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Uncontrollable and unpredictable stress with a reminder experience induces long-lasting effects on physiology and behavior

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Title:
Uncontrollable and unpredictable stress with a reminder experience induces long-lasting effects on physiology and behavior a novel approach to modeling post-traumatic stress disorder in rats
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Book
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English
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Zoladz, Phillip R
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University of South Florida
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Tampa, Fla
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Subjects / Keywords:
PTSD
Glucocorticoids
Metyrapone
AF-DX 116
Yohimbine
Dissertations, Academic -- Psychology -- Masters -- USF
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bibliography   ( marcgt )
theses   ( marcgt )
non-fiction   ( marcgt )

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Abstract:
ABSTRACT: People who endure horrific, life-threatening experiences are at risk for developing post-traumatic stress disorder (PTSD). However, only about 25% of all individuals who experience trauma develop PTSD. Recent research indicates that the presence of certain physiological conditions, such as reduced cortisol and parasympathetic inhibition, during trauma may increase one's susceptibility to developing PTSD. Thus, I attempted to develop a novel animal model of PTSD and test the hypothesis that reduced adrenal and parasympathetic activity during stress would exacerbate its long-term effects on behavior.In Experiment One, adult male rats were exposed to two stress sessions, each involving one hour of immobilization plus cat exposure. Before each session, rats were injected with vehicle, metyrapone, AF-DX 116, or both drugs. The second session occurred 10 days after the first and served to model a traumatic flashback. Stressed rats endured unstable housing conditions throughout t he experiment to add an element of daily anxiety. Three weeks after the second session, all rats underwent a battery of tests to examine the lasting effects of stress on physiology and behavior. The results indicated that stressed rats exhibited heightened anxiety on the elevated plus maze, an exaggerated startle response, and greater blood pressure, relative to controls. Moreover, metyrapone, when combined with stress, led to significant short- and long-term spatial memory impairments. Experiment Two assessed the effects of the same stress paradigm on rats' sensitivity to yohimbine, an alpha-2 adrenergic receptor antagonist. Yohimbine induces flashbacks and panic attacks in patients with PTSD; thus, I hypothesized that stressed rats would react abnormally to this agent. Stressed and unstressed rats were administered vehicle or yohimbine (1 mg/kg) 30 min prior to behavioral testing. The results indicated that stressed rats were hyperresponsive to yohimbine, as evidenced by a greater su ppression of rearing, greater avoidance of the center of the open field, and a greater suppression of activity on the elevated plus maze, relative to controls. Collectively, the findings of these studies indicate that uncontrollable and unpredictable psychological stress produces lasting changes in the physiology and behavior of rats that resemble symptoms commonly observed in people with PTSD.
Thesis:
Thesis (M.A.)--University of South Florida, 2006.
Bibliography:
Includes bibliographical references.
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System requirements: World Wide Web browser and PDF reader.
System Details:
Mode of access: World Wide Web.
Statement of Responsibility:
by Phillip R. Zoladz.
General Note:
Title from PDF of title page.
General Note:
Document formatted into pages; contains 193 pages.

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aleph - 001913971
oclc - 174964377
usfldc doi - E14-SFE0001733
usfldc handle - e14.1733
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Uncontrollable and unpredictable stress with a reminder experience induces long-lasting effects on physiology and behavior :
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ABSTRACT: People who endure horrific, life-threatening experiences are at risk for developing post-traumatic stress disorder (PTSD). However, only about 25% of all individuals who experience trauma develop PTSD. Recent research indicates that the presence of certain physiological conditions, such as reduced cortisol and parasympathetic inhibition, during trauma may increase one's susceptibility to developing PTSD. Thus, I attempted to develop a novel animal model of PTSD and test the hypothesis that reduced adrenal and parasympathetic activity during stress would exacerbate its long-term effects on behavior.In Experiment One, adult male rats were exposed to two stress sessions, each involving one hour of immobilization plus cat exposure. Before each session, rats were injected with vehicle, metyrapone, AF-DX 116, or both drugs. The second session occurred 10 days after the first and served to model a traumatic flashback. Stressed rats endured unstable housing conditions throughout t he experiment to add an element of daily anxiety. Three weeks after the second session, all rats underwent a battery of tests to examine the lasting effects of stress on physiology and behavior. The results indicated that stressed rats exhibited heightened anxiety on the elevated plus maze, an exaggerated startle response, and greater blood pressure, relative to controls. Moreover, metyrapone, when combined with stress, led to significant short- and long-term spatial memory impairments. Experiment Two assessed the effects of the same stress paradigm on rats' sensitivity to yohimbine, an alpha-2 adrenergic receptor antagonist. Yohimbine induces flashbacks and panic attacks in patients with PTSD; thus, I hypothesized that stressed rats would react abnormally to this agent. Stressed and unstressed rats were administered vehicle or yohimbine (1 mg/kg) 30 min prior to behavioral testing. The results indicated that stressed rats were hyperresponsive to yohimbine, as evidenced by a greater su ppression of rearing, greater avoidance of the center of the open field, and a greater suppression of activity on the elevated plus maze, relative to controls. Collectively, the findings of these studies indicate that uncontrollable and unpredictable psychological stress produces lasting changes in the physiology and behavior of rats that resemble symptoms commonly observed in people with PTSD.
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Uncontrollable and Unpredictable Stress with a Reminder Experience Induces Long-Lasting Effects on Physiology and Behavior: A Novel Approach to Modeling Post-Traumatic Stress Disorder in Rats by Phillip R. Zoladz 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. Paula Bickford, Ph.D. Edward Levine, Ph.D. Kristen Salomon, Ph.D. Date of Approval: August 21, 2006 Keywords: PTSD, glucocorticoids, metyrapone, AF-DX 116, yohimbine Copyright 2006, Phillip R. Zoladz

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For my parents, who have been there for me every step of the way

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Acknowledgements I would first like to thank my major professor, Dr. David Diamond, for providing his guidance and expertise and for affording me the opportunity to conduct this research. I would also like to thank my committee members, Dr. Paula Bickford, Dr. Edward Levine, and Dr. Kristen Salom on, for their input and words of encouragement from my initial proposal to the final manuscript. I am al so grateful for the assi stance of my fellow lab assistants, Dr. Collin Park, Dr. Adam Campbell, and Josh Halonen. You have each helped me become a better researcher in some way or another, and I am thankful to have you all as my colleagues. In addition, I would like to thank Tadd Patton for not only reading early drafts of the present manuscrip t, but also for offering advice and guidance throughout the process. I would al so like to thank the laboratory of Dr. Monika Fleshner at the University of Colorado for assis ting with the cortic osterone analyses. I would like to thank Dr. Bryan Raude nbush, who brought me to love research. Thank you, Bryan, for your support, wisdom, and most of all, your fr iendship. Lastly, but certainly not least, I would like to express thanks to my family. My parents have supported me through the ups and downs and ha ve always backed me, no matter what my decisions were. I owe you everything, and I hope that I have made you proud. Also, thank you, Meagan, for supporting me throughout this pr ocess. I know it ha s been difficult at times, but you have always stood by my side, and I love you for that.

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i Table of Contents List of Tables vi List of Figures vii Abstract x Chapter One: Background 1 Stress 1 Anxiety Disorders and Post-Traumatic Stress Disorder 3 Diagnostic Criteria 3 Do PTSD Patients Exhibit Heightened Startle? 7 Do PTSD Patient Display Impairments in Cognition? 10 Development of PTSD 11 Autonomic Nervous System 12 Sympathetic Nervous System and General Arousal 12 Parasympathetic Nervous System 15 Role of the Noradrenergic System in PTSD 17 The Hypothalamus-Pituitary-Adrenal Axis 19 Near the Time of Trauma 19 Baseline 20 Enhanced Negative Feedb ack of the HPA Axis 23 Can CORT Administration Help Treat PTSD? 24 Hippocampal Volume in PTSD 25 Animal Models of PTSD 27 Hypotheses of the Present Experiments 35 Chapter Two: Experiment One 37 Does a Combination of Reduced PNS and Adrenal Activity Exacerbate Rats Long-Term Response to Stress? 37 Methods 37 Rats 37 Design 38 Heart Rate/Blood Pressure Machine Habituation 38 Pharmacological Agents 39 Stress Manipulations 40 Blood Sample 40 Heart Rate and Blood Pressure 41 Completely Unstressed Control Group 41

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ii Stress Sessions 42 Randomized Housing 43 Handling, Weight, and Be havioral Testing 43 Behavioral Apparatus 44 Elevated Plus Maze 44 Startle Response 45 Radial-Arm Water Maze 46 Fear Conditioning 48 Blood Samples, Heart Rate, and Blood Pressure 50 Statistical Analysis 51 Heart Rate and Blood Pressure 52 Corticosterone 52 Elevated Plus Maze 53 Startle Response 53 Radial-Arm Water Maze 53 Fear Conditioning 54 Weight 54 Results 54 Heart Rates and Blood Pressu re during Stress Session 1 54 Heart Rates 54 Systolic Blood Pressure 56 Diastolic Blood Pressure 58 Heart Rates and Blood Pressu re during Stress Session 2 59 Heart Rates 59 Systolic Blood Pressure 61 Diastolic Blood Pressure 63 Stress Sessions Corticosterone Levels 65 Stress Session 1 Corticosterone Levels 65 Stress Session 2 Corticosterone Levels 66 Elevated Plus Maze 67 Ambulations 67 Percent Time Spent in the Open Arms 69 Percent Time Spent in the Open Arms, Controlling for Ambulations 70 Percent Time Spent in the Closed Arms 71 Movement per Unit Time in the Closed Arms 73 Distance on the Elevated Plus Maze 74 Fecal Boli 76 Comparison of the Control Groups 77 Startle Response 77 90 dB Auditory Stimuli 77 100 dB Auditory Stimuli 79 110 dB Auditory Stimuli 80 Fecal Boli 81

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iii Comparison of the Control Groups 82 Radial-Arm Water Maze 82 Acquisition 82 One-Hour Memory 83 Twenty-Four Hour Memory 85 Comparison of the Control Groups 86 Fear Conditioning 86 Contextual Fear Memory 86 Context Test Fecal Boli 87 Cued Fear Memory 88 Cue Test Fecal Boli 90 Comparison of the Control Groups 90 Final Days Heart Rate and Blood Pressure 91 Heart Rates 91 Systolic Blood Pressure 93 Diastolic Blood Pressure 94 Correlation between Cardiovasc ular Activity and Anxiety 96 Relationship between Cardi ovascular Activity during the Stress Sessions and Anxiety-Like Behavior on the Elevated Plus Maze 96 Correlation between Corticoste rone Levels and Average Startle Response 97 Relationship between Cortic osterone Levels during Stress Session One a nd Average Startle Responses 97 Final Days Corticosterone Levels 98 Weight 99 Comparison of the Control Groups 103 Discussion 103 Major Findings and Significance 103 Stress-Induced Enhancements of Anxiety and Acoustic Startle Response 104 Spatial Learning & Fear Memory 107 Long-Term Stress Effects on th e Cardiovascular System 109 Stressed Controls and the Eff ectiveness of Metyrapone and AF-DX 116 110 Why the Pharmacological Manipulations were, for the Most Part, Ineffective 112 Limitations of the Present Study 116 Chapter Three: Experiment Two 118 Does Stress Produce Dynamic Brain Changes that Increase Rats Long-Term Sensitivity to Yohimbine? 118 Methods 118 Rats 118

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iv Design 119 Stress Manipulations 119 Stress Sessions 120 Randomized Housing 120 Handling and Weight 121 Behavioral Testing 121 Behavioral Apparatus 121 Open Field 121 Elevated Plus Maze 122 Startle Response 122 Blood Samples 122 Statistical Analysis 123 Open Field 123 Elevated Plus Maze 124 Startle Response 124 Corticosterone 124 Weight 124 Results 125 Open Field 125 Ambulations 125 Rearing 126 Time Spent in the Perimeter 127 Time Spent in the Center 128 Distance in the Open Field 129 Fecal Boli 130 Elevated Plus Maze 130 Ambulations 130 Percent Time Spent in the Open Arms 131 Percent Time Spent in the Open Arms, Controlling for Ambulations 131 Percent Time Spent in the Closed Arms 132 Movement per Unit Time in the Closed Arms 133 Distance on the Elevated Plus Maze 134 Fecal Boli 135 Startle Response 135 90 dB Auditory Stimuli 135 100 dB Auditory Stimuli 136 110 dB Auditory Stimuli 137 Fecal Boli 138 Final Days Corticosterone Levels 138 Weight 140 Discussion 141 Major Findings and Significance 141 Enhanced Sensitivity to Yohimbine in the Open Field 142

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v Elevated Plus Maze and the Ma gnitude of the Stress Effect 143 Startle Response 143 Weight as an Index of Stress 144 Effects of Stress and Yohimbine on Adrenal Activity 145 Limitations of the Present Study 146 Chapter Four: General Discussion and Conclusions 148 Mechanisms Mediating the Long-Term Effects of Stress on Physiology and Behavior 148 Amygdala Plasticity 148 Serotonin 149 Noradrenergic Mechanisms 151 Relevance of the Present Findings to Understanding PTSD in Humans 151 References 155

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vi List of Tables Table 1 Average Raw Weights for all Groups in Experiment 1 101 Table 2 Sample Sizes for all Groups in Experiment 2 119 Table 3 Average Raw Weights for the Stressed Groups in Experiment 2 140

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vii List of Figures Figure 1. Schematic Diagram of the Elevated Plus Maze 45 Figure 2. Schematic Diagram of the Radial-Arm Water Maze 46 Figure 3. Heart Rates during Stress Session One 56 Figure 4. Systolic Blood Pressu re during Stress Session One 57 Figure 5. Diastolic Blood Pressu re during Stress Session One 59 Figure 6. Heart Rates dur ing Stress Session Two 61 Figure 7. Systolic Blood Pressu re during Stress Session Two 62 Figure 8. Diastolic Blood Pressu re during Stress Session Two 64 Figure 9. Corticosterone Leve ls during Stress Session One 66 Figure 10. Corticosterone Leve ls during Stress Session Two 67 Figure 11. Ambulations on the Elevated Plus Maze 68 Figure 12. Percent Time Spent in the Open Arms of the El evated Plus Maze in Experiment 1 70 Figure 13. Percent Time Spent in the Cl osed Arms of the Elevated Plus Maze in Experiment 1 72 Figure 14. Distance/Time Traveled in the Closed Arms of the Elevated Plus Maze in Experiment 1 74 Figure 15. Distance Traveled on the Elevated Plus Maze in Experiment 1 75 Figure 16. Startle Responses for the 90 dB Noise Bursts in Experiment 1 79

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viii Figure 17. Startle Responses for the 100 dB Noise Bursts in Experiment 1 80 Figure 18. Startle Responses for the 110 dB Noise Bursts in Experiment 1 81 Figure 19. Acquisition Curve in the Radial-Arm Water Maze 83 Figure 20. Arm Entry Errors on the OneHour Memory Test Trial in the Radial-Arm Water Maze 84 Figure 21. Arm Entry Errors on the Twenty-Four Hour Memory Test Trial in the Radial-Arm Water Maze 85 Figure 22. Freezing during the Cont extual Fear Memory Test 87 Figure 23. Freezing during the Cue Memo ry Test without the Presence of the Tone 88 Figure 24. Freezing during the Cue Memo ry Test with the Presence of the Tone 90 Figure 25. Heart Rates dur ing the Final Day of Be havioral Testing 92 Figure 26. Systolic Blood Pressure during the Final Day of Behavioral Testing 94 Figure 27. Diastolic Blood Pressu re during the Final Day of Behavioral Testing 95 Figure 28. Relationship between Av erage Diastolic Blood Pressure during the Stress Sessions and Percent Time Spent in the Open Arms of the Elevated Plus Maze 96 Figure 29. Relationship between Co rticosterone Levels during Stress Session One and Average Startle Response 97 Figure 30. Corticosterone Leve ls during the Final Day of Behavioral Testing in Experiment 1 99 Figure 31. Body Weight Gain ed throughout the Course of Experiment 1 102 Figure 32. Schematic Diagram of the Open Field Apparatus 122

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ix Figure 33. Percent Change of Ambu lations in the Open Field in Yohimbine-Treated Rats 125 Figure 34. Percent Change of Rearing Episodes in the Open Field in Yohimbine-Treated Rats 126 Figure 35. Percent Change in Percent Time Spent in the Perimeter of the Open Field in Yohimbine-Treated Rats 127 Figure 36. Percent Change in Percent Time Spent in the Center of the Open Field in Yohimbine-Treated Rats 128 Figure 37. Total Distance Traveled in the Open Field in Yohimbine-Treated Rats 129 Figure 38. Percent Change in Tota l Ambulations on the Elevated Plus Maze in Yohimbine-Treated Rats 130 Figure 39. Percent Time Spent in the Open Arms of the Elevated Plus Maze in Experiment 2 132 Figure 40. Percent Time Spent in the Closed Arms of the Elevated Plus Maze in Experiment 2 133 Figure 41. Distance/Time Traveled in the Closed Arms of the Elevated Plus Maze in Experiment 2 134 Figure 42. Percent Change in Total Distance Traveled on the Elevated Plus Maze in Yohimbine-Treated Rats 135 Figure 43. Startle Responses for the 90 dB Noise Bursts in Experiment 2 136 Figure 44. Startle Responses for the 100 dB Noise Bursts in Experiment 2 137 Figure 45. Startle Responses for the 110 dB Noise Bursts in Experiment 2 138 Figure 46. Corticosterone Leve ls during the Final Day of Behavioral Testing in Experiment 2 139 Figure 47. Body Weight Gained throughout the Course of Experiment 2 141

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x Uncontrollable and Unpredictable Stress with a Reminder Experience Induces Long-Lasting Effects on Physiology and Behavior: A Novel Approach to Modeling Post-Traumatic Stress Disorder in Rats Phillip R. Zoladz ABSTRACT People who endure horrific, lif e-threatening experiences ar e at risk for developing post-traumatic stress disorder (PTSD). Howeve r, only about 25% of all individuals who experience trauma develop PTSD. Recent resear ch indicates that the presence of certain physiological conditions, such as reduced cortisol and parasympathe tic inhibition, during trauma may increase ones susceptibility to developing PTSD. Thus, I attempted to develop a novel animal model of PTSD and test the hypothesis that reduced adrenal and parasympathetic activity during stress would exacerbate its long-term effects on behavior. In Experiment One, adult male rats were exposed to two stress sessions, each involving one hour of immobilization plus cat exposure. Before each session, rats were injected with vehicle, metyrapone, AF -DX 116, or both drugs. The second session occurred 10 days after the firs t and served to model a trau matic flashback. Stressed rats endured unstable housing conditions throughout the experiment to add an element of daily anxiety. Three weeks after the second se ssion, all rats underwen t a battery of tests to examine the lasting effects of stress on physiology and behavior. The results indicated that stressed rats exhibited heightened anxiety on the elevated plus maze, an exaggerated

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xi startle response, and greater blood pressure, relative to controls Moreover, metyrapone, when combined with stress, led to signifi cant shortand long-term spatial memory impairments. Experiment Two assessed the effects of the same stress paradigm on rats sensitivity to yohimbine, an 2 adrenergic receptor antagonist. Yohimbine induces flashbacks and panic attacks in patients with PTSD; thus, I hy pothesized that stressed rats would react abnormally to this agent. Stressed and unstresse d rats were administered vehicle or yohimbine (1 mg/kg) 30 min prior to behavioral testing. The results indicated that stressed rats were hyperresponsive to yohimbine, as evidenced by a greater suppression of rearing, greater avoidance of the center of the open field, and a greater suppression of activity on the elevated plus maze, relative to controls. Collectively, the findings of these studies i ndicate that uncontrollable and unpredictable psychological stress produces lasting change s in the physiology and behavi or of rats that resemble symptoms commonly observed in people with PTSD.

