological changes (Halonen, Zoladz, & Diamond, 2006 ; Zoladz et al, 2008). Rats we re randomly assigned to psychosocial stress or no psychosocial stress groups. Rats in the psychosocial stress groups were exposed to two acute stress sessions lasting one hour each. The first session took place during the light cycle (between 0800 and 1500 h) afte r the one week housing room acclimation period. The second acute stress session occurr ed 10 days later during the dark cycle (between 1900 and 0200 h) based on research indicating the enhancement of synaptic activity in the amygdala 10 days after restraint stress, indica tive of enhanced fear based processing (Mitra, Jadhav, McEwen, Vyas & Chattarji, 2005; Vyas, Mitra, Shankaranarayana, & Chattarji, 2002 ). The second stress session, in theory, served to reinforce any stress-induced changes in the br ain and behavior ini tiated during the first session. During each acute stress session, rats were placed in a box for 3 minutes. The box (25.5 x 30 x 20 cm; Coulbourn Instruments; A llentown, PA) consists of 2 aluminum sides, an aluminum ceiling, and a Plexiglass front and back. The floor of the chamber consists of 18 stainless steel rods, spaced 1.25 cm apart. During the last 30 sec of the exposure, a 30sec tone wa s played (74dB @ 2400 Hz) Immediately following box exposure, rats were immobilized in Decapic ones (Braintree Scientific; Braintree, MA)
18 and transported to a different room where th ey were individually placed in triangularshaped wedges within a circular Plexiglass pie enclosure (20 x 20 x 8 cm; Braintree Scientific; Braintree, MA) located inside of a large metal cage (61 x 53 x 51 cm). An adult female cat was placed on top of the pie enclosure and cat food was smeared on top of the enclosure to direct cat activity toward s the rats. The door to the metal cage was then securely closed. Rats were subjected to only non-tactile cues of the cat, as the pie enclosure prevents any physical contact between the two animals. As with the psychosocial stress groups, ra ts in the no psycho social stress groups were brought to the laboratory on two occasi ons separated by 10 days and placed in the box for 3 minutes, after which they were plac ed back in their home cages where they remained for one hour. In addition to the two cat exposures, rats in the psychosocial stress groups were subjected to unstable housi ng conditions (i.e. social instability) where, on each day, rats were pseudo-randomly paired with a different cage mate from the previous day. Social instabil ity manipulations started on the day of the first cat exposure, and continued daily until the in itiation of behavioral /physiological testing. Rats in the no psychosocial stress groups were housed with the same cage cohort for the duration of the experiment. Daily Social Stimulation Exposures Animals in the psychosocial stress groups were randomly assigned to one of two SES conditions (home cage or DSS). The DSS apparatus (91.44 x 63.50 x 157.48 cm; Ferret Nation; Muncie, IN) consists of thr ee interconnected levels containing plastic
19 platforms, tunnels, metal ladders, two cloth hammocks, and a climbing rope. Animals in the DSS groups were housed under standard conditions until the day after the first stress session. Twenty-four hours after the first cat exposure, this group of animals (N=10) was transported from the housing room to the labo ratory and placed into the DSS apparatus for 2 hours. These daily 2 hour exposures co ntinued until the first day of testing. Animals in the home cage groups were housed under standard conditions throughout the experiment and did not receive any exposures to the DSS a pparatus. Between sessions, all objects and platforms were removed from the DSS apparatus and cleaned with soap and tap water. All procedures adhered to the University of South Floridas ethical guidelines on the treatment of animals in re search and the University of South Florida IACUC regulations. Behavioral Testing Three weeks after the second stress session, animals were subjected to a battery of behavioral tests. Prior to the start of testing, all animals were handled for three consecutive days (2 min each). Body weights were recorded on the day of the first stress session and on the first day of testing. On each behavioral testing day, rats were transported from the housing room into the laboratory and remained in their home cages for 30 minutes in order to acclimate them to the surroundings. Fear Memory. On the first day of behavioral testing, rats were assessed for their memory of the specific context and cue temporal ly associated with the two cat exposures. Memory was behaviorally measured by asse ssing the percentage of time rats spent
20 immobile (typical fear responses in rats ) upon exposure to the c ontext and cue which were paired with the two cat exposures. To assess contextual fear memory, rats were placed in the box previously temporally paired with the two cat exposures, and remained there for 5 minutes. Approximately one hour late r, rats were tested for their memory of the cue (tone) presented in the box prior to each cat exposure. During cue testing, rats were placed in a different box (25 x 22.5 x 33 cm; Coulbourn Instruments; Allentown, PA) than the one used during contextual testing. It consists of two aluminum sides, an aluminum ceiling, and a clear Plexiglas front an d back with the shuttle door in the closed position. The floor consists of a metal plate (21.5 x 21.5 cm) to eliminate the sensation of stainless steel rods beneath their paws. Thes e conditions serve to reduce any similarities between the training context and the cue testing chamber. The cue testing period commenced with 3 three minutes without an auditory stimulus (tone), followed by 3 minutes with tone (74dB @ 2400 Hz). Tone s were presented through a speaker located on one of the sides of the shuttle box, and a hous e light was turned on inside the chamber. This procedure provided a behavioral measur e in a novel context (p re-tone period) and a more direct measure of cue-based memory (tone period). Immobility behaviors are monitored by a 24-cell infrared activity m onitor (Coulbourn Instruments; Allentown, PA) mounted on the top of the boxes which uses emitted infrared body heat images (1300 nm) from the animals to detect movement. Immobility was defined as periods of inactivity lasting 5 sec. A Microsoft Excel spreadsh eet with a macro designed to analyze immobility behavior calculated the total num ber of seconds spent immobile by each animal in 30 second epochs. This time divided by the total amount of time in the chamber
21 yielded a percentage of time immobile for each animal, thus providing behavioral indices of both context and cue-based memories. Elevated Plus Maze (EPM) The EPM has been extensively used in testing for anxiety in rodents (Korte & De Boer 2003). Twenty-four hou rs after the context and cue tests, all rats were transported to a room w ithin the laboratory and tested for anxiety-like behaviors on the EPM. The EPM (Hamilton-Ki nder; San Diego, CA.) consists of two (10.80 x 51.17 cm) open and two closed (10.80 x 51.17 cm) arms which intersect to form the shape of a plus sign. The intersection area is 10.80 by 10.80 cm, and the walls of the closed arms are 40.01 cm in height. E ach rat was placed on th e EPM apparatus for a period of 5 minutes where 48 infrared photo-beams (located along the perimeter of the open and closed arms) connected to a com puter program (Motor Monitor, HamiltonKinder, San Diego, CA) scored its behavior. The computer program monitors various types of behaviors for further assessment such as movement, total time spent in each area of the apparatus, and head dips exhibited ove r the edges of the appa ratus. Anxiety-like behaviors were assessed by measuring times spent in certain areas of the EPM. Rats which spend more time in the open arms than the closed arms are considered to be less anxious as this exposes them to open view and, in theory, possible threat. A primary dependent measure of interest, therefore, was th e percentage of total time rats spent in the open arms of the EPM. Another measure of ex ploratory, or risk-taki ng behavior, the total number of head dips (scored by the comput er program each time rats heads cross photobeam sensors along the edges and ends of the open arms) was another main focus of
22 analysis. Fecal boli was removed from th e EPM between each ten minute session and cleaned using a 25 % ethanol solution to reduce odor buildup in the apparatus. Startle Response. Approximately one hour after the EPM assessment, all rats were administered an acoustic startle response te st. These startle responses were measured using a large startle monitor cabinet (H amilton-Kinder; San Diego, CA; 36 x 28 x 50 cm). Each rat was placed inside a small Plexiglas box (19 x 10 x 10 cm) which was located inside the larger cabinet. At the be ginning of each trial, rats were placed on a sensory transducer, contained within the small Pl exiglas box, which was be connected to a computer program (Startle Monitor; Ha milton-Kinder; San Diego, CA). The program records startle responses by measuring the maximum amount of force (in Newtons) that rats exert on the sensory tran sducer for a period of 250 ms after the presentation of each auditory stimulus. Differences in body we ight were controlled for by adjusting the sensitivity settings on the sensory transducer (a range of 0-7 arbitrary units) prior to each trial. Each startle trial began with a 5 min acclimation period, followed by the presentation of 24 bursts of white noise (50 ms each) consisting of 8 bursts at 3 different auditory intensities (90, 100, 110 dB) presented in sequentia l order. The time between each noise burst was pseudorandomly varied between 25 and 55 seconds. After the start of the initial noise burst, the startle appara tus provided an uninterrupted background noise of 57 dB. Each session lasted approximately 20 minutes. Novel Object Recognition Twenty-four hours after acoustic startle response testing, rats were transported back in the laboratory where they were placed in an open field. The apparatus consists of a plastic box with black walls and an open top