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1 Chapter One: Background Stress Since the pioneering research of Hans Selye (for a review, see Selye, 1976, 1973, & 1936), it has been accepted that stress has a number of effects on physiology and behavior, some beneficial and some aver sive. Stress can invigorate organisms and provide them with the energy to act rapidly in threatening situations, or stress can be overpowering, debilitating, and lead to both physiological and behavioral impairments. Early work discussed stress as an elicito r of the well known fight-or-flight response, which leads to an increased release of neurotransmitters (e.g., epinephrine and norepinephrine) and stress hormones (e .g., cortisol) throughout the body, which helps prepare an organism for action. Science has since then focused on the adverse consequences of stress, acknowledging that in chronic forms, stress can induce adrenalgland enlargement, atrophy of the thymus and lymph nodes, increased cardiovascular tone, immune-system suppression, and ulcera tions (Sapolsky, 1992; Selye, 1976). Recent work has implicated stress as a major precipit ating factor to the development of mental disorders (Boyer, 2000; Esch, Stefano, Fri cchione, & Benson, 2002). Considering these findings, it is of primary importance to deve lop a better understanding of the mechanisms by which stress affects physiology and cognition. Both chronic stress and a persistent eleva tion of glucocorticoids (one of the main physiological markers of stress) have detrim ental effects on brain morphology, leading to

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2 neuronal atrophy and cell death in the hippocampus, a medial tem poral lobe structure that is critical for the formation of memori es (Arbel, Kadar, Silberman, & Levy, 1994; Bodnoff, Humphreys, Lehman, Diamond, Rose, & Meaney, 1995; Bremner, 1999; Conrad, Magarinos, LeDoux, & McEwen, 1999; Lambert et al., 1998; Luine, Villegas, Martinez, & McEwen, 1994; Magarinos & Mc Ewen, 1995; Magarinos, McEwen, Flugge, & Fuchs, 1996; Magarinos, Verdugo, & McEw en, 1997). Others have found that stress and elevated glucocorticoids impair syna ptic plasticity (Bodnoff et al., 1995; Diamond, Bennett, Fleshner, & Rose, 1992; Diamond & Park, 2000; Garcia, 2001; Garcia, Musleh, Tocco, Thompson, & Baudry, 1997; Garcia, Tocco, Baudry, & Thompson, 1998; Kim, Foy, & Thompson, 1996; Kim, Koo, Lee, & Han, 2005; Kim, Lee, Han, & Packard, 2001; Maroun & Richter-Levin, 2003; Mesche s, Fleshner, Heman, Rose, & Diamond, 1999; Pavlides, Nivon, & McEwen, 2002; Sacche tti et al., 2002; Sapolsky, 2003; Shors, Seib, Levine, & Thompson, 1989; Vouimba, Yaniv, Diamond, & Richter-Levin, 2004) and memory in both humans (Elzinga, Bakke r, & Bremner, 2005; Kirschbaum, Wolf, May, Wippich, & Hellhammer, 1996; Kuhlmann, Piel, & Wolf, 2005; Sauro, Jorgensen, & Pedlow, 2003) and rodents (Conrad, Galea, Kuroda, & McEwen, 1996; de Quervain, Roozendaal, & McGaugh, 1998; Diamond & Park, 2000; Diamond, Park, Heman, & Rose, 1999; Diamond, Park, & Woodson, 2004; Kim et al., 2005; Kim et al., 2001; Luine et al., 1994; Park, Campbell, & Diamond, 2001; Rashidy-Pour, Sadeghi, Taherain, Vafaei, & Fathollahi, 2004; Sandi et al., 2005; Sauro et al., 2003; Woodson, Macintosh, Fleshner, & Diamond, 2003). These impairments have been linked to the effects that stress and glucocorticoids have on N-methyl-D-aspartate (NMDA) receptors (Kim et al.,

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3 1996; Magarinos et al., 1995; Park, Fleshner, & Diamond, 2 004), neural cell adhesion molecules (Sandi, 2004; Sandi et al., 2005; Sandi, Merino, Cordero, Kruyt, Murphy, & Regan, 2003), synaptic currents (Karst & Joels, 2003), brain-derived neurotrophic factor (Marmigere, Givalois, Rage, Arancibia, & Tapia-Arancibia, 2003; Radecki, Brown, Martinez, & Teyler, 2005), and amygdala-med iated modulation of hippocampal plasticity (Abe, 2001; Akirav & Richter-Levin, 2002; Akirav & Richter-Levin, 1999; Kim et al., 2005; Kim et al., 2001; Park & Diamond, 2005; Richter-Levin, 2004). Taken together, these studies indicate that acute and chronic stress can lead to permanent modifications of brain biochemistry, physiologi cal functioning, and behavior. Anxiety Disorders and Post-Traumatic Stress Disorder Diagnostic Criteria Anxiety disorders are the mo st common form of mental illness in the United States (National Institute of Mental Health, 2002). Characte rized by extreme fear, panic, and anxiety, the disorders affect nearly 20 million adults nationwide. One of the most notable anxiety disorders is post-traumatic stress disorder (PTSD). This disorder is precipitated by some terrifying life-threatening event, such as war, rape, or a natural disaster, and instills worry, panic, fear, a nxiety, and terror in the individual for years thereafter. PTSD is unique relative to othe r disorders because its diagnostic criteria actually specify an etiologic event: exposure to trauma (McN ally, 2003). Importantly, to be considered traumatic the event must pose a threat to the individuals physical well being and cause him or her to feel a sense of horror and helplessness. According to the

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4 Diagnostic and Statistic Manual of Mental Disorders (American Psychiatric Association, 1994, p. 424-429), the diagnostic criteria for PTSD are: A. The person has been exposed to a trauma tic event in which both of the following were present: 1) The person experienced, witnessed, or was confronted with an event or events that involved actual or threatened death or serious injury, or a threat to the physical integrity of others. 2) The persons response involved intense fear, helplessness, or horror. In children, this may be expressed in stead by disorganized or agitated behavior. B. The traumatic event is persistently re-experienced in one (or more) of the following ways: 1) Recurrent and distressing recollections of the event, including images, thoughts, or perceptions. In young childr en, repetitive play may occur in which themes or aspects of the trauma are expressed. 2) Recurrent distressing dreams of the event. In children, there may be frightening dreams without recognizable content. 3) Acting or feeling as if the traumatic event were recurring (includes a sense of reliving the experience, illusions hallucinations, and dissociative flashback episodes, including those that occur on awakening or when intoxicated). In young children, trauma-specific r eenactment may occur.

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5 4) Intense psychological distress at exposure to internal or external cues that symbolized or resemble an aspect of the traumatic event 5) Physiological reactivity on exposure to internal or external cues that symbolize or resemble an aspect of the traumatic event C. Persistent avoidance of stimuli associated with the trauma and numbing of general responsiveness, as indicated by th ree or more of the following: 1) Efforts to avoid thoughts, feelings, or conversations associated with the trauma 2) Efforts to avoid activities, places, or people that arouse recollections of the trauma 3) Inability to recall an importa nt aspect of the trauma 4) Markedly diminished interest or pa rticipation in significant activities 5) Feeling of detachment or estrangement from others 6) Restricted range of affect (e.g., unable to have loving feelings) 7) Sense of a foreshortened future (e.g ., does not expect to have a career, marriage, children, or a normal life span) D. Persistent symptoms of increased arousal, as indicated by two (or more) of the following: 1) Difficult falling or staying asleep 2) Irritability or outbursts of anger 3) Difficulty concentrating

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6 4) Hypervigilance 5) Exaggerated startle response E. Duration of the disturbance (symptoms in Criteria B, C, and D) is more than one month. F. The disturbance causes clini cally significant distress or impairment in social, occupational, or other impor tant areas of functioning. Individuals diagnosed with PTSD are hypervigilant and frequently experience trouble sleeping, concentrating, and functioning regularly in their daily lives. These symptoms are further exacerbated by remi nders of the trauma through flashbacks, situational reminders, intrusive memories and nightmares (Bryant, 2003; Reynolds & Brewin, 1999). In fact, PTSD patients are not ju st reminded of the traumatic event; rather, they feel as if they actuall y relive the experience. Accordi ngly, individuals with PTSD make great efforts to avoid stimuli that remind them of their trauma. The recent acts of war and terrorism in countries across the world assure that individuals will continue to develop PTSD in the years to come. In fact, recent reports (Heffernan, 2006) have suggested that of the 170,000 Operation Iraqi Freedom veterans, over 34,000 have sought assistan ce from VA medical centers a nd been diagnosed with some type of psychological disorder, incl uding PTSD. This is an extremely high prevalence rate of psychological illness (rough ly 20%), which supports the need for more research geared towards a better understanding of PTSD.

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7 Do PTSD Patients Exhibit Heightened Startle? PTSD is the only anxiety disorder where an exaggerated startle response is listed as one of its core symptoms. Although dia gnosticians and researchers have emphasized this indication, studies examining the baseli ne startle response of PTSD patients have presented conflicting results. The startle res ponse is defined as the rapid sequence of flexor motor movements that occurs after the onset of a briefly-presented, intense stimulus (Morgan, 1997). The most frequently u tilized method to assess an individuals startle response, in both humans and rodents, has been the ac oustic startle reflex. This task consists of exposing a human or a rode nt to a loud, rapid noise burst and assessing their startle to the stimulus. In humans, this involves measuring the intensity and speed of ones eye-blink via EMG recordings, while in rats, it consists of measuring the whole bodys motor response, typically through some type of sensory transducer. Several studies have examined the baseline startle re sponse in individuals with PTSD; and, while some have found heightened startle in th ese individuals (Morga n, Grillon, Lubin, & Southwick, 1997; Orr, Lasko, Shalev, & P itman, 1995; Shalev, Peri, Orr, Bonne, & Pitman, 1997), others have found no differe nces between PTSD patients and control subjects (Elsesser, Sartory, & Tackenberg, 2004; Grillon, Morgan, Southwick, Davis, & Charney, 1996; Lipschitz, Mayes, Rasmusson, Anyan, Billingslea, Gueorguieva, & Southwick, 2005; Orr, Solomon, Peri, Pitman, & Shalev, 1997; Shalev, Orr, Peri, Schreiber, & Pitman, 1992; Siegelaar et al., 2006). Despite thes e inconsistencies, research has reliably shown that upon exposure to loud tones, indivi duals with PTSD do exhibit significantly greater au tonomic reactivity (e.g., increases in heart rate, blood pressure,

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8 and skin conductance) than controls (Orr et al., 1997; Orr et al., 1995; Shalev, Peri, Brandes, Freedman, Orr, & Pitman, 2000). Th is includes a failure to physiologically habituate to the stimuli, in addition to the el icitation of greater autonomic responses from the onset of the tones. Thus, while it is unc lear whether or not i ndividuals with PTSD exhibit a heightened baseline startle res ponse, they do tend to display exaggerated autonomic reactivity to sudden, intense stimulation. Given the inconclusive nature of these studies, Morgan and colleagues (Grillon et al., 1996; Morgan, Grillon, Southwick, Da vis, & Charney, 1995; Morgan, Grillon, Southwick, Nagy, Davis, & Charney, 1995) conduc ted a series of experiments to examine the startle response in PTSD patients. In one study, they examined the individuals startle response at baseline (no threat condition) and when they were expecting to be shocked (threat condition). In another study, they ex amined the effects of a noradrenergic drug versus a placebo on their startle response. Th e investigators found th at individuals with PTSD exhibited greater startle responses th roughout both experiments. Given that these individuals displayed exaggerate d startle in the no threat and placebo conditions of these studies, it could be reasoned that startle is elevated in indivi duals with PTSD at baseline. However, it could also be that the exaggera ted startle in these patients was due to the anxiety associated with the unfamiliar envi ronment (i.e., the laboratories of Yale University) in which the experiments took pl ace. Accordingly, in another study, Grillon et al. (1996) examined the base line startle response of Vietna m veterans with PTSD in a familiar environment, namely, the VA hospita l from which they had been recruited. In this study, the investigators found no differe nces between the startle responses of

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9 Vietnam veterans with PTSD, Vietnam veterans without PTSD, and healthy control subjects. This finding supports the argument that individuals with PTSD exhibit an exaggerated startle response only when they are already anxious or aroused. In the case of Morgan et al. (1995), the PTSD patients c ould have endured height ened anxiety due to the novelty of their surroundings. Thus, the exa ggerated startle responses that have been reported in PTSD patients could be state-dependent, rather th an a stable trait of these individuals. In support of this argument, ot her studies (Grillon & Morgan, 1999; Grillon, Morgan, Davis, & Southwick, 1998; Pole, Ne ylan, Best, Orr, & Marmar, 2003) have found that manipulations of the experimental c ontext or the presentation of explicit threat cues consistently leads to enhanced startle responses in PTSD patients. Therefore, these individuals may display exaggerated fear-poten tiated startle responses, rather than greater baseline startle responses in general. Most, if not all, of the data obtained fr om PTSD patients has been collected after the individuals experienced th e trauma. Thus, researchers ha ve been unable to determine PTSD patients pre-trauma startle responses and compare them with their post-trauma startle responses. It is well known that startle responses are remarkably consistent within subjects, but extremely variab le between subjects (Morga n, 1997). Thus, comparing the startle responses of independent samples could be problematic in and of itself. It would be optimal to obtain baseline startle responses in traumatized individu als before and after the trauma to understand whether heightened star tle is a cause of or is caused by the onset of PTSD. In light of this reasoning, Guthrie and Bryant (2005) assessed the auditory startle response of firefight ers before and after they had been exposed to trauma.

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10 Although none of the firefighters who were e xposed to trauma developed PTSD during the course of the study, they did display more symptoms (e.g., intrusive memories, avoidance) of the disorder after the trauma than firefighters who had not been exposed to a traumatic experience. More importantly, th e investigators found th at the magnitude of the pre-trauma startle response predicted th e development of acute PTSD symptoms, as measured by the Impact of Event Scale (IES). The authors emphasized that the individuals pre-trauma physio logical reactivity could have been a risk factor for the development of PTSD symptomatology after ex posure to an intense, traumatic event. These findings suggest that PTSD may not necessarily cause an exaggerated startle response; rather, it could be a pre-existing factor that increases ones susceptibility to developing the disorder. Do PTSD Patients Display Impairments in Cognition? As noted above, stress can have detrim ental effects on hippocampus-dependent learning and memory. Accordingl y, several studies (Bremner et al., 1995; Bremner et al., 1993; Gilbertson, Gurvits, Lasko, Orr, & Pitman, 2001; Golier, Yehuda, Lupien, Harvey, Grossman, & Elkin, 2002; Jenkins, Langlais, Delis, & Cohen, 1998; Moradi, Doost, Taghavi, Yule, & Dalgleish, 1999; Sachinvala et al., 2000; Uddo, Vasterling, Brailey, & Sutker, 1993; Vasterling, Constans, Brailey, & Sutker, 1998) have re ported declarative and working memory impairments, along with deficits in attenti on, in PTSD patients. Many of the investigators have attempted to relate their findings to studies reporting smaller hippocampi in individuals with PT SD (described in further detail below). However, as with much of the research in th is area, not all studies have provided similar

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11 results. In fact, some researchers have found no differences in cognitive functioning between individuals with PTSD and healthy control subjects (Barrett, Green, Morris, Giles, & Croft, 1996; Crowell, Kieffer, Side rs, & Vanderploeg, 2002; Neylan et al., 2004; Zalewski, Thompson, & Gottesman, 1994). One major criticism of the work reporting neurocognitive impairments in PTSD patients is that many of these investigations have reported high rates of major depressive diso rder (MDD) and a history of substance abuse in the PTSD groups, which makes it difficult to ascribe the cognitive deficits to PTSD alone. Indeed, MDD (Veiel, 1997) and substan ce abuse (Goldman, Brown, Christiansen, & Smith, 1991) can have major effects of their own on physiology and cognition. Moreover, many of these studies found that the individuals with PTSD had fewer years of education and a lower IQ than controls. Wh en all of these factors were carefully controlled that is, when th e individuals with MDD or substance abuse were excluded from the experiment, and the individuals with PTSD were well educated , investigators (Neylan et al., 2004) found no differences be tween the two groups on any measure of cognitive functioning, including assessments of attention. Collect ively, these findings suggest that deficits in cognitive functioni ng may not be unique to PTSD; rather, they could be due to other factors associated w ith the disorder (e.g., MDD, substance abuse, less education). Development of PTSD Not everyone who is traumatized develops PTSD. In fact, research has indicated that only about 25% of the individuals who ar e exposed to trauma eventually develop the disorder (Yehuda, 2001). This finding implies that there is some fundamental difference

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12 between those individuals who develop the disorder and those w ho do not develop the disorder. While specific characteristics of th e trauma (e.g., natural disaster, rape, combat, etc.) and developmental experience (see Heim and Nemeroff, 2001 for a review) appear to play a large role in whether or not an individual will develop PTSD, certain physiological predispositions of the person seem to be important as well. For instance, research has shown that peopl e with PTSD exhibit an abno rmally large sympathetic [e.g., increased heart rate (HR) a nd blood pressure (BP)] response (Cohen et al., 1998; Orr, 1990) and reduced adrenal steroid levels (Bremner et al., 2003a) in resp onse to the initial traumatic event than traumatized people who do not develop PTSD. Although this work examined individuals after the trauma, it is pos sible that the differences existed before the trauma and influenced the development of the disorder. Autonomic Nervous System Sympathetic Nervous System and General Arousal Shalev and colleagues (Shalev et al., 1998) found that following trauma, those individuals who exhibited th e highest HRs upon admission to the emergency room were more likely to later develop PTSD. A sim ilar study found that mo tor-vehicle-accident survivors who displayed higher HRs upon hospita l discharge were more likely to express symptoms of PTSD 6 mont hs later (Bryant, Harvey, Guthrie, & Moulds, 2000). According to Cohen et al. (1998), PTSD patients display dysfunctional autonomic activity at two distinct levels: basal tone a nd reaction to stress-related cues. Compared to control subjects, PTSD patients have been found to exhibit signifi cantly elevated basal HR and BP (Blanchard, 1990; Blanchard, Ko lb, Pallmeyer, & Gerardi, 1982; Buckley,

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13 Holohan, Greif, Bedard, & Suvak, 2004; Ge rardi, Keane, Cahoon, & Klauminzer, 1994; Kolb, 1987; Muraoka, Carlson, & Chemtob, 1998) Others have presented conflicting results, however, suggesting that PTSD pa tients do not demonstrate these elevations (McFall, Murburg, Ko, & Veith, 1990; Mc Fall, Veith, & Murburg, 1992; Murburg, McFall, Lewis, & Veith, 1995). Despite these findings, Buckley and Kaloupek (2001) published a meta-analytic review, examini ng 34 studies that investigated basal cardiovascular activity in PTSD patients. Effect sizes for HR and diastolic BP differences were significant when individuals with PTSD were compared to traumatized individuals without PTSD and healthy controls. However, effect sizes for systolic BP differences were only significant when PTSD patients were compared to healthy controls. Additionally, anticipatory, or priming, anxiety (i.e., uneasiness that participants experience in a laboratory setti ng partially because of the unc ertainty involved) was ruled out as a contributing factor to the basal HR and BP elevations observed in PTSD patients. These findings suggest that in dividuals with PTSD do exhibi t significant elevations in basal HR and BP and that the inconsistent findings across laboratori es could be due to differences in environmental surroundings or methodology. PTSD patients have also been found to re act more strongly to trauma-related cues than traumatized people who have not developed the disorder (Kolb & Mutalipassi, 1992; McFall et al., 1990; Orr, 1990). Although this sounds tautological (since the disorder itself is defined by traumatic memories having a greater lasting effect on these individuals), a large amount of effort has been put fort h to understand the underlying mechanisms for these results. Combat vete rans with PTSD have shown heightened

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14 physiological responses to simulated combat noise, intense scripts describing combat, and hypnotically-induced imagery of combat situations (Blanc hard et al., 1982; Malloy, Fairbank, & Keane, 1983; Pitman, Orr, Fo rgue, de Jong, & Claiborn, 1987). Similarly, veterans have been shown to respond to traumatic combat stimuli with significantly greater elevations of epinephrine (EPI) (M cFall et al., 1990) and norepinephrine (NE) (Blanchard, Kolb, Prins, & Gates, 1991) th an control subjects. Liberzon et al. (1999c) used single photon emission computerized to mography to show that in response to simulated combat sounds, war veterans with PTSD showed increased activation in the amygdala, an increase that was not observ ed in control subjects. Using functional magnetic resonance imaging, Lanius and coll eagues (Lanius et al., 2003) showed that PTSD patients exhibited signifi cantly less activation of the an terior cingulate gyrus when emotional scripts were read to them. Others have found that when PTSD patients were presented with traumatic scripts, they demons trated direct influences of the amygdala on the visual cortex, subcallosa l gyrus, and anterior cingulate gyrus (Gilboa et al., 2004). Again, these effects were not observed in c ontrol subjects. The amygdala is an almondshaped, temporal lobe structure that plays a major role in emotional memory and is greatly activated during stress ful situations (LeDoux, 1998). Pa st research has suggested that PTSD patients hyperreactivity to trauma -related cues and their recurrent, intrusive memories of the traumatic event may be relate d to a failure of cingul ate gyrus inhibition over a hyperresponsive amygdala and/or some general dysfunction of limbic system structures (see Gilboa et al., 2004). These findings provide further support for this argument.

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15 Parasympathetic Nervous System Although investigators have extensively described the relationship between the sympathetic nervous system (SNS) and PTSD little work has addressed the contribution of the parasympathetic nervous system (PNS) to the disorder. The PNS is often referred to as the brakes of the autonomic nervous system (ANS), or the system that slows things down and returns the body to baseli ne. Hence, it is th e negative feedback component of homeostasis (Stern, Ray, & Quigley, 2001). The vagus nerve, the tenth cranial nerve and an important part of the PN S, innervates the sinoatrial (SA) node on the right atrium of the heart, where electri cal impulses are generated to trigger cardiac contraction. By modulating the SA node, th e vagus nerve slows HR and maintains a balance between the SNS and PNS. Vagal m odulation of HR is very important for reactions to and recovery from stressful s ituations and has been considered a possible mechanism for the differences in basal HR and HR changes in response to trauma-related cues in individuals with PTSD (Sahar, Shalev, & Porges, 2001). Parasympathetic function has been m onitored in numerous studies involving PTSD patients, where heart-rate variability (HRV) was used as the primary measure. HRV is a measure of beat-to-be at alterations in heart rate or more specifically, the variability of the intervals between R wave s. The two main frequency bands that are examined during HRV assessment are the Low-Frequency (LF) band (0.04 to 0.15 Hz) and the High-Frequency (HF) band (0.15 to 0.40 Hz ) (Sahar et al., 2001). It is believed that the SNS influences the LF component, while the PNS primarily influences the HF component. Cohen and colleagues (Cohen, Benj amin, Geva, Matar, Kaplan, & Kotler,

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16 2000a; Cohen, Kotler, Matar, Kaplan, Miodownik, & Cassuto, 1997) found that, at rest, PTSD patients displayed significantly lower HR V than control subjects. Further, these patients demonstrated lower HF and higher LF HRV components than controls, suggesting enhanced sympathetic and redu ced parasympathetic tone at baseline. However, Cohen and colleagues (Cohen et al ., 2000a; Cohen et al., 1998) showed that PTSD patients did not respond to recollection of trauma with elevations of HR and LF. The investigators reasoned th at since PTSD patients exhib ited autonomic dysregulation at rest, they could not manifest further increases in sympathetic tone in response to the traumatic reminder. Sahar et al. (2001) examined PTSD patient s vagal modulation of HR in response to a mental challenge by using respiratory sinus arrhythmia (RSA) as their dependent measure. Changes in RSA have been shown to reflect activity of the vagus nerve, as RSA levels positively correlate with parasymp athetic influence on the heart (Berntson, Cacioppo, & Quigley, 1993). Sahar and colleagues (Sahar et al., 2001) found that PTSD patients and control subjects, who had prev iously experienced trauma but had not developed a stress disorder, did not differ on resting levels of parasympathetic activity. However, when faced with a challenging arithmetic task, contro l subjects showed a significant increase in RSA (which was highl y correlated with their HR), while PTSD patients showed no such increase. Thus, vagal mechanisms contributed to control subjects, but not PTSD patients, HR regulation. Similar findings were observed by Sack, Hopper, and Lamprecht (2004), who found that when PTSD patients were divided into low and high RSA groups, individuals w ith high RSA showed a greater increase in

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17 HR in response to trauma-related script. Together, these studies suggest that the mechanisms responsible for HR regulation in response to stress may be different between individuals who develop PTSD and trauma tized people who do not develop PTSD. In particular, HR regulation in PTSD patients, especially in response to stress, may be controlled in part by non-vagal means. Role of the Noradrenergic System in PTSD The noradrenergic system influences physiological arousal by supplying NE throughout an organisms central nervous system. Noradrenergic cell bodies are prominently found in hindbrain nuclei and most extensively in an ar ea of the brain known as the locus coeruleus (LC). Studies in animal s have shown that increased LC firing leads to alertness (Berridge & Foote, 1991; Foot e, Ashton-Jones, & Bloom, 1980), while a decrease in LC firing leads to lethargy (C aballero & de Andres, 1986). Furthermore, direct stimulation of the LC via electrical current or pharmacological agents elicits vigilance and fear responses in rodents (S outhwick, Bremner, Rasmusson, Morgan III, Arnsten, & Charney, 1999a). A vast amount of literature has implicated increased noradrenergic activity in individuals with PTSD (see Southwick et al., 1999a). Geracioti et al. (2001) found significant elevations of baseline cerebrospinal fluid (CSF) NE in PTSD patients, compared to healthy control subjects. More over, these individuals CSF NE levels positively correlated with the severity of th eir PTSD symptoms. Yehuda and colleagues (Yehuda et al., 1998) also found increased basal plasma NE levels in PTSD, as compared to individuals diagnosed with MDD and nonps ychiatric control subjects. In addition,

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18 Kosten, Mason, Giller, Ostroff, and Harkness ( 1987) examined urinary NE and EPI levels at two-week intervals in hospitalized pa tients suffering from PTSD, MDD, bipolar disorder (BD), paranoid schizophrenia, and undifferentiated schizophrenia. In this study, PTSD patients exhibited signifi cantly greater NE levels than all other groups, and they demonstrated significantly greater EPI levels than all groups except for those individuals with BD. Nevertheless, some investigators have been unable to show elevated levels of NE and EPI in PTSD patients (Murburg et al ., 1995; Pitman & Orr, 1990). According to Yehuda et al. (1998), the increase in baseline noradrenergic activity may be confined to PTSD patients without comorbid depression. Investigators in this study found that NE levels were significantly associated with se verity of depression; when PTSD patients were divided into two groups, those with or without comorbid depression, only the PTSD patients without comorbid depression exhibite d significantly elevated plasma NE levels. The noradrenergic system is regulated in part by 2 adrenergic receptors, which exert an inhibitory influence on the LC (S outhwick et al., 1999a). Alpha-2 adrenergic receptor antagonists, such as yohimbine, block this inhibition, which thereby leads to an increase in LC firing and ultimately enhan ced alertness and arousal. PTSD patients, unlike controls, exhibit an exaggerated respons e to these substances including flashbacks and panic attacks (Southwick, Morgan III, Charney, & High, 1999b). Southwick and colleagues (Southwick et al., 1993) found th at 70% of PTSD patients who were administered yohimbine experienced panic attacks, and 40% experienced flashbacks. PTSD patients also exhibited significantly gr eater HR, systolic BP and anxiety-related behavior in response to the drug. Investigators have linked these findings to a reduction

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19 in NE metabolism in patients suffering from PTSD (Bremner et al., 1997a). Accordingly, PTSD patients, in comparison to control subjec ts, exhibited a significa nt decrease in the metabolism of NE in neocortical brain regions when administered yohimbine. As a result of persistently elevated catecholamine levels, PTSD patients also exhibit a downregulation of 2 adrenergic receptors (Perry, 1994; Perry, Southwick, Yehuda, & Giller, 1990), a finding that is consistent with the evidence for an overactive SNS in these individuals. Comparable findi ngs have been demonstrated in animal studies, where chronic psychosocial stress has led to a down-regulation of 2 adrenergic receptors in the LC of tree shrews (Flugge, 1996). In theory, fewer 2 adrenergic receptors could underlie the enhanced sensitivity to yohimbine that has been demonstrated by PTSD patients. The Hypothalamus-Pituitary-Adrenal Axis Near the Time of Trauma Increases in stress cause the hypothala mus to send corticotrophin-releasing hormone (CRH) to the anterior pituitary gland, which subsequently releases adrenocorticotrophin (ACTH), which then stim ulates the adrenal cortex to produce and release corticosteroids (CORT) (corticosterone in rodents; cortisol in humans). These neuromodulators help coordinate an individuals ability to co pe with stress and divert energy to undersupplied tissues (d e Kloet, Oitzl, Joels, 1999). Given the role of CORT as a stress hormone, it would be intuitive to antici pate heightened levels of CORT in PTSD patients. On the contrary, a majority of the re search in this area ha s found that individuals diagnosed with PTSD exhibit attenuated CORT levels shortly after the initial traumatic event. Resnick et al. (1995) examined CORT levels in rape victims only hours after the

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20 trauma and related them to those who did and did not develop PTSD. Those who developed PTSD exhibited lower CORT levels shortly after the trauma, in comparison to those who never deve loped the disorder. In addition, Delahanty, Raimonde, and Spoonster (2000) collected urine samples 15 hrs after individual s experienced motor vehicle accidents. They assessed the development of PTSD symptomatology in these individuals 1 month later. Th eir results indicated that those who developed PTSD had significantly lower urinary CORT levels shor tly after the traumatic event than accident victims who did not develop PTSD. In addition, cortisol levels within the first few days after the accident were negatively correlated with the presence of intrusive memories of the trauma in these individuals. Together, these findings indicate that traumatized individuals who exhibit attenu ated CORT responses shortly after the traumatic event may be more susceptible to developing PTSD symptomatology. Baseline Studies comparing the baseline levels of CORT in PTSD patients and control subjects have produced mixed results. Most investigations have found that individuals with PTSD exhibit signifi cantly lower levels of CORT at baseline (Boscarino, 1996; Kanter et al., 2001; King, Mandansky, King, Fletcher, & Brewer, 2001; Mason, Giller, Kosten, Ostroff, & Podd, 1986; Yehuda, Southwick, Nussbaum, Wahby, Giller, & Mason, 1990; Yehuda, Boisoneau, Mason, & G iller, 1993b; Yehuda, Teicher, Trestman, Levengood, & Siever, 1996). Then again, others have found that these le vels are elevated (Carrion, Weems, Ray, Glaser, Hessl, & Reis s, 2002; Lemieux & Coe, 1995; Liberzon, Abelson, Flagel, Raz, & Young, 1999a; Linda uer et al., 2006; Lindley, Carlson, &

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21 Benoit, 2004; Maes et al., 1998; Pitman & O rr, 1990) or not different from controls (Baker et al., 1999; Duval et al., 2004; Yehuda, Golier, Halligan, Meaney, & Bierer, 2004). Boscarino (1996) found that, independent of PTSD symptomatology, the levels of CORT in Vietnam veterans were a function of the degree of combat exposure that they had experienced. As the veterans combat exposure increased, their levels of CORT decreased. Bremner (2001) hypothesized that the differences in basal CORT levels among PTSD patients could be dependent on the length of time that an individual has had the disorder. In other words, individuals with chronic PTSD may have had high CORT levels for such a long period of time that regulation of CORT production eventually became defective. However, Boscarino (1996) only observed low baseline CORT levels in Vietnam veterans who had current PTSD that is, they had de veloped it within the past year. Those with a lifetime diagnosis of PTSD did not have lower CORT levels than control subjects. In contrast to Bremner (2001), these invest igators reasoned that CORT hyporesponsivity could be related to the recency of PTSD, with more exaggerated effects being apparent closer to the time of PTSD onset. In any case, the HPA abnormalities observed in individuals with PTSD does not lead to impairments in CORT release during stress (Bremner et al., 2003a). In fact, so me studies have shown that PTSD patients exhibit an exaggerated CORT response to trauma-related stressors, in comparison to healthy controls (Elzinga, Schmahl, Vermetten, van Dyck, & Bremner, 2003). Cortisol is released in pulses (Deuschl e et al., 1997); its levels are highest upon awakening and lowest late in the even ing (Gunnar & Vazquez, 2001). Yehuda et al. (1996b) examined the baseline levels of CORT in combat veterans with PTSD at 30-min