23 (Hamilton-Kinder; San Diego, CA; 40 x 47 x 70 cm). The rats spent 5 minutes in the open field to habituate them to this environm ent prior to training and testing periods. All behaviors were monitored by a video feed to a computer program (Any Maze; Stoelting; Wood Dale, IL). The computer program enable s the assessment of behaviors exhibited in the open field such as total distance traveled in each area (center and perimeter), total time spent in each area, and en tries into each quadrant of th e apparatus, which provide a source of assessment for the general behavior of the rats. Twenty-four hours after habituation to the open field, rats were pl aced in the same open field containing two identical (plastic/metal) objects for 5 minutes (training phase). The objects were placed in opposite, diagonally oriented corners of the open field and secured to the flooring with tape to prevent rats from manipulating and possibly disp lacing them. The objects and their locations were counterbalanced across rats to control for place or object preferences. The testing phase commenced th ree hours later in which rats were placed back in the open field, and one of the objects used duri ng training was replaced by a novel object. A 16 cm 2 zone was specified around each object to assess the time spent with the each object, and was measured by the computer pr ogram by tracking the rats head movements in relation to the location of the object. During testing, greater time spent by rats in proximity to the novel versus familiar object was taken as an indication of intact memory for the familiar object. Physiological Testing
24 Blood Sampling and Cardiovascular Measurements. Physiological testing commenced twenty-four hours after the last day of behavioral testi ng. On this day, blood samples and heart rate (HR)/ blood pressure (BP) measurements were collected and recorded from all rats in order to assess corticosterone (CORT), a main physiological marker of stress, and cardiovascular responses respectively. For base line and stress-level blood sample collections, petroleum jelly was sp read across the saphenou s veins of rats to facilitate vein access from which blood was colle cted within a 2 minute time interval via venipuncture. For baseline blood sampling, rats were transported from the housing room to an adjacent procedure room in which a single blood sample was collected after which they were restrained in plastic Decapicone s (Braintree Scientific; Braintree, MA) and transported to the laboratory. Twenty minut es later, rats were removed from the restrainers, and another blood sample was collected to examine their CORT response to restraint stress. For HR/BP measurements, ra ts were placed in a Plexiglass tube (IITC Life Science; Woodland Hills, CA) within a warming test chamber (approximately 32 degrees Celsius) which serves to facilitate bl ood flow to the tail. Th is enables HR and BP measurements to be taken using tail cuffs fitted with photoelectric sensors (IITC Life Science; Woodland Hills, CA). Rats were th en returned to their home cages for one hour, after which a sample of trunk blood was colle cted via rapid consci ous decapitation to determine post-stress CORT levels. Following the trunk blood sample, adrenal glands, thymus glands, kidneys, and hearts were ha rvested and weighed for further analysis. After all blood collections cl otted at room temperature, they were centrifuged (3000 rpm
25 for 8 minutes) after which serum was extracted and stored at -80 C until it was shipped for assay. Statistical Analyses Experimental Design and General Analyses The current study utilized a betweensubjects, 2 x 2 factorial design. The inde pendent variables were psychosocial stress (psychosocial stress, no psychosocial stress) and environment (home cage, DSS). The majority of data were analyzed utiliz ing between-subjects, two-way ANOVAs with psychosocial stress and environment serving as the between subjects factors. Planned comparisons (independent samples t -tests) were also conducted between groups that were predicted to differ a priori based on previous findings (Seetharaman, Zoladz & Diamond, 2008; Zoladz et al, 2008). For all statistical analyses, alpha was set at .05, and LSD post hoc tests were employed when applicable. Fear Memory. Contextual and cue-based fear memory tests were analyzed separately. Contextual fear memory, as m easured by immobility, was analyzed utilizing two-way, between subjects ANOVAs. The num ber of fecal boli pr oduced by rats during both context and cue tests were also an alyzed separately. For each assessment, psychosocial stress and environment served as the between subjects factors. For analysis of the cue fear test, a three-way mixed m odel ANOVA was utilized with tone (before tone, during tone) serving as the within subjects factor, and psychosocial stress, and environment serving as the between subjects factors.
26 Elevated Plus Maze. The amount of time rats spent in the open arms of the EPM was calculated as a percent of the total 5 minute trial time (i.e. percent time spent in the open arms). To further examine anxiety-like behaviors in rats, the percent time that animals spent in specific areas of the open arms on the EPM was examined. The percent time spent in both the near open arms (close to the intersection leading to the closed arms) and far open arms (further away from the intersection leading to the closed arms) were analyzed to further examine rats anxiety-like behavior on the EPM. Mean head dips, which provide an index of exploratory-like behavior were also analyzed. The percent time rats spent in the closed arms of the EPM was assessed in order to complete the overall analysis of behaviors relati ng to anxiety, exhibited by animals on the apparatus. Additionally, motor movement was assessed by examining overall ambulations made on the EPM, as well as the velocity of animals in the open and closed arms of the apparatus. All measures were a ssessed separately utilizing between-subjects, two-way ANOVAs with psychosocial stress an d environment serving as the between subjects factors. Startle Response. Startle responses for each of the three auditory stimulus intensities (90, 100, and 110 dB) were anal yzed separately. For each assessment, between-subjects, two-way ANOVAs were employed to analyze the data, with psychosocial stress and environment serving as the between-subjects factors. Novel Object Recognition. Single ratio time values were calculated for each rat by dividing the time spent with the novel object by the time they spent with the familiar object (ratio time = time spent with novel objec t / time spent with familiar object) for the
27 entire 5 minute testing trial, as well as th e first minute. These data, obtained from behavior measured in the open field were subjected to betweensubjects two-way ANOVAs with psychosocial Stress and environment serving as th e between-subjects variables. Heart Rate and Blood Pressure. S eparate between-subj ects, two-way ANOVAs were utilized to examine differences in HR and BP, where psychosocial stress and environment served as the be tween-subjects variables. Growth Rate. Growth rate was calculated by di viding rats total weight gained during the course of the study by the number of total days in the experiment (i.e. 31 days). A two-way, between subjects ANO VA was employed to examine differences between groups, with psychosocial stress and environment serving as the between subjects factors. Organ Weights. Adrenal, thymus, heart, and kidn ey weights were harvested from all rats on the last day of te sting. All organs were weighed and expressed as milligrams per 100 grams of body weight (mg/100g b.w.) A two-way, between subjects ANOVA was employed to examine differences in body weights with psychosocial stress and EE serving as the between subjects factors. Results Fear Memory Context Test Immobility. For the analysis of imm obility during the 5 minute context test, there were no significant main effects of either psychosocial stress, F (1,31) =
.129, or environment, F (1,31) = .292, and no significant psychosocial stress x environment interaction, F (1, 31) = .194 ( p values > .05) indicating no significant between-groups differences in immobility to the context associated with the cat exposures (see Figure 1). 28 % Time Immobile 0 10 20 30 40 50 Effects of Psychosocial Stress and Environment on Immobility to the Cat-Associated Context DSS Stress DSS No Stress Home Cage No Stress Home Cage Stress Figure 1. Effects of psychosocial stress and environment on immobility during the context test. The data are presented as mean percent time immobile SEM. Context Test Fecal Boli. As depicted in Figure 2, the analysis of fecal boli produced by rats during the context test revealed no signifi cant main effects of either
psychosocial stress, F (1,35) = .253, or environment, F( 1,35) = .999, and no significant psychosocial stress x environment interaction, F (1,35) = .999 ( p values > .05). Effects of Psychosocial Stress and Environment on Boli Produced During the Context Test 29 Boli 0 1 2 3 4 5 6 Home Cage No Stress Home Cage Stress DSS Stress DSS No Stress Figure 2. Effects of psychosocial stress and environment on fecal boli produced during the contextual fear test. Data are presented as mean boli produced SEM. Cue Test Immobility For the analysis of immobility during the cue fear test, there was no significant main effect of tone, F (1,28) = 1.81, p >.05. There were also no significant main effects of either psychosocial stress, F (1,28) = 2.77, or environment,
30 F (1,28) = 3.00 ( p values > .05). Analysis did reveal a significant tone x psychosocial stress interaction, F (1,28) = 5.49, p < .05, as well as a significant tone x environment interaction, F (1,28) = 5.32 ( p values < .05). There was no significant three-way tone x psychosocial stress x environment interaction, F (1,28) = 2.50, p > .05. Post-hoc analyses revealed that immobility exhibited by the home cage psychosocial stress group during the tone was significantly greater than before th e tone. Also, these animals were significantly more fearful to the tone than all other groups, indicated by significantly higher immobility levels relative to them. Additionally the data indicated that this significant stress-induced increase in immobility to the tone was prevented with DSS, as shown by the DSS-psychosocial stress group exhibiting si gnificantly less immobility compared to the home cage-psychosocial stress group during th e tone. These behavioral data suggest that, in contrast to all ot her groups, the home cage psychosocial stress animals were more fearful when re-exposed to the salient cue (t one), which was temporally associated with the cat sessions, compared to before the t one. The finding that ther e were no significant differences in immobility before and during the tone in the DSS-psychosocial stress group suggests that DSS may have ameliorate d fear-responses produced by the specific cue temporally associated with cat exposures, as was shown by the home cagepsychosocial stress group. This is further ev idenced by data indicati ng that, relative to non-psychosocially stressed controls, th e home cage-psychosocial stress group spent significantly greater time in immobility during the tone, and that this behavioral increase in fear-related memory was prevented with DSS (see Figure 3).