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22 intervals over a 24-hr period of bed rest. Thei r results revealed that the individuals with PTSD had significantly lower le vels of CORT duri ng the late evening and early morning hours, which appeared to result from a prol onged nadir and shorter peak response in the cycle of CORT release. Because further analys es revealed a greater signal-to-noise ratio in PTSD patients, the inves tigators reasoned that HPA functioning in these individuals could provide optimal conditions for response to stress-related cu es. As Yehuda et al. (1996b, p. 86) suggested, the enhanced signal-to -noise ratio describe s a system with a maximally low background and, accordingly, a pote ntially greater capacity to respond to the environment. This could help explain why individuals diagnosed with PTSD are more reactive to their surroundings. In suppor t of this theory, Liberzon et al. (1999a) found that in response to combat sounds, ve terans with PTSD displayed significantly greater plasma CORT than controls. Investigators have also s hown that the production of the precursors to CORT is atypical in PTSD patients. Bremner a nd colleagues (Bremner et al., 1997b) found significantly elevated levels of CSF CRH in Vietnam veterans with PTSD. By using a serial CSF sampling technique, Baker and colle agues (Baker et al., 1999) replicated the findings of Bremner et al. ( 1997b) and correspondingly reported greater levels of CRH in combat veterans with PTSD. These findings cr eate a paradox that is, why do a majority of PTSD patients exhibit lower levels of CORT if they have significantly elevated levels of CRH? Well, Smith and colleagues (Smith et al., 1989) found that in response to CRH treatment, PTSD patients produced significantly less ACTH than heal thy control subjects, which would at least help expl ain the low levels of CORT ob served in individuals with

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23 PTSD. It is possible that persistent eleva tions of CRH desensitize CRH receptors in the anterior pituitary, l eading to a blunted AC TH release. In addition, Lerer, Ebstein, Shestatsky, Shemesh, and Greenberg (1987) f ound lower levels of cyclic adenosine monophophate (AMP) in individu als with PTSD. Since cyclic AMP is the second messenger for CRH stimulation of ACTH in the pituitary, these fi ndings could explain why PTSD patients exhibit a blunted ACTH response to CRH administration and why they can have greater levels of CRH and lower levels of cortisol simultaneously. Enhanced Negative Feedback of the HPA Axis A review of the literature suggests that the CORT differences in PTSD patients could be attributed to an enhanced negativ e feedback of the HPA axis. When CORT is released into the blood, it exerts a negative feedback on the HPA axis by binding to glucocorticoid receptors (GRs) in the brai n. One of the primary regions where GRbinding takes place is the hippocampus. In fact, this medial temporal lobe structure has one of the highest numbers of glucocortico id receptors in the mammalian brain (Kim & Diamond, 2002). Research has shown that PT SD patients display an increased number and sensitivity of glucocorti coid receptors (Yehuda, Bo isoneau, Lowy, & Giller, 1995; Yehuda et al., 1993b; Yehuda, Lowy, Southwick, Shaffer, & Giller, 1991). In addition, some studies have found an increased suppr ession of CORT (Grossman et al., 2003; Stein, Yehuda, Koverola, & Hanna, 1997b; Yehuda et al., 1995; Yehuda, Southwick, Krystal, Bremner, Charney, & Mason, 1993c) and ACTH (Duval et al., 2004; Yehuda et al., 2004) in PTSD patients in response to the administration of dexamethasone (DEX), a synthetic glucocorticoid. Theoretically, the DEX is capable of producing more negative

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24 feedback in PTSD patients, which leads to a greater suppression of CORT and ACTH. Nevertheless, other studies have failed to re plicate these findings (Kosten, Wahby, Giller, & Mason, 1990; Lindley et al., 2004). Investigators have also observed increased pituitary activation in PTSD patients following the administration of metyrapone, a gl ucocorticoid antagonist that blocks the conversion of 11-deoxycortisol to cortisol (or 11-deoxycorticote rone to corticosterone in rodents) (Kanter et al., 2001; Yehuda, Le vengood, Schmeidler, Wilson, Guo, & Gerber, 1996a). Since it prevents the production of cortisol, metyrapone hinders the negative feedback component of the HPA axis and cons equentially leads to gr eater levels of CRH, ACTH, and 11-deoxycortisol. These findings ther efore provide further support the theory that PTSD patients have enhanced negative feedback of the HPA axis. Can CORT Administrati on Help Treat PTSD? If a blunted CORT response to stress in creases an individual s susceptibility to develop PTSD, then perhaps the administrati on of CORT could prevent the onset of the disorder. Schelling et al. (1999) tested the hypothesis that CORT administration at the time of trauma would reduce the incidence of PTSD in the sample. These investigators examined the effects of CORT administration on patients with septic shock, a major systemic infection that ultimately leads to multiple organ dysfunctions and a considerable amount of physical and emotional stress. Sinc e a large percentage (upwards of 50%) of individuals who suffer from septic shock eventu ally develop PTSD, this group of subjects was a premier candidate for testing this hypot hesis. In the study, some patients received stress doses of hydrocortisone in addition to the standard treatment for the pathology.

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25 Administration of hydrocortisone during septic shock significa ntly reduced the incidence of PTSD in patients, as only 19% of the patients who received hydroc ortisone as part of their treatment developed the disorder, compared to the 59% of control patients who developed PTSD symptomatology. Other invest igators have examined the efficacy of CORT treatment in individuals who have al ready developed PTSD. Aerni et al. (2004) found that one month of low-dose cortisol administration (10 mg/day) resulted in a reduction of reexperiencing symptoms associated with traumatic memories. In addition, Soravia et al. (2006) even showed that the administration of cortisol reduces phobic fear in humans. Collectively, these studies suggest that the administra tion of CORT may not only help prevent the onset of PTSD, but al so serve as a therapeutic agent as well. Hippocampal Volume in PTSD Studies have reported smaller hippocampa l volumes in individuals who developed PTSD after combat exposure (Bremner et al ., 1995; Gurvits et al., 1996), firefighting (Shin et al., 2004), police work (Lindauer et al., 2006; Linda uer et al., 2004), childhood abuse (Bremner et al., 2003b; Bremner et al ., 1997c; Stein et al., 1997a), and mixed types of events, such as motor vehicle accidents a nd assaults (Villarreal et al., 2002; Wignall et al., 2004). Many of these studies have observed smaller hippocampi in individuals with PTSD even after adjusting for participants total brain volume and age. The debate over the mechanisms underlying these effects is still very active (Bremner, 2001; Pitman, 2001; Yehuda, 2001), and there is some dispute over the significance of these findings in relation to the disorder. For one, the diffe rences in hippocampal volume between PTSD patients and controls have ra nged from 5% to > 20%, and some may question whether or

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26 not these findings are biologica lly relevant to understandi ng the disorder. Other major problems with these studies include the como rbidity of PTSD with other disorders and the substance abuse that is often observed in these individuals. For instance, many of these studies have not controlled for comorb idity of PTSD with MDD. Major depressive disorder is in many key ways different from PTSD and could very well influence individuals hippocampal volumes. Despite the reports of smaller hippocampi in PTSD patients, some investigations (Bonne et al., 2001; De Bellis, Hall, Boring, Frustaci, & Moritz, 2001; De Bellis et al., 1999; Fennema-Notestine, Stein, Kennedy, Arch ibald, & Jernigan, 2002; Pederson et al., 2004; Schuff et al., 2001; Yamasue et al., 2003) have been unable to replicate these findings. These investigators, in other word s, have found no differences in hippocampal volume between the individuals diagnosed with PTSD and control subjects. The question arises, then, as to whether or not a smaller hippocampal volume is a necessary characteristic of all individuals with PTSD or if it simply increases susceptibility to developing the disorder in select individua ls. One study (Wignall et al., 2004) examined the hippocampal volumes of individuals with recent-onset PTSD and compared them to non-traumatized controls. After adjusting for total brain volume and participants age, the investigators found that the PTSD patients had significantly smaller right hippocampal volume than controls. Given that the average time between the trauma and the experiment was 158 days, these findings suggested that e ither (1) the hippocampi of PTSD patients atrophies quickly after the onset of the disorder or (2 ) a smaller hippocampus increases ones susceptibility to developing PTSD.

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27 To help resolve this issu e, Gilbertson and colleague s (Gilbertson et al., 2002) examined the hippocampal volumes of identical twins (all males). In each twin pair, one of the individuals had been exposed to th e Vietnam war, while the other had not. In addition, some of these individuals developed PTSD and some did not. The investigators found that hippocampal volumes of the trau matized individuals who developed PTSD was not different from those of their twin brothers; however, both of these individuals hippocampal volumes were smaller than those of the traumatized individuals who did not develop PTSD and their twin brothers. In addition, there was a negative relationship between the hippocampal volumes of the indivi dual with PTSD and his twin brother and PTSD symptom severity of the twin brothe r with the disorder. Thus, not only did the individual with PTSD have a smaller hippo campus than controls but so did his twin brother. These findings suggest that a small hippocampus is a risk factor for developing PTSD rather than a result of the disorder. Animal Models of PTSD Although PTSD remains a disorder that is unique to humans, the limitations of human research have compelled investigators to attempt to model the disorder in nonhuman animals. A valid animal model of PT SD would allow scien tists to study many aspects of the disorder, including (1) the f actors that contribute to the disorders development, (2) the neurobiological progression of the diso rder, and (3) the effects of novel therapeutic agents on trea tment of the disorder. Indeed, many researchers have used stressors such as electric s hock, immobilization (i.e., restrain t stress), underwater trauma, and predator stress to produce physiological and behavioral effects in rodents that are

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28 comparable to those observed in humans with PTSD. However, many of these models are inadequate, either because they lack face va lidity and ethological relevance or because they do not encompass the entire range of symp toms that are observed in PTSD patients. Rachel Yehuda, a well-known researcher who studies PTSD in humans, presented five criteria that all animal mode ls of PTSD should meet before they are accepted by the scientific community: (1) Even very brief stressors should be capable of induci ng biological and behavioral sequelae of PTSD. (2) The stressor should be capable of producing the PTSD-like sequelae in a dosedependent manner. (3) The stressor should produce biological alterations that persist over time or become more pronounced with the passage of time. (4) The stressor should induce bi obehavioral alterations th at have the potential for bidirectional expression (i.e., enhan ced and reduced responsiveness to different aspects of the environment). (5) Interindividual variability in response to a stressor should be present either as a function of experience (e.g., prio r stress history and poststressor adaptations), genetics, or an intera ction of the two (Yehuda & Antelman, 1993a, p. 480-482). These points are well warranted and should be kept in mind throughout the following review of the most relevant animal models of PTSD.

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29 Although PTSD is a disorder than can develop after very brief exposure to trauma, some investigators have likened the long-term effects of chronic stress in rodents to the disorder. Several studies have shown that chronic restraint stress (6 hrs/day for 21 days) leads to atrophy of hippocampal dendrite s (Magarinos et al ., 1996; Magarinos et al., 1995) and impairments of hippocampus-dep endent, spatial memory (Conrad et al., 1996; Luine et al., 1994). These effects have be en linked with the pe rsistent increase in glucocorticoids and excitatory amino acids (EAAs) that accompanies stress (Magarinos et al., 1995). In fact, the effects of restra int stress on hippocampal morphology can be blocked via the administration of NMDA recepto r antagonists (Magarinos et al., 1995) or pharmacological agents, such as phenytoin, th at reduce extracellular levels of EAAs (Watanabe, Gould, Cameron, Daniels, & Mc Ewen, 1992). Other work has investigated the long-term effects of milder psychosocial stress on rats func tioning. For instance, Park and colleagues (Park et al., 2001) examin ed the effects of chronic cat exposure and unstable housing conditions on rats behavior and sensit ivity to yohimbine. The investigators found that stress led to a significant impairment in rats spatial memory and an increased sensitivity to yohimbine, as evidenced by greater immobility in the open field following its administration. Similarly, Song, Che, Min-wei, Murakami, & Matsumoto (2006) found that unpredictable, chronic mild stress led to significant impairments in spatial memory, as did Gerg es and colleagues (Gerges, Alzoubi, Park, Diamond, & Alkadhi, 2004), who used 4-6 w eeks of unstable housing as the primary stressor in their study.

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30 Other investigators have studi ed the effects of a small nu mber of stress sessions or a single stress session with pe riodic reminders of the traum a on long-term behavior in rodents. Pynoos and colleagues (Pynoos, Ritzmann, Steinberg, Goenjian, & Prisecaru, 1996) exposed mice to 10 sec of footshock (2 mA) and then assessed their behavioral response 1, 21, or 42 days later. Some of th e stressed mice were reminded of the initial experience weekly throughout th e experiment by placing them back in the apparatus in which they received the footshocks. The inves tigators found that all of the stressed mice, regardless of whether or not they received the reminders, exhibited increased locomotor activity 24 hrs after being shocked. However, only those mice that were reminded of the shock on a weekly basis exhibited increased anxiety on the elevated plus maze and a heightened startle response during behavioral testing. In fact, these rats displayed increased anxiety on the plus maze 1, 21, and 42 days after being shocked, but they did not demonstrate an exaggerated startle response until six weeks post-stress. Similar to these findings, Servatius and colleagues (S ervatius, Ottenweller, Bergen, Soldan, & Natelson, 1994; Servatius, Ottenwelle r, & Natelson, 1995) observed a delayed sensitization of startle following exposure to repeated stress. Se rvatius et al. (1994) exposed rats to either 1 or 3 days of 2-hr stress sessions that in volved being restrained and exposed to 40, 2-mA tailshocks. The investigators found that the rats exposed to 3 days of stress exhibited a he ightened startle response 4 da ys, but not 1 or 10 days, after cessation of the stress. Servatius et al. (1995) used the same methodology to assess the effects of 1 or 3 days of stress on startle 4, 7, and 10 days after the stress. In contrast to their earlier findings, the results indicated that 1 day of stress led to a greater startle

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31 response 7 days post-stress, while the rats exposed to 3 days of stress exhibited exaggerated startle 10 days post-stress. Th ese findings were inconsistent with the investigators prior findings that 3 days of stress led to an exaggerated startle response 4 days after the stressor. Thus stress may induce a delayed se nsitization of rats startle response, but the timeline for this effect seems to be unclear. Robert Adamec and colleagues have exte nsively investigated the long-term effects of cat exposure on rats behavior. In a series of experiments, Adamec and Shallow (1993) found that a single 5-min exposure to a cat led to heightened anxiety in rats, as indicated by a reduction in ope n-arm exploration on the elevat ed plus maze, up to three weeks later. Further work by Adamec and colleagues (Adamec, Burton, Shallow, & Budgell, 1999; Blundell, Adamec, & Burton, 2005) supported the argument that these effects are mediated, in part, by NMDA-dependent plasticity in the brain. When rats were administered MK-801, AP7, or CPP (all co mpetitive NMDA-receptor antagonists) 30 min prior to cat exposure, the rats did not s how a lasting increase in anxiety. However, these drugs were incapable of blocking the stress-induced increas e in anxiety if they were administered 30 min after cat exposure, suggesti ng that they had to be present at the time of the stress to be effectiv e. In theory, it is NMDA-dependent plasticity in the amygdala that is in some ways responsible for the la sting effects of cat e xposure on anxiety. Studies have observed NMDA-dependent synaptic plasticity in the amygdala as a result of fear conditioning (Bauer, Schafe, & LeDoux, 2002; Rogan, Staubli, & LeDoux, 1997), and the administration of NMDA receptor antagon ists prevents the formation of fear memories in rodents (Fanselow, Kim, Yi pp, & De Oca, 1994; Kim, DeCola, Landeira-

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32 Fernandez, & Fanselow, 1991; Kim, Fansel ow, DeCola, & Landeira-Fernandez, 1992; Maren, Aharonov, Stote, & Fanselow, 1996). Thes e findings support the idea that stress induces NMDA-dependent plasticity within the amygdala, which results in increased anxiety-like behavior. To model PTSD, most investigators expos e rodents to some form of stress and then assess the effects of that stress on long-term physiology and behavior. The investigators typically present the results as mean values and therefore compare the entire stressed groups to control animals. Cohe n, Zohar, and Matar (2003) argued that, in practice, this stressed gro up of animals is not a homogenous population. Rather, some animals appear to be more vulnerable to the stress than others, which supports the fifth criterion in Yehuda & Antelmans (1993a) manu script (see above). Given this argument, Cohen et al. (2003) examined the differentia l response of rats to intense stress. The investigators exposed 150 rats to a cat for a period of 10 min and then examined their behavior on the elevated plus maze one week later. As a group, the stressed rats did exhibit greater levels of anxiet y on the elevated plus maze, re lative to controls. However, within the stressed group of rats there were some rats that di d not show elevated levels of anxiety and freely explored the open arms of the maze. Therefore, the investigators used cutoff behavioral criteria to divide the st ressed rats into well -adapted (WA) or maladapted (MA) rats. A rat was considered WA if it spent < 1 min in the closed arms of the plus maze and made > 8 entries into the open arms; a rat was considered MA if it spent the entire 5-min trial in the closed arms and made no entries into the open arms. These groups of stressed rats were then co mpared on additional physiological measures.