% Time Immobile 0 5 10 15 20 25 30 Before Tone During Tone Home Cage No Stress Home Cage Stress DSS Stress DSS No Stress Effects of Psychosocial Stress and Environment on Immobilit y Durin g the Cue Fear Test Figure 3. Effects of psychosocial stress and environment on immobility before and during the tone in the cue fear test. Data are presented as mean percent time immobile SEM. = p < .05 relative to all other groups; = p < .05 relative to within group immobility before tone Cue Test Fecal Boli. The analysis of fecal boli produced during the 6 minute cue test revealed a significant main effect of psychosocial stress, F (1,29) = 33.55, p < .05, but not of environment, F (1,29) = 1.19, p > .05. Results also indicated a significant 31
psychosocial stress x environment interaction, F (1,29) = 20.03, p <.05. Post hoc tests showed that animals in the home cage-psyc hosocial stress group produced significantly more boli compared to all other groups. Also, this stress-induced increase in boli was prevented with DSS, as evidenced by a significantly lower defecation level in the DSSpsychosocial stress group compared to home cag eno psychosocial st ress controls (see Figure 4). Boli 0 1 2 3 4 5 6 Home Cage No Stress Home Cage Stress DSS Stress DSS No Stress *Effects of Psychosocial Stress and Environment on Boli Produced During the Cue Fear Test Figure 4. Effects of psychosocial stress and environment on fecal boli produced during the cue fear test. Data are presente d as mean boli produced SEM. = p < .05 relative to home cage-no psychosocial stress group; = p < .05 relative to home cagepsychosocial stress group. 32
Elevated Plus Maze Percent Time in Open Arms. The analysis of percent time spent in the open arms of the apparatus revealed a significant main effect of psychosocial stress, F (1,28) = 5.86, environment, F (1,28) = 33.01 ( p values < .05), and no signifi cant psychosocial stress x environment interaction, F (1,28) = 1.34, p > .05. Planned comparisons indicated that animals in the home cagepsychosocial stress group spent significantly less percentage of time in the open arms compared to the no psychosocial stress controls, t (12) = 2.33, p < .05 and that the stress-indu ced increase in anxiety-like behavior was prevented with DSS, t (14) = -4.84, p < .05 (see Figure 5). % Time in Open Arms 0 10 20 30 40 50 60 70 80 90 100 Home Cage No Stress Home Cage Stress DSS Stress DSS No Stress Effects of Psychosocial Stress and Environment on Percent Time in the Op en Arms on the EPM Figure 5. Effects of psychosocial stress and envi ronment on percent time spent in the open arms of the elevated plus maze. Data are presented as mean percent time in open 33
34 arms SEM. = p <.05 relative to all other groups. = p < .05 relative to home cageno psychosocial stress group. Percent Time in Closed Arms. As shown in Figure 6, the analysis of percent time spent in the closed arms revealed signifi cant main effects of bot h psychosocial stress, F (1,30) = 9.23, and environment, F (1,30) = 29.34 ( p values < .05). There was no significant psychosocial stress x environment interaction, F (1,30) = 2.92, p > .05. A planned comparison showed that the hom e cage-psychosocial stress group spent a significantly greater percentage of time in the closed arms compared to the no psychosocial stresshome cage control group, t (12) = -3.01. In addition, this significant increase was prevented with DSS, as shown by the DSS-psychosocial stress group spending significantly less percentage of time in the closed arms compared to the home cage-psychosocial stress group, t (15) = 4.99 ( p values < .05).
% Time in Closed Arms 0 40 60 80 100 DSS Stress DSS No Stress Home Cage No Stress Home Cage Stress *Effects of Psychosocial Stress and Environment on Percent Time in the Closed Arms on the EPM Figure 6. Effects of psychosocial stress and envi ronment on percent time spent in the closed arms on the EPM. Data are presented as mean percent time in closed arms SEM. = p < .05 relative to all other groups; = p < .05 relative to ho me cageno psychosocial stress. Movement Results assessing movement on th e EPM revealed no significant main effect of psychosocial stress, F (1,33) = .722, p > .05. There was, however, a significant main effect of environment, F (1,33) = 20.71, p < .05, where both DSS groups exhibited significantly more ambulati ons, suggestive of increased motor activity on the EPM relative to controls. There was no significant psychosocial stress x environment interaction, F (1,33) = 1.64, p < .05. These findings suggest that DSS produced more movement relative to co ntrols (see Figure 7). 35
Movement (ambulations) 0 150 200 250 300 350 Home Cage No Stress Home Cage Stress DSS Stress DSS No Stress Effects of Psychosocial Stress and Environment on Movement on the EPM Figure 7. Effects of psychosocial stress and e nvironment on ambulation made on the EPM. Data are presented as mean percent time in closed arms SEM. = p < .05 relative to home cage-no psychosocial stress; = p < .05 relative to home cage psychosocial stress. Velocity. In order to further investigate movement on the EPM, velocity calculations for each rat were made by divi ding the total time sp ent in the open and closed arms by respective distances travelled. A one-way ANOVA was employed to analyze group differences on behavior on th e open versus closed arms. Results did showed an overall significant ANOVA, F (7,68) = .015,but no significant within group differences, indicating that groups did not travel at significan tly different rates when in 36
the open and closed arms. Findings did, how ever, indicate that both the home cagepsychosocial stress, and DSS-no psychosocial stress groups exhibited greater velocities on the open arms of the EPM re lative to home cage controls. In addition, DSS seemed to increase velocity in the closed arms compared to home cageno psychosocial stress controls (see Figure 8) Velocity (cm / sec) 0 2 4 6 8 10 12 14 16 Open Arms Closed Arms Effects of Psychosocial Stress and Environment on Velocity on the EPM Home Cage No Stress Home Cage Stress DSS Stress DSS No Stress Figure 8. Effects of psychosocial stress and envi ronment on velocity in the open and closed arms on the EPM. Data are presented as mean velocity SEM. = p < .05 relative to home cage-no psychosocial stress, open arms; = p < .05 relative to home cage psychosocial stress, closed arms 37
Head Dips Analysis of head dips exhibited during the entire 5 minute test on the apparatus revealed no significant main effect of psychosocial stress, F (1,30) = .002, p <.05, a significant main effect of environment, F (1,30) = 35.33, but no significant psychosocial stress x environment interaction, F (1,30) = .374 ( p values > .05). These results indicate that both DSS groups exhibi ted significantly greater head dips on the EPM compared to the home cage groups. Thes e behaviors provide another index which suggests that animals given DSS exposures were more exploratory than those in the home cage conditions (see Figure 9). Effects of Psychosocial Stress and Environment on Head Di p s on the EPM Head Dips 0 5 10 15 20 Home Cage No Stress Home Cage Stress DSS Stress DSS No Stress 38
39 Figure 9. Effects of psychosocial stress and environment on head dips made on the EPM. Data are presented as mean head dips SEM. = p < .05 relative to home cageno psychosocial stress controls. = p < .05 relative to home cag epsychosocial stress group. Startle Response 90dB Acoustic Stimuli. At the 90dB stimulus level of the startle response test, there were no significant main effects of psychosocial stress, F (1,33) = .914, environment, F (1,33) = .790, and no significant psyc hosocial stress x environment interaction, F (1,33) = .843 ( p values > .05). 100dB Acoustic Stimuli For the 100 dB stimulus analysis, there was no significant main effect of stress, F (1,30) = .220, a significant main effect of housing, F (1,30) = .025, and no significant psychosoc ial stress x environment interaction, F (1,30) = 5.58 ( p values > .05). 110dB Acoustic Stimuli Analysis of the 110 dB aud itory stimulus of the startle response test revealed no significant main effect of psychosocial stress, F (1,28) = 4.57, a significant main effect of environment, F (1,28) = 3.61, p <.05, and a significant psychosocial stress x environment interaction, F (1,28) = 12.76 ( p values < .05). Post hoc tests revealed that animal s in the home cagepsychosocial stress group exhibited significantly greater startle re sponses relative to both home cage groups, but not the DSSno psychosocial stress group. These data indica te that the psychosocial stress-induced increase in startle response was prevented with DSS, evidenced by animals in the DSS-
psychosocial stress group exhibi ting significantly lower startle responses compared to the home cagepsychosocial st ress group (see Figure 10). Auditory Stimulus Intensity 90 dB100 dB110 dBStartle Response (Newtons) 0.0 0.5 1.0 1.5 2.0 2.5 3.0 Home Cage-No Stress Home Cage-Stress EE-No Stress EE-Stress Effects of Psychosocial Stress and Environment on Startle Response Figure 10. Effects of psychosocial st ress and environment on star tle response. Data are presented as the mean startle response (N ewtons) to the 90, 100, and 110 dB acoustic stimuli SEM. = p < .05 relative to all other groups at 110 dB stimulus intensity. Novel Object Recognition 40
Ratio Time5 Minute Test. Statistical analysis of memory measured by a ratio time revealed no significant main effects of either psychosocial stress, F (1,25) = .016 or environment, F (1,25) = .120, and the psychosocial stress x environment interaction was also not significant, F (1,25) = .504 ( p values > .05) indicating no significant differences with regards to the amount of time spent with the novel and fam iliar objects between groups during the 5 minute test (see Figure 11). Ratio Time 0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 2.0 Home Cage No Stress Home Cage Stress DSS Stress DSS No Stress Effects of Psychosocial Stress and Environment on Novel Object Recognition -5 Minute Test Figure 11. Effects of psychosocial stress and en vironment on memory, measured by a recognition index in the NOR task. Data are pr esented as the mean ratio time SEM. Ratio TimeFirst Minute of Test Similar to the 5 minute test results, analysis of ratio time during the first minute of the testi ng period also revealed no significant main 41
effects of either psychosocial stress, F (1,25) = .190 or environment, F (1,25) = .015, and no significant psychosocial stre ss x environment interaction, F (1,25) = 1.35 ( p values > .05) (see Figure 12). Ratio Time 0.0 0.5 1.0 1.5 2.0 2.5 Effects of Psychosocial Stress and Environment on Novel Object Recognition -First Minute of Test DSS Stress DSS No Stress Home Cage No Stress Home Cage Stress Figure 12. Effects of psychosocial stress and en vironment on memory, measured by ratio time in the NOR task during the first minute of the test. Data are presented as mean ratio time SEM. Growth Rate 42
The analysis of growth rate revealed no significant main effects of psychosocial stress, F (1,33) = .948 or environment, F (1,33) = .850 ( p values > .05), but there was a significant psychosocial stress x environment interaction, F (1,33) = 9.45, p < .05. Post hoc analyses of the data show ed a significantly lower grow th rate of the home cagepsychosocial stress group compared to nonps ychosocially stressed controls. Also, this stress-induced reduction in growth rate was prevented with DSS. This was indicated by the DSS psychosocial stress group demonstrat ing a significantly higher growth rate compared to their home cagepsychosocially stressed controls. These data indicated that DSS blocked chronic stress-induced reductions in growth rate. Growth Rate 0.0 4.0 4.5 5.0 5.5 6.0 DSS Stress DSS No Stress Home Cage No Stress Home Cage Stress Effects of Psychosocial Stress and Environment on Growth Rate 43
Figure 13. Effects of psychosocial st ress and environment on growth rate. Data are presented as mean growth rate SEM. = p < .05 relative to home cageno psychosocial stress controls. = p < .05 relative to home cagepsychosocial stress group. Cardiovascular Testing Heart Rate. Analysis of between group effects on heart rate revealed no significant main effects of psychosocial stress, F (1,21) = 1.25, environment, F (1,21) = .390, and no significant psychosocial st ress x environment interaction, F (1,21) = .082 ( p values > .05) indicating no si gnificant differences between groups on heart rate (see Figure 14). 44 Heart Rate (bpm) 0 100 200 300 400 500 DSS Stress DSS No Stress Home Cage No Stress Home Cage Stress Effects of Psychosocial Stress and Environment on Heart Rate
Figure 14. Effects of psychosocial stress and en vironment on heart rate. Data are expressed as mean heart rate (bpm) SEM. Systolic Blood Pressure Systolic blood pressure level analysis produced no significant main effects of psychosocial stress, F (1,21) = .693, environment, F (1,21) = .045, and no significant psychosocial st ress x environment interaction, F (1,21) = .000 ( p values > .05) indicating no si gnificant differences between groups on systolic blood pressure (see Figure 15). 45 Systolic Blood Pressure (mmHG) 0 20 40 60 80 100 120 140 DSS Stress DSS No Stress Home Cage No Stress Home Cage Stress Effects of Psychosocial Stress and Environment on Systolic Blood Pressure
Figure 15. Effects of psychosocial stress and en vironment on systolic blood pressure. Data are presented as mean blood pressure (mmHg). Diastolic Blood Pressure. For the analysis of diastolic blood pressure, there was no significant main effects of psychosocial stress, F (1,21) = .044, environment, F (1,21) = .141, and no significant psychosocial stress x environment interaction, F (1,21) = .427 ( p values > .05) indicating no significant differences betw een groups on diastolic blood pressure (see Figure 16). 46 Figure 16. Effects of psychosocial stress and en vironment on diastolic blood pressure. Data are presented as mean blood pressure (mmHg). Diastolic Blood Pressure (mmHG) 0 20 40 60 80 100 120 140 DSS Stress DSS No Stress Home Cage No Stress Home Cage Stress Effects of Psychosocial Stress and Environment on Diastolic Blood Pressure
47 Organ Weights Adrenal Gland. Analysis of adrenal weights (see Figure 17), expressed as milligram per 100g body weight, revealed a ma rginally significant main effect of psychosocial stress, F (1,31) = 3.68, p = .07, a significant main effect of environment, F (1,31) = 5.95, p < .05, indicating that the DSS groups had significantly larger adrenal glands than animals in the home cage groups, Th is result confirms previous findings that stimulating environments produces larger ad renals (Bakos, Duncko, Makatsori, Pirnik, Kiss & Jezova, 2006) compared to animals hous ed under standard conditions. There was no significant psychosocial stre ss x environment interaction, F (1,31) = 2.91, p > .05. A planned comparison performed based on predictions made a priori revealed that the home cagepsychosocial stress group had signi ficantly heavier adrenal glands compared to the home cageno psychosocial stress control group, t (14) = -4.13, p < .01, confirming previous findings of work by our group wh ich utilized a similar chronic psychosocial stress paradigm (Zoladz et al, 2008).