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33 Results of these analyses indicated that the MA rats exhibited greater levels of CORT and ACTH shortly after the stress than the WA rats. Moreover, the MA rats displayed lower HRV, with a higher HF and lower LF com ponent of their HRs, indicating greater sympathetic and lower vagal tone, respectiv ely. Cohen and colleagues have replicated and extended this work by reporting similar e ffects on other behavioral measures, such as the acoustic startle response, and using anot her type of stress underwater trauma (Cohen, Zohar, Matar, Kaplan, & Geva, 2005; Cohen, Zohar, Matar, Zeev, Loewenthal, & Richter-Levin, 2004). Collectively, these findings support the not ion that stress does not affect all rodents the same; rather, some appear to be more vulnerable to the effects of stress. These studies relate to humans in that not every traumatized individual reacts the same way to the stress. Other work has focused on developing a m odel of PTSD that compares well with the HPA alterations observed in human patients. For instance, Liberzon, Krstov, and Young (1997) found that exposing rats to a si ngle prolonged stress session (SPS) and restressing them 7 days later led to enhan ced negative feedback of the HPA axis. These investigators conducted additional work (Lib erzon, Lopez, Flagel, Vazquez, & Young, 1999b) using the SPS procedure and found that it not only led to enhanced negative feedback of the HPA axis but a differentia l regulation of GR a nd mineralocorticoid receptor (MR) mRNA in the hippocampus as well. Specifically, there was an upregulation of GR mRNA and a down-regulation of MR mRNA that lasted for at least 2 weeks in the stressed rats. These results provide insight into the mechanisms that may underlie the enhanced negative feedback of the HPA axis th at has been observed in

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34 humans with PTSD. As noted above, these individuals show greater numbers of GRs than controls, which could be responsible for a lower baseline level of CORT. Another group of researchers used a stress -restress paradigm si milar to that of Liberzon et al. (1997) to pr oduce PTSD-like physiological an d behavioral symptoms in rats. Harvey, Naciti, Brand, and Stein (2003) ex posed rats to several sequential stressors in one day, restressed them 7 days later, and then examined th eir physiological and behavioral responses one week later. The stre ssed rats displayed significant learning and memory impairments in a hippocampus-depende nt spatial memory task and had lower baseline levels of CORT, compared to contro ls. This is a finding that coincides with a majority of the literature on HPA functioni ng in human PTSD patients, providing support for the use of a stress-restress paradigm to model the disorder in non-human animals. Cohen et al. (2006) recently hypothesized that a blunted HPA axis response to stress may increase the susceptibility of rats to develop PTSD-like symptoms. These investigators tested the hypothesis by taking adva ntage of a strain of rats (Lewis rats) that naturally fails to show a stress-induced incr ease in CORT. They exposed the Lewis rats and two other strains of rats (Fisher and Sprague-Dawley ra ts, both produce the typical stress-induced increase in CORT) to a well-soil ed cat litter for 10 min and examined their behavioral responses 7 days later. While the Fisher and Sprague-Dawl ey rats exhibited a significant increase in CORT levels from ba seline to stress exposure, the Lewis rats showed no such elevation. The Lewis rats also exhibited more PTSD-like symptoms than the other strains, including a la rger startle response and grea ter anxiety on the elevated plus maze. Given these findings, the e xperimenters tested the hypothesis that

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35 administering CORT to the Lewis rats would am eliorate these effects. The results of this manipulation indicated that when the Lewis ra ts were given CORT prior to stress, they demonstrated fewer PTSD-like symptoms, a lo wer startle response, and less anxiety on the plus maze than the Lewis rats that were treated with vehicle. These findings suggest that a blunted CORT response during stress incr eases the susceptibility of rats to develop PTSD-like symptoms. However, there are some caveats for these results. For one, in this work, the Lewis rats displayed a significantly greater startle response and heightened anxiety, compared to the other rat strains, at base line. Therefore, the a bnormal HPA functioning exhibited by these rats may not just create greater PTSD-like symptoms in response to stress; it could produce them at baseline. In addition, the experimenters could not replicate the stress-induced increase in anxietylike behavior and star tle in the Lewis rats in the second experiment (the one in which CORT was administered to the Lewis rats). These problems, in addition to the short delay be tween stress and behavior al testing (7 days), demand further consideration. Hypotheses of the Present Experiments The following experiments were designed to mimic a subset of physiological features in rats that may c ontribute to the development of PTSD in humans. In the first experiment, rats were administered the pharmacological agents AF-DX 116 and metyrapone to respectively enhance HR and BP and inhibit the stress-induced rise in circulating CORT prior to two intense st ress sessions. Rats long-term behavioral responses to these manipulations were examined three weeks later. The second

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36 experiment extended the findings of the first experiment and assessed rats sensitivity to yohimbine three weeks after th e second stress session. I hypothesized that stress alone woul d result in long-term behavioral abnormalities, including enhancements of anxiety, startle, and fear conditioning and impairments of learning and memory. I al so hypothesized that a combination of decreased PNS activity (induced by AF-DX 116) and adrenal activity (induced by metyrapone) would exacerbate the effects, lead ing to a greater expr ession of behavioral abnormalities in these rats. Furthermore, I ex pected the stress to induce dynamic brain changes that would increase rats sensitivity to yohimbine.

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37 Chapter Two: Experiment One Does a Combination of Reduced PNS and Adre nal Activity Exacerbate Rats Long-Term Response to Stress? The initial experiment was designed to examine the long-term effects of a unique stress paradigm on rats physiology and behavi or. In addition, the study served to assess whether a combination of decreased PNS and adrenal activity at the time of stress could exacerbate these effects. Prio r to each of two stress sessi ons, rats were administered pharmacological agents to reduce their PNS and adrenal activity, conditions which are believed to contribute to the development of PTSD in humans. The second stress experience served to remind rats of the ini tial stress session and exacerbate the stressinduced effects on brain and behavior. It was also inte nded to add elements of unpredictability and uncontrollability to the stress experience. Three weeks after the second stress session, all rats were tested for the long-term effects of the stress experience on anxiety, startle, fear, and learning and memory through use of various behavioral assessments. Methods Rats Adult male Sprague-Dawley rats (225-250 g upon delivery) obtained from Charles River laboratories were used for th e present experiment. The rats were housed two to a cage (standard Plexiglas 46 x 25 x 21 cm), maintained on a 12-hr light-dark

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38 cycle (lights on at 0700), and had access to f ood and water ad libitum. Upon arrival, all rats were afforded a habituation period of one week to acclimate to the housing room and cage changes before any experimental manipulations took place. All procedures were approved by the Institutional Animal Care a nd Use Committee at the University of South Florida. Design The present study employed a 2 x 2 x 2 factorial design. The three manipulated factors were stress (stress, no stress), metyrapone (metyra pone, vehicle), and AF-DX 116 (AF-DX 116, vehicle). Each cell of the factorial had a samp le size of n = 10, except for the stressed rats treated with both vehi cles, which had a sample size of n = 8. Heart Rate/Blood Pressure Machine Habituation The measurement of heart rate and blood pressure (HR/BP) required immobilizing rats in a Plexiglas tube and placi ng their tails in a tail cuff sensor. The tube was designed to prevent excessive movement during testing, which would help eliminate movement artifacts during HR/BP recordi ng. Since rats find even such subtle immobilization stressful, it was necessary to habituate the rats to the procedure prior to the stress sessions. This would prevent (1 ) excessive movement artifacts during the testing sessions that could not be eliminat ed by the restraint al one and (2) misleading increases in the unstressed rats that were simply a result of the stress of the HR/BP measurement. All rats, regardless of group, were therefore transported to the laboratory one day prior to the first st ress session and exposed to the HR/BP apparatus for 10 min

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39 each. Afterwards, all rats were returned to the housing room, where they were left undisturbed for the remainder of the day. Pharmacological Agents The day after HR/BP habituation, all rats were divided into stress and no stress groups. The rats were transported to the laboratory where they sat for 30 min. Next, each rat was tail-marked with a black permanent marker and received an i.p. injection of metyrapone (50 mg/kg) or vehi cle. Metyrapone is a potent glucocorticoid synthesis inhibitor that blocks the conversion of 11-deoxycorticosterone to corticosterone. Previous work in our lab has shown that when administered at this dose, metyrapone significantly blunts the stressinduced CORT increase in rats (Park, Campbell, Woodson, Smith, Fleshner, & Di amond, 2006). The drug was dissolved in 40% polyethylene glycol soluti on and administered at a vo lume of 1 ml/kg. After the injection, all rats were left undisturbed for 1 hr. Subsequently, each rat received an i.p. injection of AF-DX 116 (2 mg/kg) or vehicle. AF-DX 116 is a well-studi ed presynaptic muscarinic M 2 receptor antagonist that has been found to significantly elevate heart rate and blood pre ssure in rats (Hata, Itoh, Funakami, Ishida, & Uchida, 2001). The drug works specifically on ca rdiac receptors by reducing PNS modulation of hear t rate (Giachetti, Micheletti, & Montagna, 1986). AF-DX 116 is poorly soluble in aqueous solu tions and was therefore dissolved in 0.1 N HCl. This solution was brought to a stable pH of 7.4 by adding 0.1 N NaOH and 0.9% saline and then administered at a volume of 1 ml/kg. After the injection, all rats were left

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40 undisturbed for 30 min, as resear ch indicates that AF-DX 116 begins having a significant effect on blood pressure and heart rate 30-60 min after administrati on (Hata et al., 2001). Stress Manipulations Thirty minutes after the administration of AF-DX 116 or vehicle, rats in the stress group were restrained in plastic DecapiCones (Braintree Scientific; Braintree, MA) and placed in a perforated wedge-shaped Plexiglas enclosure (Braintree Scientific; Braintree, MA; 20 x 20 x 8 cm). Rats in th e no stress group remained in their home cages until the HR/BP measurements describe d below. After 15 min, the stressed rats, restrained in the plastic DecapiCones and resting in the perforated, wedge-shaped Plexiglas enclosure, were placed in a metal cage (24 x 21 x 20 in) with an adult female cat for 45 min in a room located adjacent to the rat housing rooms. The cat posed no threat to the physical wellbe ing of the rats, as the plas tic DecapiCones and Plexiglas enclosure prevented any contact between th e two organisms. Also, canned cat food was placed on top of the Plexiglas enclosure to di rect cat activity towards the rats. After 45 min had elapsed, the rats were returned to the laboratory. Blood Sample Immediately after cat expos ure (or a yoked time period of 1 hr in the control animals), a blood sample was taken from all rats to examine the level of circulating CORT. Rats were placed in a wire mesh rest rainer, and a 2 mm tail snip was made with a sterile razor blade. A 0.5 cc sa mple of blood was collected in a microcentrifuge tube. After the blood had been collect ed, lidocaine and gauze were placed on the tip of the tail, and the rats were removed from the restrain er. All blood samples were collected within

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41 2-3 min after the rats were restrained in or der to obtain the samples before a rise in CORT could be detected in the serum. Once th e blood had clotted at room temperature, it was centrifuged (3000 rpm for 8 min), and the se rum was extracted and stored at -80 C until shipped for assay. Heart Rate and Blood Pressure After the blood sample was obtained, all rats HR and BP were assessed. Rats were immobilized in a Plexiglas tube w ithin a warming test chamber (ambient temperature = ~32 C) to increase their body temperature. This allowed for an enhancement of blood flow to the tail, which permitted HR and BP to be assessed using tail cuffs with photoelectric sensors (IITC Li fe Science; Woodland Hills, CA). After the rats body temperature had increased to a le vel suitable for HR/BP measurement (this took an average of 10 min), I attempted to obt ain three HR/BP recordings from each rat. For each recording, the tail cuff was inflated with a manual BP inflator, and the measurements were obtained via computer software that was provided by IITC Life Science, Inc. If three recordings could not be obtained, I attained as many recordings as possible (i.e., one or two recordings). In some instances (e.g., rat would not stop moving, HR/BP equipment was unable to acquire a valid reading, etc.), I could not acquire any recordings. These cases were simply rem oved from the analysis of HR/BP data. Completely Unstressed Control Group After each stress session, all rats were restrained to obtain HR/BP recordings and blood samples for analysis of CORT leve ls. These manipulations were undoubtedly stressful and could have had lasting effect s on the rats in the no stress conditions.

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42 Therefore, one group of unstressed rats (n = 10) were not exposed to either of these manipulations and simply received vehicle in jections during the stress sessions. Planned comparisons were made between the behavi or of this group and the vehicle-treated, unstressed rats that were rest rained during the stress sessions to determine the effects of the HR/BP and blood sampling manipulations on the rats long-term behavior. Stress Sessions All of the manipulations during the firs t stress session were performed during the rats light cycle, be tween 0700 and 1500 hrs. All rats that were stressed in the initial session were exposed to a second stress session 10 days later, as research has shown that 10 days of repeated immobilization stress si gnificantly modifies brain morphology (Vyas, Mitra, Shankaranarayana Rao, & Chattarji, 2002). The second stress session took place during the rats dark cycle to add elements of unpredictability and uncontrollability to the stress experience, as well as to reinforce stress-induced changes in brain and behavior initiated by the first stress session. During the second stress session, all rats were transported to th e laboratory. Here, the same pharmacological manipulations that were performed prior to the first stress session were performed again. Rats received th e same pharmacological agents that they received the first time, depending on the group to which they belonged. Then, the procedure that was followed in the first stress session was employed once more. The stress rats were restrained and exposed to th e cat, while the no stress rats remained in their home cages. Afterwards, another blood sample and HR/BP measurement was obtained from all rats. Upon completion of the st ress session, all rats were placed in their

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43 home cages and returned to the vivarium. All manipulations took place between 1900 and 0300 hrs. Randomized Housing After the initial stress se ssion, the stressed rats were exposed to unstable housing conditions until the commencement of behavior al testing. Previous research has found that social support aids in recovery from traumatic stress (Solom on, Mikulincer, & Flum, 1989; Tarrier & Humphreys, 2003). I therefore hypothesized that preventing a stable social environment in the stre ssed rats would prevent them from adapting appropriately to the stress experiences. All stress ed rats were still housed tw o per cage, but every day, the cage mate of each rat was changed. The random ization of the stressed rats occurred within groups, so each rat was exposed to ev ery other rat within its own group between 3 to 4 times prior to behavioral testing. No rat had the same cage mate on two consecutive days, and the randomization manipulati ons took place between 0800 and 1200 hrs. Handling, Weight, and Behavioral Testing Before behavioral testing began, all rats were handled for three consecutive days. The handling procedure involved transporting th e rats to the laboratory, placing them in a room with attenuated light a nd sound, and letting them sit for 1 hr. After 1 hr, each rat was handled for 2-3 min each. On the third and final day of handling, each rat was tail marked again, as the initial markings had wo rn down considerably by this time. All rats were weighed prior to the firs t stress session, prior to the second stress session, on the last day of handling, and on the last day of behavioral testing.

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44 3 weeks after the second stress session, all rats were te sted for fear, anxiety, and learning and memory through use of several behavioral assessments. I chose this time point because PTSD patients show long-term changes in these types of behaviors, and research involving rats has found that a single exposure to a predator can have significant effects on behavior up to 3 weeks later (Adamec & Shallow, 1993). All behavioral testing took place between 0700 and 1500 hrs. Behavioral Apparatus Elevated plus maze (EPM; see Figure 1) Twenty-four hrs afte r the last day of handling, all rats were transported to the la b and subjected to the EPM assessment. The EPM (Hamilton-Kinder; San Diego, CA) is an ap paratus that has been used extensively to study anxiety in rodents (Korte & DeBoer 2003). It consists of one open arm (10.80 x 51.17 cm) and one closed arm (10.80 x 51.17 cm) that intersect each other to form the shape of a plus sign. The in tersection area is 10.80 by 10.80 cm, and the walls of the closed arms are 40.01 cm high. The more time ra ts spend in the closed arms, the more anxious they are assumed to be. In other word s, time spent in the open arms is considered risk-taking behavior, as it plac es the rat in open view and su sceptible to danger. Each rat was placed on the EPM for 10 min, and its behavior was monitored by 48 infrared photobeams connected to a computer program (Motor Monitor) that analyzed the behavior. The program enabled the experiment er to assess the rats total ambulations, distance traveled in each area of the maze, di stance traveled overall, and time spent in each area of the maze. The primary measuremen t of concern was the percentage of time that each rat spent in the open arms, as compared to the closed arms. The EPM was

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45 wiped down with 25% ethanol solution between each testing session to reduce odor buildup in specific areas of the maze. Figure 1. Schematic diagram of the elevated plus maze. Startle response. Approximately 1 hr after the EPM assessment, all rats were subjected to a test of their startle reflex. Each rat was placed inside a restraint box that was inside a larger startle monitor cabinet (Hamilton-Kinder; San Diego, CA; 35.56 x 27.62 x 49.53 cm). Within the restraint box, the rats sat on a sensory transducer, which recorded their startle reflexes. The startle trial began with a 5-min acclimation period, followed by the presentation of 24 noise bursts, eight from each of three auditory intensities (90, 100, and 110 dB). The noise bursts were presented in sequential order (i.e. 8 bursts at 90 dB, followed by 8 bursts at 100 dB, etc.), and the time between each noise burst varied between 25 and 55 sec. Upon the commencement of the first noise burst, the startle apparatus provided an uninterrupted background noise of 57 dB. Each startle reflex was recorded in Newtons, and the complete session lasted approximately 20 min.

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46 Radial-arm water maze (RAWM; see Figure 2). Twenty-four hrs after the EPM and startle reflex assessments, all rats underwent RAWM training to assess their learning and memory. The RAWM consists of a black galvanized round tank (168 cm diameter, 56 cm height, 43 cm depth) filled with water (21-22 C). Using 6 V-shaped stainless steel walls (54 cm height, 56 cm length), the tank was divided into six arms radiating from an open central area. A black plastic platform (12 cm diameter) was placed 1 cm below the surface of the water at the end of one arm (the goal arm). Figure 2. Schematic diagram of the radial-arm water maze. The black arrows represent a path that rats might take to locate the hidden platform. All rats received a total of 12 acquisition trials to learn the location of the hidden platform. At the beginning of each trial, rats were released at the top of one arm (the start arm) and given 60 sec to find the hidden platform. If the rat could not locate the platform within the 60 sec time frame, I guided it to the platform. Once a rat found or was guided to the platform, it was left there undisturbed for 15 sec. For each trial, I recorded the

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47 number of errors made by each rat and how long the rat took to find the platform. An error consisted of (1) an en try into one of the arms that did not contain the hidden platform or (2) an entry into the arm that c ontained the hidden platform but an inability to locate it. The latter type of entry error was extremely rare over 98% of arm entry errors were entries into arms that did not contain the hidden platform. An arm entry was defined as the rat passing halfway down the arm. In addition, if a rat turn ed around and entered the arm in which it was released (i.e., the start arm), it was not c ounted as an error. However, if the rat left the start arm (i.e., en tered the open area) afte r being released, then a subsequent entry into the start arm was counted as an arm entry error. The hidden platform was placed in a different arm for each rat. This eliminated the possibility of odor cues building up in one arm and attracting rats to the goal arm solely for this reason. The goal arms for each rat were assigned randomly without replacement, and between trials, all f ecal boli were removed from the water. Additionally, the start arms va ried randomly as the trials progressed. All six arms were used as start arms before repeating a start arm a second time. The sequential order of the start arms was also random. All rats received 12 acquisition trials (T1T12) to learn the location of the hidden platform. Afterwards, they were dried and returned to their home cages where they spent a 1-hr delay period. The rats were then given a single short-term (1 hr) memory test trial (T13). Then, the rats were dried and returned to their home cages. To assess long-term memory, all rats were returned to the laborat ory 24 hrs later and gi ven one retention trial

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48 in the RAWM. Under control conditions, rats ha ve been shown to exhibit excellent 24-hr memory in this training paradigm. Fear conditioning Twenty-four hrs after the RAWM retention trial, all rats underwent fear conditioning. Fear conditioni ng training took place in a dark fear conditioning chamber (25.5 x 30 x 29 cm; Coul bourn Instruments; Allentown, PA) that consisted of two aluminum sides, an alumin um ceiling, and a Plexiglas front and back. The floor consisted of 18 stainless steel r ods, spaced 1.25 cm apart, through which shock could be delivered. Auditory stimuli were presented through a speaker located on one side of the chamber. The chamber was wipe d down with 25% ethanol solution after each training and retention session. The fear conditioning training paradigm consisted of placing the rat in a fear conditioning chamber. After a 2-min acclimation period had elapsed, a 10-sec tone (74 dB; 2500 Hz) was presented, which co-ter minated with a 2-sec, 0.4 mA footshock. Rats then endured a 50-sec post-shock pe riod, with the presence of no stimulus. Subsequently, another 10-sec tone was presented, which again co-terminated with a 2-sec, 0.4 mA footshock. Afterwards, there was a 30-sec final pos t-shock period, after which the experimenter removed the rat and retu rned it to its home cage. The entire fear conditioning training session lasted 240 sec (4 min). Fear conditioning retention tests were conducted 24 hrs after the training session. Rats freezing behavior served as an indicator of the rats memory. Rats behavior was measured by a 24-cell infrared activity mon itor (Coulbourn Instruments; Allentown, PA), mounted on the top of the fear conditioning ch amber, which uses the emitted infrared

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49 body heat image (1300 nm) from the animal to detect movement. Freezing was defined as periods of inactivity lasting 3 sec. A Microsoft Excel spreadsheet with a macro designed to analyze freezing be havior was used to calculate the total number of seconds spent freezing by each animal at 30-sec epoc hs. These parameters have been employed by laboratories elsewhere (Lee & Kim, 1998) and have been shown to significantly correlate with time sampling observer methods often employed to assess freezing behavior (Kim et al., 1991; Lee & Kim, 1998) This procedure allowed for accurate measurement of the rats freezing behavior without requiring the presence of the experimenter, which could interfere w ith the rats naturalistic behavior. To assess contextual fear conditioning, which is dependent upon the hippocampus and the amygdala (Phillips & LeDoux, 1992), rats were returned to the same context in which they were originally shocked. All ra ts remained in the shock context (without receiving shock) for 5 min, and freezing scor es were calculated for every 30-sec epoch. Freezing was presented as a percentage score, defined as the time of inactivity divided by the total time. An hour and a half after a ssessing contextual fear conditioning, the rats cue-based fear conditioning (i.e., fear of th e tone), which is dependent on the amygdala but independent of the hippocampus (Phillip s et al., 1992), was assessed. Rats were placed in the light side (25 x 22.5 x 33 cm ) of a shuttle box (Coulbourn Instruments; Allentown, PA) that consisted of two alum inum sides, an aluminum ceiling, and a Plexiglas front and back. A house light was turned on, and a metal plate (21.5 x 21.5 cm) was placed on the floor of the shuttle box to e liminate the sensation of the stainless steel rods beneath their paws. The rats remained in the light side of the shuttle box for a total

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50 of 6 min. During the first 3 min, no tone was presented; during the last 3 min, a tone (74 dB; 2500 Hz) was introduced to the rat. All rats were expected to show little or no freezing during the first 3 min (since it was a different context), and their freezing was expected to increase dramatically at the commencement of the tone. Therefore, rats freezing behavior in response to the tone serv ed as a measure of their memory for the tone-shock association, independe nt of the context, and the extent of freezing indicated the strength of this memory. Blood samples, heart rate, and blood pressure Twenty-four hrs after the fear conditioning retention tests, 3 blood samples and a HR/BP measurement were obtained from all rats to assess the long-term eff ects of the drug and stress manipulations on ANS and adrenal activity. All rats were placed in a wire mesh re strainer, and a 2 mm tail snip was made with a sterile razor blade. A 0.5 cc sample of blood was collected in a microcentrifuge tube. Lidocaine was then pl aced on the tip of th e tail. The first blood sample was the baseline measure of CORT in all rats and was collected within 2-3 min after rats were removed from their home cages to obtain the sample before a rise in CORT could be detected in the plasma. After obtaining this sample, all rats remained in the restrainer for 20 min. Then, the wound on th e tip of the tail wa s gently opened with sterile gauze, thereby making another tail snip unnecessary. Another 0.5 cc sample of blood was collected in a microcen trifuge tube. This stress measure of CORT served to examine rats hormonal response to restraint st ress. After collecting the sample, lidocaine and sterile gauze were placed on the tip of th e tail, and the rats were removed from the restrainer. Then, the rats were placed in a Plexiglas tube within a warming test chamber

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51 to increase their body temperatur e. The rats tails were plac ed in a tail cuff sensor, and once the rats body temperature was increased appropriately, I attemp ted to obtain three HR/BP recordings from each rat. The rats we re then returned to their home cages and remained undisturbed for 1 hr. Afterwards, one last blood sample was collected by placing rats in a wire mesh restrainer, openi ng the tail wound gently with sterile gauze, and obtaining 0.5 cc of blood in a microcentrif uge tube. Lidocaine was placed on the tip of the tail once the blood had been collected. This sample examined how well rats CORT levels recovered and returned to baseline after restraint stress. Once all of the blood had clotted at room temperature, it was centrifuged (3000 rpm for 8 min), and the serum was extracted and stored at -80 C until shipped for assay. Statistical Analysis Most data were analyzed with between-subjects, three-way analyses of variance (ANOVAs); mixed-model ANOVAs were employe d when repeated measures variables were a part of the behavior al assessment. Post-hoc comparisons were made through use of Bonferroni-corrected t -tests. Since I originally hypothesi zed that stress alone would have effects on behavior, planned comparisons were made between the st ressed rats treated with both vehicles and the unstressed rats treated with both vehicles in cases when the omnibus F test did not reveal a suspected difference between these two groups. Independent samples t -tests were used for these analyses. It was also of biological importance to determine whether or not each of the other groups was different from the unstressed rats treated with both vehicles. This group was

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52 considered a reference group, or prim ary control group, and therefore enabled differences from baseline to be detected with in the unstressed and stressed groups of rats. These differences could highlight specific drug effects of interest for future work. These comparisons were strictly post-hoc and were made using Bonferroni-corrected t -tests. Data points that were greater than three standard devi ation units beyond the exclusive mean were considered outliers and removed from the analyses. Less than 1% of the data were outliers, and no more than one data point was re moved from any single group of rats. All data are expressed as means SEM. Heart rate and blood pressure Three separate three-way ANOVAs were utilized to examine group differences in HR, systolic BP, and diastolic BP. Stress (stress, no stress), Metyrapone (50 mg/kg, vehicle), a nd AF-DX 116 (2 mg/kg, vehicle) served as the between-subjects factors at each time point (first stress session, second stress session, last day of behavioral testing). Corticosterone. Only those blood samples from groups that provided physiological and/or behavioral effects were assayed for CO RT levels. All of the blood samples from stress session one were a ssayed. Therefore, a three-way ANOVA was employed to compare the CORT levels betw een the groups. Stress, Metyrapone, and AF-DX 116 served as the betw een-subjects factors for the an alysis. Only some of the blood samples from stress session two were assayed. Since the AF-DX 116 manipulations resulted in little or no behavioral effects, this factor was dropped from the CORT analysis for stress session two. Thus, a two-way ANOVA was employed to compare the CORT levels between the gr oups. Stress and Metyrapone served as the

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53 between-subjects factors for the analysis. In addition, only some of the blood samples from the final day of testing were assa yed. Again, AF-DX 116 was dropped from this analysis. A mixed-model ANOVA was employed to analyze the CORT samples. Stress and Metyrapone served as the be tween-subjects factors, and ti me (baseline, stress, returnto-baseline) served as the within-s ubjects factor. It is important to note that the AF-DX 116 factor was kept in the analysis for stress session one for the sole purpose of determining if administration of th e drug affected rats CORT levels. Elevated plus maze (EPM) Each dependent measure acquired from the EPM was subjected to a three-way ANOVA, with Stre ss, Metyrapone, and AF-DX 116 serving as the between-subjects factors. Startle response. For each rat, there were eight startle responses at each of three auditory intensities. These eight responses we re averaged to create one data point per intensity per rat. The data from each audito ry stimulus intensity were analyzed using separate three-way ANOVAs, with Stress, Metyrapone, and AF-DX 116 serving as the between-subjects factors for each analysis. Radial-arm water maze (RAWM) Separate analyses were conducted on the acquisition trials, the 1-hr memory test trial, and the 24-hr retention trial for the RAWM data. A mixed-model ANOVA was used to co mpare arm entry errors between the groups during the acquisition trials. Stress, Metyrapone, and AF-D X 116 served as the betweensubjects factors, and trial (T1-T12) served as the within-subjects factor. Data from the 1-hr and 24-hr retention trials were anal yzed using three-way ANOVAs, with Stress, Metyrapone, and AF-DX 116 serving as the be tween-subjects factors for each analysis.

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54 Fear conditioning The fear conditioning retention tests (contextual fear conditioning, cue-based fear conditioning) were analyzed separately. Contextual fear conditioning freezing percentages were compared using a three-way ANOVA, with Stress, Metyrapone, and AF-DX 116 serving as the between-subjects factors. The cuebased fear conditioning freezing percentages were compared using a mixed-model ANOVA, with Stress, Metyrapone, and AFDX 116 serving as the between-subjects factors and tone (no tone, tone) se rving as the within-subjects factor. Weight. Although each group of rats was ordered for the same weight range, a one-way ANOVA indicated there we re weight differences between some of the groups at the time of the first stress session, F (7,71) = 8.65, p < .001. Nevertheless, all of the groups reached adult weight status by the ti me of stress session one (see Table 1). In order to examine group differences in weight over the course of the experiment, the amount of weight that rats gained between stress session one and each subsequent time point (i.e., stress session two, last day of handling, last day of behavioral testing) was analyzed using a mixed-model ANOVA, with Stress, Metyrapone, and AF-DX 116 serving as the between-subjects factors and time (i.e. first stress sess ion to second stress session, first stress session to the last day of handling, first stre ss session to th e last day of behavioral testing) serving as the within-subjects factor. Results Heart Rates and Blood Pressure during Stress Session 1 Heart rates (see Figure 3) The analysis of rats HR s during stress session one revealed no main effect of stress, F (1,57) = 0.94, p > .33. There was a main effect of

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55 metyrapone, F (1,57) = 9.19, p < .01, indicating that metyrapone-treat ed rats (435.36 6.79 bpm) had lower HRs than ve hicle-treated rats (463.32 6.24 bpm). In contrast, rats treated with AF-DX 116 ( 464.92 6.79 bpm) had higher HRs than vehicletreated rats (433.77 6.25 bpm), F (1,57) = 11.41, p < .001. The Stress x Metyrapone, F (1,57) = 0.59, p > .44, Metyrapone x AF-DX 116, F (1,57) = 0.04, p > .84, and Stress x Metyrapone x AF-DX 116, F (1,57) = 2.54, p > .11, interactions were not significant. There was a significant Stress x AF-DX 116 interaction, F (1,57) = 4.09, p < .05. Post hoc analyses indicated that AF-DX 116 increased HRs of rats that were not stressed (469.76 9.00 bpm), relative to vehicle-injected controls (419.97 9.00 bpm). However, the drug did not lead to a greater increas e in HR (460.07 10.16 bpm) than that which was produced by stress alone (447.57 8.66 bpm) ( ps < .05). A planned comparison revealed a trend, sugge sting that the stressed rats treated with both vehicles (466.27 12.23 bpm) displa yed greater HRs than the unstressed rats treated with both vehi cles (431.03 13.90 bpm), t (18) = 1.90, p = .07. The stressed rats treated with metyrapone, t (16) = -0.10, AF-DX 116, t (14) = 1.36, or both drugs, t (15) = 1.45, did not differ from the uns tressed rats treated with both vehicles ( p s > .05). The unstressed rats tr eated with AF-DX 116 (493.72 10.85 bpm) exhibited greater HRs than the unstresse d rats treated with both vehicles, t (18) = 3.56, p < .05. The unstressed rats treated with metyrapone, t (15) = -1.27, or both drugs, t (15) = 0.83, did not differ from the unstressed rats treated with both vehicles ( p s > .05).

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56 Heart Rate (bpm) 0390420450480510 AF-DX 116 Vehicle StressNo StressMetyraponeVehicleMetyraponeVehicle #**Heart Rates during Stress Session One Figure 3. Heart rates (+ SE) during stress session one. AF-DX 116 only increased heart rates in the unstressed rats. # p = .07 compared to unstressed rats treated with both vehicles; p < .05 compared to respective groups of unstressed rats treated with the vehicle for AF-DX 116. Systolic blood pressure (see Figure 4). There was a main effect of stress, F(1,55) = 14.05, p < .001, indicating that the stressed rats (139.46 1.97 mm Hg) had greater systolic BP than the unstressed rats (129.37 1.84 mm Hg). In addition, rats that were treated with AF-DX 116 (141.89 2.01 mm Hg) had greater systolic BP than vehicle-treated rats (126.05 1.79 mm Hg), F(1,55) = 30.76, p < .001. There was no main effect of metyrapone, F(1,55) = 0.04, p > .84, and the Stress x Metyrapone, F(1,55) = 0.05, p > .82, Stress x AF-DX 116, F(1,55) = 2.06, p > .15, Metyrapone x AF-DX 116, F(1,55) = 0.01, p > .93, and Stress x Metyrapone x AF-DX 116, F(1,55) = 1.50, p > .22, interactions were not significant.

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57 A planned comparison indicated that the stressed rats treated with both vehicles (132.73 3.12 mm Hg) exhibited greater systolic BP than the unstressed rats treated with both vehicles (121.48 4.57 mm Hg), t(17) = 2.07, p = .05. Systolic Blood Pressure (mm Hg) 0105120135150 AF-DX 116 Vehicle MetyraponeVehicleMetyraponeVehicle StressNo Stress****Systolic Blood Pressure during Stress Session One Figure 4. Systolic blood pressure (+ SE) during stress session one. Stress and AF-DX 116, in general, led to greater systolic blood pressure. p < .05 compared to respective rats treated with the vehicle for AF-DX 116; # p < .05 compared to unstressed rats treated with both vehicles. The stressed rats treated with metyrapone (135.13 2.46 mm Hg), t(15) = 2.54, AF-DX 116 (147.33 3.61 mm Hg), t(13) = 4.06, or both drugs (142.67 2.15 mm Hg), t(13) = 3.58, all displayed greater systolic BP than the unstressed rats treated with both vehicles (ps < .05). The unstressed rats treated with AF-DX 116 (137.21 3.30 mmHg), t(15) = 2.73, or both drugs (140.33 2.54 mm Hg), t(15) = 3.48, exhibited greater systolic BP than the unstressed rats treated with both vehicles (ps < .05). The unstressed

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58 rats treated with metyrapone did not differ from the unstressed rats treated with both vehicles, t (15) = -0.42, p > .68. Diastolic blood pressure (see Figure 5) There was a main effect of stress, F (1,57) = 45.00, p < .001, indicating that the stressed ra ts (99.54 1.35 mm Hg) had greater diastolic BP than the unstre ssed rats (87.32 1.22 mm Hg). In addition, rats that were treated with AF-DX 116 (97.20 1.36 mm Hg) had greater di astolic BP than vehicletreated rats (89.66 1.21 mm Hg), F (1,57) = 17.18, p < .001. The Stress x AF-DX 116 interaction was also significant, F v = 8.43, p < .01. Post hoc analyses indicated that AF-DX 116 increased diastolic BP in rats that were not stresse d (93.74 1.75 mm Hg), relative to vehicle-injected controls (80.91 1.71 mm Hg). However, the drug did not lead to a greater increase in diastolic BP (100.67 2.08 mm Hg) than that which was produced by stress alone (98.41 1.71 mm Hg) ( ps < .05). There was no main effect of metyrapone, F (1,57) = 0.10, p > .75, and the Stress x Metyrapone, F (1,57) = 0.20, p > .65, Metyrapone x AF-DX 116, F (1,57) = 0.00, p > .96, and Stress x Metyrapone x AF-DX 116, F (1,57) = 0.01, p > .90, interactions were not significant. A planned comparison indicated that the st ressed rats treated with both vehicles (98.60 2.86 mm Hg) exhibited gr eater diastolic BP than the unstressed rats treated with both vehicles (80.07 1.10 mm Hg), t (18) = 6.05, p < .001. The stressed rats treated w ith metyrapone (98.21 3.03 mm Hg), t (16) = 6.13, AF-DX 116 (100.72 2.28 mm Hg), t (14) = 9.19, or both dr ugs (100.61 1.70 mm Hg), t (14) = 10.64, displayed greater diastolic BP than unstressed rats treated with both vehicles ( p s < .05). Unstressed rats treated with AF-DX 116 (93.19 2.41 mmHg), t (17)