Effects of Psychosocial Stress and Environment on Adrenal Gland Wei g hts Adrenal Weight (mg/100g body weight) 0 8 9 10 11 12 13 14 DSS Stress DSS No Stress Home Cage No Stress Home Cage Stress Figure 17. Effects of psychosocial stress and en vironment on adrenal gland weights. Data are presented as mean adrena l weight (mg / 100g body weight). = p < .05 relative to home cageno psychosocial stress group (planned comparison). = p < .05 relative to home cage-no psychosocial stress controls (ANOVA main effect of environment). Thymus Gland. For the analysis of thymus gl ands (see Figure 18), there were significant main effects of psychosocial stress, F (1,28) = 5.96, and environment, F (1,28) = 7.54 ( p values < .05). There was no signifi cant psychosocial stress x environment interaction, F (1,28) = 1.03, p > .05. Interestingly, these data indicated that both stress 48
and DSS produced smaller thymus glands co mpared to no stress-home cage controls. A planned comparison also revealed that the home cagepsychosocial stress group had lower thymus weights compared to their no psychosocial stress control animals, t (14) = 2.39, p < .05, confirming previous findings of wo rk by our group which utilized a similar chronic psychosocial stress pa radigm (Zoladz et al, 2008). 49 Figure 18. Effects of psychosocial st ress and environment on thym us gland weight. Data are presented as mean thymus weight (mg / 100g body weight) SEM. = p < .05 relative to home cageno psychosocia l stress group (planned comparison). = p < .05 relative to home cageno psychosocia l stress controls (ANOVA main effect) Thymus Weight (mg/100g body weight) 0 80 90 100 110 120 DSS Stress DSS No Stress Home Cage No Stress Home Cage Stress Effects of Psychosocial Stress and Environment on Thymus Gland Weight
Kidney. Analysis of between group effect s of kidney weights revealed no significant main effects of either psychosocial stress, F (1,33) = .646, environment, F (1,33) = .086, and no significant psychosoc ial stress x environment interaction, F (1,33) = .398 ( p values > .05) (see Figure 19). Kidney Weight (mg/100g body weight) 0 100 200 300 400 500 600 700 Home Cage No Stress Home Cage Stress DSS Stress DSS No Stress Effects of Psychosocial Stress and Environment on Kidne y Wei g hts Figure 19. Effects of psychosocial st ress and environment on kidney weights. Data are expressed as mean kidney weight (mg / 100g body weight). 50
Heart For the analysis of heart weights, there was no significant main effect of psychosocial stress, F (1,32) = .989, p > .05. There was, however, a significant main effect of environment indicating that, co mpared to home cage-no psychosocial stress controls, animals in the DSS groups had significantly heavier hearts, F (1,32) = 4.79, p < .05. There was no significant psychosocial stress x environment interaction, F (1,32) = 1.43, p > .05 (see Figure 20). Heart Weight (mg/100g body weight) 0 250 275 300 325 Home Cage No Stress DSS Stress DSS No Stress Home Cage Stress Effects of Psychosocial Stress and Environment on Heart Weight Figure 20. Effects of psychosocial stress and en vironment on heart weight. Data are expressed as mean heart we ight (mg / 100g body weight). = p < .05 relative to home cageno psychosocial stress control group. 51
52 A summary of the above results is show n in the table below. The table includes initial hypotheses of each behavi oral and physiological measure, as well as the actual outcome on those measures at testing. An indi cation as to whether initial hypotheses were satisfied or not with regards to each measure is also given. Table 1: Measures, Hypotheses, and Outcomes Behavioral/ Physiological Measure Initial Hypothesis Hypothesis Met? Contextual Fear Memory Stress would increase % time immobile to context relative to controls, blocked by DSS NO Boli During Context Test Stress would increase boli production relative to controls, blocked by DSS NO Cue Fear Memory Stress would increase % time immobile to cue relative to controls, blocked by DSS YES Boli During Cue Test Stress would increase boli production relative to controls, blocked by DSS NO Anxiety: % time in open arms Stress would decrease % time spent in open arms of EPM relative to controls, blocked by DSS YES Anxiety: % time in closed arms Stress would increase % time spent in closed arms of EPM relative to controls, blocked by DSS YES Ambulations DSS would increase ambulations relative to controls YES Head Dips DSS would increase Head Dips on the EPM relative to controls YES Startle Response Stress would increase acoustic startle response relative to controls, blocked by DSS YES @ 110dB Novel Object Recognition Stress would decrease time spent with novel object NO
53 relative to controls, blocked by DSS Growth Rate Stress would decrease growth rate relative to controls, blocked by DSS YES Heart Rate No predictions made Blood Pressure No predictions made Adrenal Gland Weight Stress would increase adrenal gland weight, blocked with DSS NO Thymus Gland Weight Stress would decrease thymus gland weight, blocked with DSS NO Kidney Weights Stress would increase kidney weights, blocked with DSS NO Discussion Summary of Major Findings The major findings of this study showed that providing animals with 2 hours of daily DSS prevented the development of se veral behavioral and physiological changes analogous to those observed in patients with PTSD. Data obtained from behavioral testing indicated that chronic psychosocial stress produced robust increases in cue fear memory, anxiety, startle responses, and recognition memory, replicating earlier findings from our group utilizing a similar chronic ps ychosocial stress paradigm (Zoladz et al, 2008). Additionally, DSS prevented all of thes e psychosocial stress-induced changes in behavior, suggesting that these environmental manipulations were effective in mitigating PTSD-like behavioral outcomes, simila r to those found in preliminary work (Seetharaman, Zoladz & Diamond, 2008). Results also revealed that groups subjected to
54 the chronic psychosocial stress paradigm exhi bited physiological cha nges similar to those observed in previous findings by our group (Zol adz et al, 2008), and others (Magarinos & McEwen, 1995; Vyas et al, 2002) with regard s to a significant ch ronic stress-induced enlargement of adrenal gland weights. Speci fically, psychosocially stressed animals were found to have enlarged adrenal glands, which secrete CORT, a main physiological marker of stress relative to non-psychosocial stress controls. Relative to control animals, psychosocially stressed anim als also exhibited significan tly smaller thymus glands, indicative of inhibited immune function, also observed in previous chronic stress work (Magarinos & McEwen, 1995; Zoladz et al, 2008 ). Interestingly, da ta suggested that DSS and stress may produce similar physiological outcomes. For instance, both psychosocial stress and DSS animals had enlarg ed adrenal glands. Some work has shown that animals provided with environmental enri chment exhibit a significant enlargement of adrenal (Bakos et al, 2006) and re duction of thymus gland weight (Tsai,Pachowsky,Stelzer & Hackbarth, 2002) relative to those hous ed under standard conditions. Also, similar to psychosocial stress, DSS groups exhibited smaller thymus glands relative to controls, complementing previous reports (Tsai et al, 2002). These findings suggest that stress and DSS may produce physiological outcomes, but differ when it comes to behavioral measures associated with PTSD. That is, this study suggests that, although DSS and stress produce simila r physiological profiles, they may be beneficial and detrimental, re spectively, in terms of the brai n as it relates to behavior. Examining the possible neurobiological and neurochemical mechanisms underlying the efficacy of DSS in preventing the onset of PTSD-like behavioral and physiological
55 changes seen in this study may provide insi ght into identifying ri sk factors of and nonpharmacological treatments for PTSD, and possi ble directions for future experimental research in this area. Possible Mechanisms of Ac tion Underlying Findings Blunted Activation of Stress-Response System Stress induces th e activation of the hypothalamic-pituitary-adrenal (HPA) axis. In this system CORT is secreted by the adrenal cortex, and is elevated under stre ss. When CORT is released into the bloodstream, it participates in a negative feedback loop on the HPA axis by binding to glucocorticoid receptors (GRs). This negativ e feedback mechanism attempts to restore homeostasis in an organism. Previous work has shown that EE produces morphological changes relating to the HPA axis. Specifically, some findings indica ted that EE resulted in an up regulation of GR mRNA ex pression in the hippocampus (Mohammed, Henrikkson, Soderstrom, Ebendal, Ol sson & Seckl, 1994; Olsson, Mohammed, Donaldson, Henriksson & Seckl, 1994). An up regulation of GR receptors produced by environmental stimulation may serve to faci litate a more effici ent negative feedback control, thereby preventing elevations in CORT (Olsson et al, 1994). This theory is supported by evidence indicating significantl y higher baseline CORT levels in mice given EE for 6 weeks but did not significant increases after re-exposure to the context where they received shock (Ben aroya-Milshtein et al, 2004) re lative to standard housing controls. This finding suggest s that EE may produce elevatio ns in basal CORT, but these animals are not as reactive to stress compared to those under standard housing conditions.
56 Some human work supporting this theory s howed that social s upport suppressed CORT responses to psychological stre ssors, as well as significantl y decreasing cardiovascular and corticosteroid responses to laboratory stre ssors (DeVries et al, 2003). Intriguingly, an examination of the PTSD literature reveals that patients with the disorder exhibit similar changes. A majority of research indicates that PTSD patients express an increased number of GRs (Rohleder, Love & Bennet, 2004; Stein Yehuda, Koverola & Hanna, 1997b; Yehuda, Boisoneau, Lowy & Giller, 1995), indicative of enhanced negative feedback. These changes observed in PTSD patients correspond to a blunted HPA axis indicated by attenuated CORT levels shortly after trauma, such as in victims of rape (Resnick, Yehuda, Pitman & Foy, 1995), and motor vehicle accidents (Raimonde & Spoonster, 2000). This apparent paradox between observations in PTSD and social stimulation lends itself to the suggestion that diverse environmental experiences, through the repeated introduction of nove lty and opportunities to explore, may act as mild chronic stressors. In this fashion, or ganisms may be better prepared for future severe stressors and, in turn, exhibit a reduction of stress-indu ced responses in behavi or (Fox et al, 2006). More work is needed in order to elucidat e the differential outcom es of environmental stimulation and chronic stress, including th eir interactions with the development of PTSD, but may be an underlying mechanism of the behavioral and physiological findings of this study. Prefrontal Cortex: Extinction Learni ng and Suppression of Fear-Like Responses.
57 Neurobiological Changes in the Prefrontal Cortex. Findings of this study indicated that psychosocially stressed anim als demonstrated robust fear memory when exposed to the cue temporally associated with the two cat exposures, replicating previous findings by our group utilizing a similar chronic stress paradigm (Zoladz et al, 2008). There were, however, no significant differen ces between groups upon re-exposure to the context temporally associated w ith the acute stress sessions. It is important to note that, for the duration of the entire study, all groups were transported from the housing room into the laboratory for two hour periods. Th e difference between groups, with regards to daily manipulations, was that, after trans portation from the housing room into the laboratory, the DSS groups were placed in th e stimulating apparatus, while the control animals remained in their home cages during this time. Since there were no significant differences between groups in immobility during the contextual fear memory test, there may have been a degradation of the contex t-cat association over time due to the continuous re-exposure of the psychosocially stressed animals into the laboratory, where the box was located. In addition to the act ual box itself, there may have been an association created between the room c ontaining the box and the subsequent cat exposures during fear conditioning training. Upon continuous transportation to the laboratory, which contained the box, there was an extinction of the box-cat association in the psychosocial stress group. In other wo rds, the actual dail y transportation of psychosocially stressed animals into the labora tory, in theory, served as extinction trials, in which the association between the condi tioned stimulus (box) and the unconditioned stimulus (cat) was, over time, degraded due to extinction-based learning. Previous work
58 has shown that, over time, re-exposing animals to the CS without presentation of the US extinguishes the conditioned response (Myers & Davis, 2007) which, in this case, was measured by immobility in the box, suggestive of fear. Due to the enhanced ability of DSS to extinguish contextual fear, there we re no significant differences in immobility between groups when re-exposed to the traini ng context during testi ng. There are certain brain structures which play a role in extin ction learning. Specifi cally, The prefrontal cortex (PFC), a frontal lobe structure involved in cogniti ve flexibility, decision making, and overall executive controls, has also been identified as being part of the neural circuitry of extinction learni ng. This is evidenced by work showing that damage to the medial prefrontal cortex (mPFC) interferes with the extinction of conditioned fear responses (Lebron, Milad & Quirk, 2004; Morgan, Romanski & LeDoux, 1993; Quirk, Garcia & Gonzalez-Lima, 2006). In contrast to the contextual fear memory findings, the psychosocial stress demonstrated robust immobility to the cue (t one) temporally associated with the cat exposures relative to home cageno psychos ocial stress controls. Additionally, this increase in fear-related behavior was prevented with DSS, indicated by the DSSpsychosocial stress group being significantl y less immobile during the cue relative to home cage-psychosocial stress. These beha vioral findings showed that re-exposing psychosocially stresses animals to the cue te mporally associated with the cat exposures was effective in producing robus t increases in fear to no ps ychosocial stress controls. The robust cue-based memory demonstrated by the psychosocially stressed animals is indicative of this memory not being subjected to degradation over time since the cue was,
59 in theory, more salient than the contextual in formation close in temporal proximity to the cat exposures. The daily transportation into the laboratory may have not reactivated the memory of the cue-cat association as read ily as the context. Thus, at testing, the presentation of the cue acted as a more sp ecific, salient reminder of the cat exposures which, in turn, produced the observed behavioral increases in fear. Findings of the cue fear test also revealed that the robust increase in immobility in the psychosocially stressed animals was prevented with DSS, indicated by significantly lower immobility in the DSS-psychosocial stress group relative to their home cage counterparts. The differential expressions of fear-related beha vior may be explained by changes occurring in the brain under stress and DSS conditi ons. Some work has shown dendritic hypertrophy in the amygdala in animals subject ed to chronic stress (Vyas et al, 2002), suggestive of enhanced neuronal processing in this structure. Cue-based fear conditioning is predominantly governed by the amygdala relative to the hippocampus, as lesions to the amygdala interfere with the conditioning of fear responses to the cue and context associated with shock, but lesions to the hi ppocampus only interfere with contextual fear conditioning (Phillips & LeDoux, 1992). Th e finding, therefore, that chronic psychosocial stress produced robust cue fear conditioning in the current study is not surprising. Some work has suggested that the effects of environmental stimulation may be governed by neurobiological and neurochemical modifications in the functioning of the prefrontal cortex PFC. Examination of studies illustrating such modifications may explain the finding that DSS was effective in preventing the psychosocial-stress induced increase in cue fear. Specifically, some fi ndings indicated that, re lative to standard
60 housing, rats given 3 months of enrichment demonstrated significant increases in dendritic spine density in the mPFC, indi cating enhanced neuronal connectivity and enhanced processing of this brain struct ure (Kolb, Gorny, Soderpalm, & Robinson, 2003). The mPFC has direct projections to the amygdala, which governs emotional behaviors, including in response to stress (McDonald, Mascagni & Guo, 1996). Studies examining the interactions be tween the two structures have suggested that the mPFC exerts inhibitory projections on the amygdala such that the stimulation of the mPFC inhibits the demonstration of robust f ear-related responses (Zbrozyna & Westwood, 1991; Quirk, Garcia & Gonzalez -Lima, 2006), governed predom inantly by the amygdala. In contrast, selective lesions of the mPFC seem to disinhibit amygdala activation, resulting in a perseveration of aff ective behaviors (Morgan & LeDoux, 1995). Other work also indicates that rats exposed to chronic restraint stress exhibit significant shortening of dendrites in the mPFC in c onjunction with dendritic hypertrophy in the amygdala, suggestive of a relative impairment of the mPFC to suppr ess the response of the amygdala and, in turn, behavioral responses to stress activation (Radley, Sisti, Hao, Rocher, McCall, Hof, McEwen & Morrison, 2004). These studies suggest that, in theory, SES enhanced mPFC inhibitory projections onto the amygdala which, in turn, prevented the expression of cue fear-related behavior s in psychosocially stressed animals given daily SES in the current study. Additionally, enhanced mPFC processing relative to standard housing may have also facilitated in creased capabilities to extinguish contextual fear.
61 Neurochemical Changes in the Prefrontal Cortex. Some recent work has examined neurochemical changes in the PFC which may underlie the actions of environmental stimulation on fear memor y. Specifically, EE for 6, 12, and 24 months prevented handling stress-induced increases in dopamine (DA) levels in the PFC compared to standard housing (Segovia, De l Arco, de Blas, Garrido & Mora, 2008). In addition, animals under enriched conditions have been found to express a significant decrease in the functioning of the dopamine transporter in the medial pre-frontal cortex (mPFC) ( Zhu, Apparsundaram, Bardo & Dwoskin, 2004), and decreased expression of dopamine receptors in the PFC (Del Arco, Segovia, Canales, Garrido, de Blas, GarciaVerdugo & Mora, 2007) relative to isolated an imals. Such examples of enrichmentinduced decreases in DA functioning in the mPFC may reflect a de sensitization of the brain to stress, as, relative to controls, DA le vels are significantly increased in the mPFC in rats exposed to stressors such as in termittent tail shock (A bercrombie, Keefe, DiFrischia & Zigmond, 1989), 30 minutes of tail pressure (Finlay, Zigmond & Abercrombie, 1994), and repeated foot shoc k (Meiergard, Schenk & Sorg, 1997). It may be that environmental stimulation acts as a form of mild stress through repeated exposures of animals to novelty (Fox et al, 2006; Stairs & Bardo, 2009) which desensitizes the brain to mo re severe stress and, in tu rn, ameliorates stress-induced increases in behavior relating to fear and anxiety upon exposure to more severe stressors, as observed in the current study. Decreased dopaminergic functioning in the mPFC of enriched animals, therefore, may be an underlying mechanism which prevents the expression of deleterious stress -induced behavioral changes.