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59 = 5.13, or both drugs (94.29 2.02 mm Hg), t(16) = 6.53, exhibited greater diastolic BP than the unstressed rats treated with both vehicles (ps < .05). The unstressed rats treated with metyrapone did not differ from unstressed rats treated with both vehicles, t(16) = 0.54, p > .60. Diastolic Blood Pressure (mm Hg) 07590105 AF-DX 116 Vehicle MetyraponeMetyraponeVehicleVehicleStressNo Stress**Diastolic Blood Pressure during Stress Session On e Figure 5. Diastolic blood pressure (+ SE) during stress session one. Stress, in general, led to greater diastolic blood pressure, but AF-DX 116 only increased diastolic blood pressure in unstressed rats. p < .05 compared to respective unstressed rats; # p < .05 compared to unstressed rats treated with both vehicles. Heart Rates and Blood Pressure during Stress Session 2 Heart rates (see Figure 6). There was no main effect of stress, F(1,56) = 0.13, p > .71, or metyrapone, F(1,56) = 0.45, p > .50. There was a main effect of AF-DX 116, F(1,56) = 26.10, p < .001, indicating that rats treated with AF-DX 116 (473.26 6.22 bpm) had greater HRs than rats treated with vehicle (431.68 5.25 bpm). The Stress x Metyrapone interaction was not significant, F(1,56) = 0.08, p > .78. There was a

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60 significant Stress x AF-DX 116 interaction, F (1,56) = 15.89, p < .001. Post hoc analyses indicated that AF-DX 116 increased HRs of rats that were not stressed (469.76 9.00 bpm), relative to vehicle-injected c ontrols (419.97 9.00 bpm). However, the drug did not lead to a greater increase in HR (455.57 9.84 bpm) than that which was produced by stress alone (446.43 7.21 bpm) ( ps < .05). The Metyrapone x AF-DX 116 interaction was also significant, F (1,56) = 5.12, p < .05. Post hoc analyses indicated that only a combination of the two drugs result ed in greater HRs (479.75 9.61 bpm) than controls (445.29 10.40 bpm) (ps < .05). Lastly, the Stre ss x Metyrapone x AF-DX 116 interaction was significant, F (1,56) = 7.42, p < .01. As shown in Figure Four, a general pattern emerged from the results, indicati ng that AF-DX 116 produced relatively greater HRs compared to the respective vehicle-treate d rats. However, this was not the case for the stressed rats that had been administered the metyrapone vehicle. In this case, the opposite pattern was evident that is, AFDX 116 actually led to lower HRs than its respective vehicle. A planned comparison indicated that the st ressed rats treated with both vehicles (470.59 11.09 bpm) exhibited greater HRs than the unstressed rats treated with both vehicles (416.63 12.10 bpm), t (13) = 3.25, p < .01. The stressed rats treated with both drugs (472.00 17.76 bpm) displayed greater HRs than the unstressed rats treated with both vehicles, t (10) = 2.61, p < .05. The stressed rats treated with metyrapone, t (16) = 0.33, or AF-DX 116, t (13) = 1.38, did not differ from the unstressed rats treated with both vehicles ( ps > .05). The unstressed rats treated with AF-DX 116 (494.41 13.13 bpm), t (15) = 4.32, or both drugs (487.50 4.11 bpm),

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61 t(14) = 5.55, exhibited greater HRs than the unstressed rats treated with both vehicles (ps < .05). The unstressed rats treated with metyrapone did not differ from the unstressed rats treated with both vehicles, t(15) = 0.04, p > .96. Heart Rate (bpm) 0390420450480510 AF-DX 116 Vehicle MetyraponeMetyrapone VehicleVehicleStressNo Stress**###Heart Rates during Stress Session Two Figure 6. Heart rates (+ SE) during stress session two. AF-DX 116 led to greater heart rates in the unstressed rats. # p < .05 compared to unstressed rats treated with both vehicles; p < .05 compared to rats treated with the vehicle for AF-DX 116. Systolic blood pressure (see Figure 7). There was a main effect of stress, F(1,55) = 4.15, p < .05, indicating that the stressed rats (139.07 2.17 mm Hg) had greater systolic BP than the unstressed rats (133.21 1.90 mm Hg). In addition, rats that were treated with metyrapone (132.66 2.11 mm Hg) had lower systolic BP than vehicle-treated rats (139.62 1.96 mm Hg), F(1,55) = 5.85, p < .05. There was no main effect of AF-DX 116, F(1,55) = 1.01, p > .31, and the Stress x Metyrapone, F(1,55) = 3.16, p > .08, Stress x AF-DX 116, F(1,55) = 3.26, p > .07, and Metyrapone x AF-DX 116, F(1,55) = 0.78, p > .38 interactions were not significant. The Stress x Metyrapone x

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62 AF-DX 116 interaction, however, was significant, F(1,55) = 5.82, p < .05. As shown in Figure 7, the only time that AF-DX 116 produced a significant increase in systolic BP relative to vehicle-treated rats is when unstressed rats were administered the metyrapone vehicle as well (ps < .05). A planned comparison indicated that the stressed rats treated with both vehicles (148.46 4.80 mm Hg) exhibited greater systolic BP than the unstressed rats treated with both vehicles (125.33 5.64 mm Hg), t(13) = 3.07, p < .01. Systolic Blood Pressure (mm Hg) 0120130140150 AF-DX 116 Vehicle MetyraponeMetyrapone StressVehicleVehicleNo Stress**Systolic Blood Pressure during Stress Session Two Figure 7. Systolic blood pressure (+ SE) during stress session two. Stress, in general, led to greater systolic blood pressure. AF-DX 116 only produced greater systolic blood pressure in the unstressed rats that had been administered the vehicle for metyrapone. p < .05 compared to unstressed rats treated with both vehicles. The stressed rats treated with metyrapone, t(16) = 1.11, AF-DX 116, t(13) = 2.19, or both drugs, t(10) = 1.05, did not differ from the unstressed rats treated with both vehicles (ps > .05). The unstressed rats treated with AF-DX 116 (142.93 3.21 mm Hg)

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63 exhibited greater systolic BP than the uns tressed rats treated with both vehicles, t (15) = 2.79, p < .05. The unstressed rats treated with metyrapone, t (16) = 1.20, or both drugs, t (14) = 1.03, did not differ from the unstres sed rats treated with both vehicles (p s > .05). Diastolic blood pressure (see Figure 8) There was a main effect of stress, F (1,56) = 38.06, p < .001, indicating that stressed rats (100.40 1.28 mm Hg) displayed greater diastolic BP than unstressed rats (89.97 1.10 mm Hg). There was also a main effect of metyrapone, F (1,56) = 12.31, p < .001, indicating that rats treated with metyrapone (92.22 1.23 mm Hg) exhibited lowe r diastolic BP than rats tr eated with vehicle (98.15 1.16 mm Hg). There was no main effect of AF-DX 116, F (1,56) = 0.34, p > .56. The Stress x Metyrapone, F (1,56) = 8.55, p < .01, and Stress x AF-DX 116, F (1,56) = 40.17, p < .001, interactions were significant. Both of these interactions revealed that the stressed rats treated with ve hicle (either that of metyra pone or AF-DX 116 ) had greater diastolic BP than all other groups ( p s < .05). The Metyrapone x AF-DX 116 interaction was significant, F (1,56) = 5.23, p < .05. Post hoc analyses indicat ed that rats treated with metyrapone and the vehicle for AF-DX 116 (89.79 1.46 mm Hg) had lower diastolic BP than all other groups ( ps < .05). The Stress x Mety rapone x AF-DX 116 was also significant, F (1,56) = 8.53, p < .01. As shown in Figure 8, stre ssed rats that were treated with both vehicles showed the most prominent increase in diastolic BP ( ps < .05). A planned comparison indicated that the st ressed rats treated with both vehicles (115.08 2.66 mm Hg) exhibited greater diastolic BP than the unstressed rats treated with both vehicles (84.08 1.84 mm Hg), t (13) = 9.31, p < .001.

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64 Stressed rats treated with metyrapone (95.42 2.08 mm Hg), t(16) = 3.97, AF-DX 116 (96.57 3.04 mm Hg), t(13) = 3.62, or both drugs (94.50 2.57 mm Hg), t(10) = 3.28, displayed greater diastolic BP than the unstressed rats treated with both vehicles (ps < .05). Unstressed rats treated with AF-DX 116 (96.85 1.97 mm Hg), t(15) = 4.70, or both drugs (94.79 1.44 mm Hg), t(14) = 4.58, exhibited greater diastolic BP than the unstressed rats treated with both vehicles (ps < .05). The unstressed rats treated with metyrapone did not differ from the unstressed rats treated with both vehicles, t(16) = 0.03, p > .97. Diastolic Blood Pressure (mm Hg) 08090100110120 AF-DX 116 Vehicle MetyraponeMetyrapone VehicleVehicleStressNo Stress****###*Diastolic Blood Pressure during Stress Session Two Figure 8. Diastolic blood pressure (+ SE) during stress session two. Stress, in general, led to greater diastolic blood pressure. However, AF-DX 116 only produced greater diastolic blood pressure in the unstressed rats. # p < .05 compared to respective groups of unstressed rats treated with the vehicle for AF-DX 116; p < .05 compared to unstressed rats treated with both vehicles.

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65 Stress Sessions Corticosterone Levels Stress Session 1 Corticosterone Levels (see Figure 9) The analysis of CORT levels during stress session one revealed a main effect of stress, F (1,67) = 218.58, p < .001, indicating that the stressed rats ( 29.38 0.84 g/dL) had greater levels of CORT than the unstressed rats (11.46 0.88 g/dL). There was a trend effect of metyrapone, F (1,67) = 3.61, p = .06, suggesting that the rats treated with metyrapone (19.27 0.88 g/dL) had lower leve ls of CORT than the rats tr eated with vehicle (21.57 0.84 g/dL). There was no main effect of AF-DX 116, F (1,67) = 0.16, p > .69. The Stress x Metyrapone inte raction was significant, F (1,67) = 62.65, p < .001. Post hoc analyses indicated that the stressed rats tr eated with metyrapone (23.43 1.20 g/dL) had lower CORT levels than the stressed rats treated with vehicle (35.33 1.17 g/dL). However, these rats still had greater CORT le vels than the unstressed rats treated with metyrapone (15.11 1.27 g/dL) or vehicl e (7.82 1.20 g/dL), indicating that metyrapone did not completely block the st ress-induced increase in CORT levels. As expected, the stressed rats treat ed with vehicle exhibited greater CORT levels than the unstressed rats treated with metyrapone or vehicle. Surprisingl y, the unstressed rats treated with metyrapone displayed greater CORT levels than the uns tressed rats treated with vehicle ( ps < .05). The Stress x AF-DX 116, F (1,67) = 0.12, p > .73, Metyrapone x AF-DX 116, F (1,67) = 0.06, p > .80, and Stress x Metyrapone x AF-DX 116, F (1,67) = 1.51, p > .22, interactions were not significant.

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66 Corticosterone (g/dL) 010203040 AF-DX 116 Vehicle StressNo StressMetyraponeVehicleVehicleMetyrapone ******##%%%%Corticosterone Levels during Stress Session One Figure 9. Corticosterone levels (+ SE) during stress session one. Stress led to increased levels of corticosterone, and pre-treatment with metyrapone blunted this elevation. p < .05 compared to unstressed rats treated with the both vehicles or AF-DX 116; # p < .05 compared to stressed rats treated with vehicle or AF-DX 116; % p < .05 compared to unstressed rats treated with metyrapone or both drugs. Stress session 2 corticosterone levels (see Figure 10). The analysis revealed a main effect of stress, F(1,32) = 215.37, p < .001, indicating that the stressed rats (31.50 0.97 g/dL) had greater levels of CORT than the unstressed rats (10.73 1.04 g/dL). There was no main effect of metyrapone, F(1,32) = 0.01, p > .90. The Stress x Metyrapone interaction was significant, F(1,32) = 49.76, p < .001. Post hoc analyses indicated that the stressed rats treated with metyrapone (26.60 1.40 g/dL) had lower CORT levels than the stressed rats treated with vehicle (36.42 1.33 g/dL). However, these rats still had greater CORT levels than the unstressed rats treated with metyrapone (15.80 1.59 g/dL) or vehicle (5.65 1.33 g/dL), indicating that metyrapone did not

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67 completely block the stress-induced increase in CORT. As expected, the stressed rats treated with vehicle exhibited greater CORT levels than the unstressed rats treated with metyrapone or vehicle. Surprisingly, the unstressed rats treated with metyrapone again displayed greater CORT levels than the unstressed rats treated with vehicle (ps < .05). Stress Corticosterone (g/dL) 010203040 Metyrapone Vehicle No StressCorticosterone Levels during Stress Session Two***#%% Figure 10. Corticosterone levels (+ SE) during stress session two. Stress led to increased corticosterone levels, and pre-treatment with metyrapone blunted this elevation. p < .05 compared to unstressed rats treated with both vehicles; # p < .05 compared to stressed rats treated with vehicle; % p < .05 compared to unstressed rats treated with metyrapone. Elevated Plus Maze Ambulations (see Figure 11). Ambulations consist of beam breaks on the EPM and can serve as a useful measure of general locomotor activity. Analysis of total ambulations revealed a trend effect of stress, F(1,65) = 3.35, p = .07, suggesting that the stressed rats (374.15 15.67 ambulations) made fewer ambulations than the unstressed rats (414.48 15.48 ambulations). There was also a main effect of metyrapone, F(1,65) =

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68 14.03, p < .001, indicating that the rats treated with metyrapone (353.06 15.43 ambulations) made fewer ambulations than the rats treated with vehicle (435.57 15.72 ambulations). There was no main effect of AF-DX 116, F(1,65) = 2.81, p > .09, and the Stress x Metyrapone, F(1,65) = 0.24, p > .87, Stress x AF-DX 116, F(1,65) = 2.49, p > .12, Metyrapone x AF-DX 116, F(1,65) = 0.89, p > .34, and Stress x Metyrapone x AF-DX 116, F(1,65) = 2.39, p > .12, interactions were not significant. A planned comparison indicated that the stressed rats treated with both vehicles (387.38 16.52 ambulations) made fewer ambulations on the EPM than the unstressed rats treated with both vehicles (499.90 45.95 ambulations), t(16) = 2.09, p = .05. Total Ambulations 0225300375450525600 AF-DX 116 Vehicle StressNo StressMetyraponeVehicleVehicleMetyrapone ***Ambulations on the Elevated Plus Maze Figure 11. Ambulations (+ SE) on the elevated plus maze. Stress tended to result in fewer ambulations on the plus maze, as did the administration of metyrapone. p < .05 compared to unstressed rats treated with both vehicles. The stressed rats treated with both drugs (306.11 31.25 ambulations) made fewer ambulations on the EPM than the unstressed rats treated with both vehicles, t(17) =

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69 -3.41, p < .05. The stressed rats treated with metyrapone, t (17) = -2.41, or AF-DX 116, t (18) = -0.97, did not differ from the unstr essed rats treated with both vehicles ( p s > .05). The unstressed rats treated with both dr ugs (342.33 26.87 ambulations) made fewer ambulations on the EPM than the unstress ed rats treated with both vehicles, t (17) = -2.87, p < .05. The unstressed rats treated with metyrapone, t (18) = -1.82, or AF-DX 116, t (16) = -1.43, did not differ from the unstressed rats treated with both vehicles ( ps > .05). Percent time spent in the open arms (see Figure 12) Time spent in the open arms of the EPM was converted into a percent of to tal time score. The analysis of these scores revealed a main effect of stress, F (1,68) = 10.09, p < .01, indicating that the stressed rats (14.98 3.26 %) spent less percent time in the open arms than the unstressed rats (29.39 3.16 %). There was no main effect of metyrapone, F (1,68) = 3.00, p > .08, or AF-DX 116, F (1,68) = 0.37, p > .54. The Stress x AF-DX 116 interaction was significant, F (1,68) = 9.03, p < .01. Independent of being stressed (20.42 4.40 %) or not (21.20 4.53 %), rats that we re treated with AF-DX 116 spent a comparable amount of time in the open arms. However, stressed rats that were treated vehicle (9.54 4.79 %) spent less percent time in the open arms than unstressed rats that were treated with vehicle (37.59 4.41 %) ( ps < .05). The Stress x Metyrapone, F (1,68) = 0.64, p > .42, Metyrapone x AF-DX 116, F (1,68) = 0.05, p > .82, and Stress x Metyrapone x AF-DX 116, F (1,68) = 0.94, p > .33, interactions were not significant. A planned comparison indicated that the st ressed rats treated with both vehicles (9.97 2.09 %) spent less percent time in the open arms than the unstressed rats treated with both vehicles (46.03 5.32 %), t (16) = 5.75, p < .001.