62 These enrichment-induced neurobiological and neurochemical changes in the mPFC may be candidates for further investigation into the possible mechanisms underlying the effectiveness of DSS in pr eventing the psychosocial-stress induced increases in cue fear memory observed in the current study. This fits well with the theory that enriched animals seem to, behavior ally speaking, cope better under stressful situations ( Fox et al, 2006; Sale, Berardi, & Maffei, 2009). In hibitory projections of the PFC onto the amygdala may serve to explain several behavioral outcomes of this study. That is, the observation that DSS was eff ective in preventing, not only psychosocial stress-induced increases in cue fear memo ry, but also increased startle responses, heightened anxiety, and impaired recogniti on memory, may all be explained by the enhancement of PFC inhibito ry actions on the amygdala. Conditioned Fear & PTSD Examining social and environmental fact ors which facilitate the suppression of conditioned fear responses may be clinically applicable in the treatment of PTSD, where conditioned responses to reminders of patient s trauma are thought to be compromised (Quirk et al, 2006; Yehuda & LeDoux, 2007). There is some evidence in the human PTSD work which shows patients to demonstr ate attenuated activation of the mPFC in response to personalized trauma script s (Shin, McNally, Kosslyn, Thompson, Rauch Alpert et al, 1999; Yehuda & LeDoux, 2007) or combat sounds ( Bremner, Staib, Kaloupek, Southwick, Soufer & Charney, 1999; Yehuda & LeDoux, 2007), which may underlie their inability to suppr ess fear to cues associated with their trauma due to the
63 suppressed ability of the mPFC to inhibit these amygdala-mediated responses. Enhanced DSS-induced neural processing capabilities of the mPFC could, not only have played a role in facilitating the extin ction of contextual fear, but also suppressed the robust increase in psychosocial stress-induced cu e fear observed in this study. There are, however, other brain structures that may play a role with re gards to the effectiveness of DSS to suppress psychosocial stress-induced increases in anxiety and enhance risktaking, and exploratory-like beha viors observed in this study. Nucleus Accumbens: Decreased Anxiety and Increased Exploratory-Like Behavior Behavioral outcomes of this study showed that psychosocially stressed rats exhibited heightened anxiety, evidenced by significantly less time spent in the open arms of the EPM relative to contro l animals. This psychosocial stress-induced elevation in anxiety was prevented with DSS. Findings also revealed that animals in the DSS groups exhibited significantly more head dips on the EPM relative to the home cage groups. These behaviors are indicative of greater risk -taking, or exploratory-like behaviors as they subject the animal, in theo ry, to greater danger in that objects located over the edges of the apparatus are unknown. The nucleus ac cumbens (NAcc), a brain structure found in the ventral striatum, has been identified as governing the re gulation of behaviors related to risk-taking, anxiety, and exploratory-like behaviors, shown in studies, for instance, utilizing animal models of drug abuse ( Robinson & Kolb, 1997; Robinson, Gorny, Mitton & Kolb, 2000). Interestingly, similar to drug abuse, environmental stimulation increases dendritic growth in the NAcc relative to st andard housing (Kolb et al, 2003), suggesting
64 enhanced neuronal connectivity a nd efficiency in functioning of this brain structure. This suggests that, in addition to the PFC, incr eased neuronal processing in the NAcc may have played a role in the anxi olytic and exploration facilita ting effects on behavior which DSS seemed to exert in this study. In th e current study, psychosocially stressed animals demonstrated heightened anxiety, which was prevented with DSS. Examinations of changes in the NAcc may in future studies may be of use in identifying possible mechanisms responsible for behavioral m easures of anxiety a nd exploratory-like behavior observed in this study. Hippocampus: Recognition Memory In this study, animals were tested fo r hippocampus-dependent memory in a novel object recognition test. Rats which spent more time with the novel versus the familiar object was taken as an indication of memo ry for the familiar object. Findings did not reveal, however, any significant differences be tween groups on this test. An examination of the literature reveals that, relative to standard housing, en riched environments produce significant changes in the hippocampus of anim als shown by elevations in physiological correlates of memory including LTP (Duffy et al, 2001), neurogenesis (Van Praag et al, 2000), neurotrophic growth factors (Ickes et al, 2000) and morphological changes, such as enhanced dendritic growth (Faher ty et al, 2003; Leggio et al, 2003). The majority of studies in this field provide animals with enriched environments on a twenty-four hour basis. Since, in this study, rats were only given DSS for 2 hours daily, it can be posited that perhaps this time period was not l ong enough to produce significant changes in
65 hippocampal morphology affecting memory pro cessing. As a result, this could explain why there were no differences between groups on the NOR hippocampus-dependent task. Antidepressant-Like C hanges in the Brain There is some evidence which indicates that environmental stimulation may exert its influence by inducing antidepressant-like ch anges in the brain. Research supports the observation that SES animals we re more exploratory and risk -seeking relative to animals housed under standard conditions. Specificall y, rats housed under enriched conditions for 30 days expressed significantly higher le vels of serotonin receptor (5-HT1A) mRNA in the hippocampus (Rasmuson, Olsson, Henrikkson, Kelly, Holmes, Seckl & Mohammed, 1998), similar to studies exam ining the effects of anti-depressant administration on 5-HT1A activity. This suggests a possible involvement of the serotoninergic system in the behavioral e ffects observed in some enrichment studies relating to anxiety and exploration. One study, for instan ce, showed that chronic treatment with the antidepressant buspirone for 3 weeks significantly increased 5-HT1A mRNA levels in the dentate gyrus of the hi ppocampus in rats relativ e to vehicle-treated animals (Chen, Zhang, Rubinow & Chuang, 1995). These studies are complemented by human work indicating that buspirone admini stration is effective in the treatment of patients with anxiety (Eison & Temple, 1986). The anxiolytic effects of this 5-HT1A agonist were also illustrate d in a study showing that ra ts given intra-hippocampal administrations of buspirone spent significantly more time in the open arms of an EPM relative to controls, indicating an anxiolytic effect of th e drug (Kostowski, Plaznik, & Stefanski, 1989). Interesti ngly, these studies indicate th at there may be similar
66 neurobiological changes produced by such an tidepressants and DSS. Examining the similarities between antidepressants and DSS on the brain and behavior may be useful in identifying possible mechanisms underlying DSS, and its ability to prevent the development of elevations in, for instan ce, anxiety and startle response produced by chronic psychosocial stress obs erved in this study. Physiological Testing. Prevention of Psychosocial Stress-Induced Changes in Organ Weights. Intriguingly, both DSS and ps ychosocial stress produced sim ilar physiological outcomes in this study. Animals subjected to the chronic psychosocial stress paradigm expressed significant increases in adrenal gland wei ghts relative to home cag e control animals, replicating previous work by our group (Zol adz et al, 2008) and others. Similar to psychosocial stress, findings indicated that SES also produced si gnificant increases in adrenal weights, relative to controls. Thes e results, although intere sting, are perhaps not surprising, as stress-induced increases in adrenal weights has been shown in a study examining rats (Bakos et al, 2006), but not in another assessing the effects of EE on adrenal weights of 3 strains of mice (Tsai, Pachowsky, Stelzer & Hackbarth, 2002), suggesting species-specific changes in phys iology. These findings are also not particularly surprising considering the eviden ce which indicates that enriched animals demonstrate significant increases in endogenous glucocorticoids, such as CORT (Tsai et al, 2002;Webster, Tonelli & Sternberg, 2002; Marashi et al, 2003) measured in mice. It should be taken into consideration that, even though DSS animals in this study were not
67 provided with running wheels, the apparatus may have provided increases opportunities for physical activity, relative to the much smaller home cages. This increased activity level could explain the driving force behind th e enlargement of adrenal glands, as well as hearts of DSS animals observed in this study, as physical activity alone has been found to produce significantly larger adrenals and hearts compared to controls (Anderson, Eckburg & Relucio, 2002). Similar to psyc hosocial stress, DSS produced a robust decrease in thymus gland weight, which play a role in immune func tion, relative to home cage control animals. Other studies have suggested that EE produces thymic atrophy in female mice (Tsai et al, 2002) similar to stre ss in male rats (Zoladz et al, 2008), and a reduction in immunological parameters (Maras hi et al, 2003) in ma le mice relative to standard housing. The precise influence of environmental stimulation on immune parameters is complicated in that some st udies have shown 3 months of EE to produce significant increases in natura l killer cells, indicative of enhanced immune functioning (Benaroya-Milshtein et al, 2004), which facilitate resistance to viral infections, and tumor growth. Prevention of Psychosocial Stress-Indu ced Reduction in Growth Rate. Similar to other chronic stress rese arch, and previous findings in our laboratory (Zoladz et al, 2008), the psychosocially stressed animals demons trated significantly lower growth rates relative to no psychosocial stress control anim als. Again, this psychosocial stress-induced reduction in growth rate wa s prevented with DSS.
68 Heart Rate and Blood Pressure Findings suggested that there were no significant differences between groups on heart rate or bl ood pressure. It was surprising to note that there were no differences between the hom e cage psychosocial stress group and their no psychosocial stress counterparts. Previous work by our group has shown chronic psychosocial stress to significantly increase bl ood pressure, and lowe r heart rate (Zoladz et al, 2008). The lack of significant differe nces on these parameters may be due to differential methods between the two studies. In this study, all animals were transported from the housing room into the laboratory da ily. This differential experience, compared to the work conducted by Zoladz and collea gues, could have affected the outcomes of these measures. General Conclusions The purpose of this study was to test the hypothesis that providing rats with social and environmental stimulation would preven t the onset of PTSD-like behavioral and physiological changes in rats exposed to chro nic psychosocial stress. Similar to previous findings from our group (Zoladz et al, 2008), I found that rats subjec ted to the chronic psychosocial stress paradigm, consisting of tw o acute cat exposures, in conjunction with social instability, produced significant behavi oral changes analogous to those observed in patients with PTSD, supporting initial hypotheses and pr. The psychosocially stressed animals exhibited elevations in cued fear, a nxiety, and startle response. Also supporting initial predictions, this study s howed that these deleterious effects of psyc hosocial stresson behavior were prevented in animals pr ovided with DSS. Additionally, I found that
69 psychosocially stressed anim als exhibited significant changes in various physiological measurements. Specifically, rats subjected to the chronic psychosocial stress paradigm had significantly heavier adrenal glands, sm aller thymus glands, and heavier hearts. Interestingly, these psychosocia l stress-induced changes in or gan weights were similar to those observed in rats in the DSS groups. DSS seemed to prevent the detrimental effects of chronic psychosocial stress on behavior but produced similar changes in organ weights to psychosocially stressed rats re lative to home cage -no psychosocial stress controls. Findings of this study seem to s upport the theory, and othe r empirical evidence, that DSS may have, in a sense, inoculated rats by facilitati ng neurobiological and neurochemical changes in the brain, similar to those observed in reports examining the effects of chronic antidepressant ad ministration on analogous changes. Implications and Clin ical Relevance In this study, DSS was initiated twenty-f our hours after the first cat exposure session and continued daily for 2 hour period for the duration of the 31 day chronic psychosocial stress paradigm. This study was designed to mimic studies which examined the effects of pharmacological treatments on PTSD after trauma exposure. Interestingly, findings of this study seem to be similar to preliminary data in our laboratory indicating that chronic treatment with the atypical antidepressant tianeptine blocked the development of PTSD-like behavioral and phys iological changes when initiated twentyfour hours after the first cat exposure and ende d prior to testing, an alogous to methods of DSS exposures in this study.
70 Clinical studies have examined the eff ects of various pharmacological treatments on PTSD, with mixed results. For instance, PTSD patients treated with selective serotonin reuptake inhibitors (SSRIs) exhib ited significant improvement in depressivelike symptoms but had little improvement on the memory and anxiety-related symptoms of the disorder (Van der Kolk, 2001). Alt hough tricyclic antidepressants have been found to exert positive effects on PTSD-related sy mptoms (Bisson, 2007), other reports showed that desipramine reduced symptoms of depression in PTSD patients, but, again, was ineffective in alleviating anxiety-related symptoms, central to the disorder (Reist, Kauffman, Haier, Sangdahl, Demet, Chi cz-DeMet, 1989). These studies may provide insight into the importance of furtheri ng research examining the effects of nonpharmacological treatments of PTSD, such as group therapy, which, in contrast to drug treatment, do not produce undesired physic al side effects. Additionally, nonpharmacological treatments may serve to address the entire pr ofile of symptoms associated with PTSD, in contrast to dr ug treatments which may only target a few. Limitations and Future Directions This study does not distinguish between the efficacy of the social and environmental components of the DSS manipula tions. Future studies should attempt to parse out the differential effects of the envir onmental and social aspect inherent in the DSS manipulations. This could be accomp lished by adding more experimental groups. For instance, placing groups of animals toge ther in the apparatus without any objects would perhaps serve to elucidate the effect s of purely social stimulation on outcomes
71 observed in our model. Additionally, objects coul d be placed in rats home cages in order to investigate whether environmentally enriching the home cage exerts the same influences relative to placing a group of an imals in the large apparatus with objects. Another, perhaps overlooked, limitation in th is study are the actual reasons underlying the behavioral and physiological effects observe d. Part of the hypothesis of this study was that DSS would serve to neutralize the add itive component of daily social instability to the model and, as a result, ameliorate deleterious PTSD-like changes produced by the entire chronic psychosocial stress paradigm. This thought was based on previous findings by our group which showed that the daily soci al instability component of the model was necessary in order to produce PTSD-like behavioral and physiological changes (Zoladz et al, 2008). It is unknown in this study, how ever, whether DSS actually served to neutralize the additive effects of social inst ability manipulations to the model. Future studies should address this question by utilizing between group pseudo-randomization with a unique set of ra ts. In other words, instead of switching the cage mates of rats within a group on a daily basis, future work should pair each rat with another rat which it has not encountered before, either in the stimulating apparatus, or in its home cage. This may elucidate the true efficacy of DSS to rem ove or neutralize the detrimental additive effects of daily social instability to the en tire chronic psychosocial stress paradigm. It may be important for future studies to examine the influence of DSS on brain morphology and its interactions with chroni c stress. Specificall y, morphological, and neurochemical changes in the PFC would be interesting to examine in terms of
72 elucidating possible mechanisms by which D SS exerted its influence in preventing the development of the behavioral and physio logical changes obser ved in this study. The human social support literature suggests and strong relationship between social support and lower rates of PTSD, and PTSD-related symptoms. Examining the literature closely, however, reveal s that it is not necessarily the social support itself which may be exerting its protective influences on PTSD development but, rather, the nature of the support individuals receive after being exposed to trauma which may decrease their chances of developing the disorder. For instance, some work suggests th at it is not simply the number of social interactions, or number of individuals in ones social network that is important but, rather, the positive quality of those social interactions which act to buffer the detrimental effects of stress on health (Cohen & Wills, 1985; Solomon et al, 1987). In other words, the love from a supportive s pouse, or from a few family members may be as beneficial compared to the actual social network sizes of individuals in terms of being associated with lower rates of PTSD, and ge neral health after trauma exposure (Cohen & Wills, 1985; Keane et al, 1985; King et al, 1998). In addition, perceived available support (i.e. the perception that an i ndividual is loved and can count on others for support) may be more strongly correlated with long-term health relative to the number of people available in ones network to offer suppor t (Collin & Feeney, 2004). Interestingly, a study conducted on Vietnam veterans indicated that both structural (size and complexity of a given social network) and functiona l (emotional sustenance) correlated with lowering PTSD in men, but only functional supp ort was predicted lower rates of PTSD in women (King et al, 1998), suggesting possible gender differences with regards to
73 interactions between stress a nd social factors on PTSD development after trauma. The assessment of the quality of social rela tionships in human research is based on individuals perceptions of the positive nature of thei r interactions. Being able to empirically assess the quality of social intera ctions within the constraints of an animal model would be very difficult, if not impossibl e, based on the simple fact that perceptions cannot be assessed in rats. Utilizing an animal model is useful in th at the opportunities for social interactions can be experimentally ma nipulated, as was done in this study, but does not allow for the drawing of empirical c onclusions based on the quality of social relationships experienced by organisms, which may be important to consider in human work. Concluding Remarks Findings of this study revealed that providing animals with DSS blocked the development of several PTSD-like respons es in adult rats exposed to chronic psychosocial stress. Importantly, this study, in contrast to the majority of studies in this field, showed that providing adult rats with brief, daily stimulation may exert a profound protective influence on deleteri ous effects of chronic psychos ocial stress on the behavior and physiology of rats when initiated shortly after an acute stress experience. These results are also consistent with human rese arch suggesting that social stimulation may confer resistance of a subset of the trauma tized population to develop PTSD. This level of analysis in an animal model of PTSD se rves to underlie the importance of clinical
74 research examining social factors in identi fying risk factors for PTSD, as well as nonpharmacological treatments (e.g. social support systems) for the disorder.
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