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70 The stressed rats treated with metyrapone (9.12 3.03 %), t(18) = -1.76, or both drugs (16.61 5.54 %), t(18) = -3.83, spent less percent time in the open arms than the unstressed rats treated with both vehicles (ps < .05). The stressed rats treated with AF-DX 116 did not differ from the unstressed rats treated with both vehicles, t(18) = -2.41, p > .05. The unstressed rats treated with AF-DX 116 (15.87 5.39 %), t(17) = -3.97, or both drugs (18.15 5.99 %), t(17) = -3.49, spent less time in the open arms than the unstressed rats treated with both vehicles (ps < .05). The unstressed rats treated with metyrapone did not differ from the unstressed rats treated with both vehicles, t(18) = -1.91, p > .05. % Time 0102030405060 AF-DX 116 Vehicle No StressMetyraponeVehicleVehicleMetyrapone Stress*****Percent Time Spent in the Open Arms of theElevated Plus Maze Figure 12. Percent time spent in the open arms (+ SE) of the elevated plus maze in Experiment 1. Stress, in general, led to a reduction of open-arm exploration on the plus maze. p < .05 compared to unstressed rats treated with both vehicles. Percent time spent in the open arms, controlling for ambulations. Since there was a trend effect of stress for the analysis of total ambulations, one could argue that the

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71 stressed rats spent less time in the open arms because they moved less. Therefore, a threeway analysis of covariance (ANCOVA) was used to examine group differences in the percent of time spent in the open arms, with total ambulations on the EPM serving as the covariate. The analysis revealed that the main effect of stress remained significant, F (1,62) = 5.48, p < .05. There were, however, no main effects of metyrapone, F (1,62) = 0.01, p > .93, or AF-DX 116, F (1,62) = 1.61, p > .20. The Stress x AF-DX 116 interaction also remained significant, F (1,62) = 19.72, p < .001. The Stress x Metyrapone, F (1,62) = 0.00, p > .96, Metyrapone x AF-DX 116, F (1,62) = 1.87, p > .17, and Stress x Metyrapone x AF-DX 116, F (1,62) = 1.28, p > .26, interactions we re not significant. A one-way ANCOVA was also run to cont rol for total ambulations and compare the percent time that the stressed and unstres sed rats treated with both vehicles spent in the open arms. The analysis indi cated that even when total ambulations were controlled for, the stressed rats treated with both vehicles spent less percent time in the open arms than the unstressed rats treated with both vehicles, F (1,15) = 21.15, p < .001. Percent time spent in the cl osed arms (see Figure 13) Time spent in the closed arms of the EPM was converted into a percent of total time sc ore. The analysis of these scores revealed a main effect of stress, F (1,68) = 8.58, p < .01, indicating that the stressed rats (74.20 3.06 %) spent more percent time in the closed arms than the unstressed rats (61.54 3.05 %). There were no main effects of metyrapone, F (1,68) = 1.19, p > .27, or AF-DX 116, F (1,68) = 3.17, p > .08. The Stress x AF-DX 116 in teraction was significant, F (1,68) = 11.43, p < .001. Independent of being stress ed (70.74 4.20 %) or not (72.69 4.43 %), rats that were treated with AF-DX 116 spent a comparable amount of time in the

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72 closed arms. However, stressed rats that were treated with vehicle (77.66 4.46 %) spent more percent time in the closed arms than unstressed rats that were treated with vehicle (50.39 4.20 %) (ps < .05). The Stress x Metyrapone, F(1,68) = 0.03, p > .85, Metyrapone x AF-DX 116, F(1,68) = 0.12, p > .73, and Stress x Metyrapone x AF-DX 116, F(1,68) = 3.04, p > .08, interactions were not significant. A planned comparison indicated that the stressed rats treated with both vehicles (78.72 3.20 %) spent more percent time in the closed arms than the unstressed rats treated with both vehicles (43.14 4.56 %), t(16) = 6.07, p < .001. % Time 020406080100 AF-DX 116 Vehicle StressNo StressMetyraponeVehicleVehicleMetyrapone*****Percent Time Spent in the Closed Arms of theElevated Plus Maze Figure 13. Percent time spent in the closed arms (+ SE) of the elevated plus maze in Experiment 1. Stress, in general, increased the amount of time that rats spent in the closed arms. p < .05 compared to unstressed rats treated with both vehicles. The stressed rats treated with metyrapone (76.61 5.28 %), t(18) = 4.80, or both drugs (75.74 6.31 %), t(18) = 4.19, spent more percent time in the closed arms than the unstressed rats treated with both vehicles (ps < .05). The stressed rats treated with

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73 AF-DX 116 did not differ from unstressed rats treated with both vehicles, t (18) = 2.48, p > .05. The unstressed rats treated with AF-DX 116 (74.46 5.59 %), t (17) = 4.38, or both drugs, (70.93 6.69 %), t (17) = 3.49, spent more percen t time in the closed arms than unstressed rats treated with both vehicles ( p s < .05). The unstressed rats treated with metyrapone did not differ from unstressed rats treated with both vehicles, t (18) = 1.74, p > .05. Movement per unit time in the closed arms (see Figure 14) The distance that each rat traveled in the closed arms was divided by the amount of time it spent in the closed arms to produce a distance/time score. This score represented the distance that each rat traveled in the closed arms per the amount of time that it spent in the closed arms. The analysis of this data reveal ed a trend effect of stress, F (1,64) = 3.61, p = .06, suggesting that the stressed rats (11.88 0.39 cm/sec) trav eled less distance/time in the closed arms than the unstressed rats ( 12.92 0.39 cm/sec). Rats that were treated with metyrapone (11.76 0.38 cm/sec) traveled le ss distance/time in the closed arms than rats that were treated with vehicle (13.04 0.40 cm/sec), F (1,64) = 5.34, p < .05. There was a main effect of AF-DX 116, F (1,64) = 4.69, p < .05, indicating that rats treated with AF-DX 116 (11.80 0.38 cm/sec) traveled less distance/time in the closed ar ms than rats treated with vehicle (13.00 0.40 cm/sec). The Stress x Metyrapone, F (1,64) = 0.32, p > .57, Stress x AF-DX 116, F (1,64) = 0.23, p > .63, Metyrapone x AF-DX 116, F (1,64) = 1.02, p > .31, and Stress x Metyrapone x AF-DX 116, F (1,64) = 0.80, p > .37, interactions were not significant.

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74 A planned comparison indicated that the stressed rats treated with both vehicles (12.47 0.44 cm/sec) traveled less distance/time in the closed arms than the unstressed rats treated with both vehicles (14.72 0.86 cm/sec), t(16) = -2.16, p < .05. The stressed rats treated with metyrapone (11.94 0.59 cm/sec), t(18) = -2.67, or both drugs (10.70 0.95 cm/sec), t(18) = -3.14, traveled less distance/time in the closed arms than the unstressed rats treated with both vehicles (ps < .05). The stressed rats treated with AF-DX 116 did not differ from the unstressed controls, t(18) = -1.65, p > .05. The unstressed rats treated with metyrapone, t(18) = -1.25, AF-DX 116, t(17) = -1.35, or both drugs, t(18) = -1.76, did not differ from the unstressed controls (ps > .05). Distance Traveled per Time (cm/sec) 024681012141618 AF-DX 116 Vehicle StressNo StressMetyraponeVehicleVehicleMetyrapone***Distance/Time Traveled in the Closed Armsof the Elevated Plus Maze Figure 14. Distance/time traveled in the closed arms (+ SE) of the elevated plus maze in Experiment 1. Stress tended to decrease the distance/time that rats traveled in the closed arms, as did metyrapone. p < .05 compared to unstressed rats treated with both vehicles. Distance on the elevated plus maze (see Figure 15). There was a main effect of stress, F(1,68) = 5.22, p < .05, indicating that the stressed rats (6926.79 173.31 cm)

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75 traveled less distance on the EPM than the unstressed rats (7485.94 172.74 cm). In addition, rats that were treated with metyrapone (6791.50 170.45 cm) traveled less distance on the EPM than rats treated with vehicle (7621.23 175.56 cm), F(1,68) = 11.50, p < .001. There was also a main effect of AF-DX 116, F(1,68) = 6.95, p < .01, indicating that rats treated with AF-DX 116 (6883.89 172.74 cm) traveled less distance on the EPM than rats treated with vehicle (7528.84 173.31 cm). The Stress x Metyrapone, F(1,68) = 0.23, p > .63, Stress x AF-DX 116, F(1,68) = 0.11, p > .73, Metyrapone x AF-DX 116, F(1,68) = 1.69, p > .19, and Stress x Metyrapone x AF-DX 116, F(1,68) = 0.31, p > .57, interactions were not significant. Total Distance (cm) 0525060006750750082509000 AF-DX 116 Vehicle StressNo StressMetyraponeVehicleVehicleMetyrapone #**Distance Traveled on the Elevated Plus Maze Figure 15. Distance traveled on the elevated plus maze (+ SE) in Experiment 1. Stress, AF-DX 116, and metyrapone, in general, resulted in less distance traveled on the elevated plus maze. # p = .07 compared to unstressed rats treated with both vehicles; p < .05 compared to unstressed rats treated with both vehicles.

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76 A planned comparison suggested that the stressed rats tr eated with both vehicles (7337.38 197.61 cm) traveled less distance on th e EPM than the unstressed rats treated with both vehicles (8231.60 377.89 cm), t (16) = 1.94, p = .07. The stressed rats treated with or both drugs (6061.50 495.46 cm) traveled less distance on the EPM than the unstresse d rats treated with both vehicles, t (18) = -3.48, p < .05. The stressed rats treated with metyrapone, t (18) = -2.43, or AF-DX 116, t (18) = -1.85, did not differ from the unstressed rats treated with both vehicles, ( p s > .05). The unstressed rats treated with both drugs (6558.11 216.76 cm) traveled less distance on the EPM than the unstressed rats treated with both vehicles, t (17) = -3.73, p < .01. The unstressed rats treated with metyrapone (7467.50 294.29 cm), t (18) = -1.60, or AF-DX 116 (7686.56 324.74 cm), t (17) = -1.08, did not differ from the unstressed rats treated with both vehicles ( ps > .05). Fecal boli The amount of fecal boli that a rat deposits in an apparatus is often a good measure of its autonomic reac tivity to the test procedure. The analysis of fecal boli deposited on the EPM revealed a main effect of stress, F (1,69) = 12.81, p < .001, indicating that the stressed ra ts (3.10 0.27 boli) defecated mo re than the unstressed rats (1.74 0.27 boli). Moreover, ra ts that were treated with metyrapone (3.00 0.26 boli) defecated more than rats that were treated with vehicle (1.84 0.27 boli), F (1,69) = 9.34, p < .01. There was also a main effect of AF-DX 116, F (1,69) = 8.53, p < .01, indicating that rats treated with AF-DX 116 (1.87 0.27 bol i) defecated less than rats treated with vehicle (2.98 0.27 boli). The Stress x Metyrapone, F (1,69) = 1.46, p > .23, Stress x AF-DX 116, F (1,69) = 0.06, p > .81, Metyrapone x AF-DX 116, F (1,69) = 0.06, p > .81,

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77 and Stress x Metyrapone x AF-DX 116, F (1,69) = 1.07, p > .30, interactions were not significant. A planned comparison indicated that the st ressed rats treated with both vehicles (3.50 0.46 boli) defecated more in the EPM than the unstressed rats treated with both vehicles (1.20 0.51 boli), t (16) = 3.25, p < .01. Comparison of the control groups Independent samples t -tests were used to compare the behavior of the completely unstressed controls and the experimental unstressed controls on the EPM. There were no differences between the two groups when comparing their total ambulations, t (18) = -1.85, percent time spent in the open arms, t (18) = -1.22, entries into the open arms, t (18) = -1.84, percent time spent in the closed arms, t (18) = 1.44, distance/time trav eled in the closed arms, t (18) = -1.16, entries into the closed arms, t (18) = 0.52, total di stance traveled, t (18) = -1.76, and fecal boli deposited on the plus maze, t (18) = 0.11 (all ps > .05). Startle Response 90 dB auditory stimuli (see Figure 16) The analysis of startle responses to the 90 dB auditory stimuli revealed a main effect of stress, F (1,64) = 10.76, p < .01, indicating that the stressed ra ts (0.27 0.02 Newtons) exhibited a greater startle response than the unstressed rats (0.19 0.02 Newtons). There was also a main effect of AF-DX 116, F (1,64) = 14.76, p < .001, indicating that the rats treated with AF-DX 116 (0.18 0.02 Newtons) displayed a smaller startle response than the rats that were treated with vehicle (0.27 0.02 Newtons). Ther e was no main effect of metyrapone, F (1,64) = 2.76, p > .10. The Stress x Metyrapone intera ction was marginally significant, F (1,64) =

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78 3.58, p = .06. Post hoc analyses sugges ted that the stressed rats that were treated with vehicle (0.31 0.03 Newtons) exhibited greater startle responses than all other groups ( ps < .05). The Stress x AF-DX 116 interaction was significant, F (1,64) = 9.27, p < .01. Post hoc analyses indicated th at stressed rats treated w ith vehicle (0.35 0.03 Newtons) displayed greater startle res ponses than all other groups ( ps < .05). The Metyrapone x AF-DX 116 interacti on was significant, F (1,64) = 12.40, p < .001. Post hoc analyses indicated that the rats treated with both vehicles (0.34 0.03 Newtons) exhibited greater startle responses than all other groups ( ps < .05). The Stress x Metyrapone x AF-DX 116 interaction was significant, F (1,64) = 9.65, p < .01. Post hoc analyses indicated that the stressed rats that were treated with both vehicles (0.47 0.07 Newtons) displayed greater startle responses than all other groups ( ps < .05). Collectively, these effects indicated that metyrapone, AF-DX 116, and a combinati on of both pharmacological agents blocked the stress-induced enhancement of startle. A planned comparison indicated that the st ressed rats treated with both vehicles (0.47 0.07 Newtons) exhibited a greater startle response to the 90 dB auditory stimuli than unstressed rats treated with both vehicles (0.20 0.04 Newtons), t (15) = 3.55, p < .01.

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79 Stress Startle Response (Newtons) 0.00.10.20.30.40.50.6 AF-DX 116 Vehicle No StressMetyraponeVehicleVehicleMetyrapone *Startle Responses for the 90 dB Noise Bursts Figure 16. Startle responses (+ SE) for the 90 dB noise bursts in Experiment 1. Stressed rats treated with both vehicles show the greatest startle responses. p < .05 compared to all other groups. 100 dB auditory stimuli (see Figure 17). The analysis of startle responses to the 100 dB auditory stimuli revealed a trend effect of stress, F(1,69) = 3.40, p = .07, suggesting that the stressed rats (1.40 0.12 Newtons) exhibited a greater startle response than the unstressed rats (1.09 0.12 Newtons). There was a main effect of AF-DX 116, F(1,69) = 4.00, p < .05, indicating that the rats treated with AF-DX 116 (1.08 0.12 Newtons) displayed a smaller startle response than the rats that were treated with vehicle (1.42 0.12 Newtons). There was no main effect of metyrapone, F(1,69) = 1.85, p > .17. The Stress x Metyrapone, F(1,69) = 2.87, p > .09, Stress x AF-DX 116, F(1,69) = 0.21, p > .64, Metyrapone x AF-DX 116, F(1,69) = 0.02, p > .89, and Stress x Metyrapone x AF-DX 116, F(1,69) = 3.03, p > .08, interactions were not significant.

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80 A planned comparison indicated that the stressed rats treated with both vehicles (2.03 0.35 Newtons) exhibited a greater startle response to the 100 dB auditory stimuli than the unstressed rats treated with both vehicles (1.06 0.30 Newtons), t(16) = 2.12, p = .05. Startle Response (Newtons) 0.00.51.01.52.02.5 AF-DX 116 Vehicle StressNo StressMetyraponeVehicleVehicleMetyrapone *Startle Responses for the 100 dB Noise Bursts Figure 17. Startle responses (+ SE) for the 100 dB noise bursts in Experiment 1. Stressed rats treated with both vehicles show the greatest startle responses. p = .05 compared to unstressed rats treated with both vehicles. 110 dB auditory stimuli (see Figure 18). The analysis of startle responses to the 110 dB auditory stimuli revealed no main effects of stress, F(1,69) = 2.42, p > .12, metyrapone, F(1,69) = 2.35, p > .13, or AF-DX 116, F(1,69) = 0.39, p > .53. The Stress x Metyrapone interaction was significant, F(1,69) = 4.40, p < .05. Post hoc analyses indicated that while the stressed rats treated with vehicle (3.07 0.23 Newtons) exhibited a greater startle response than the unstressed rats treated with vehicle (2.27 0.21 Newtons), the stressed rats treated with metyrapone (2.28 0.21 Newtons)

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81 did not show this enhancement (ps < .05). The Stress x AF-DX 116, F(1,69) = 1.46, p > .23, Metyrapone x AF-DX 116, F(1,69) = 0.06, p > .81, and Stress x Metyrapone x AF-DX 116, F(1,69) = 0.93, p > .33, interactions were not significant. A planned comparison indicated that the stressed rats treated with both vehicles (3.35 0.25 Newtons) exhibited a greater startle response to the 110 dB auditory stimuli than the unstressed rats treated with both vehicles (2.07 0.33 Newtons), t(15) = 2.85, p < .01. Startle Response (Newtons) 01234 AF-DX 116 Vehicle StressNo StressMetyraponeVehicleVehicleMetyrapone *Startle Responses for the 110 dB Noise Bursts Figure 18. Startle responses (+ SE) for the 110 dB noise bursts in Experiment 1. Stressed rats treated with both vehicles show the greatest startle responses. p < .05 compared to unstressed rats treated with both vehicles. Fecal boli. The analysis of fecal boli deposited in the startle box revealed no main effect of stress, F(1,67) = 0.01, p > .92. However, there was a main effect of metyrapone, F(1,67) = 8.83, p < .01, indicating that rats treated with metyrapone (1.81 0.32 boli) defecated less than rats that were treated with vehicle (3.16 0.32 boli). There was no

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82 main effect of AF-DX 116, F (1,67) = 0.00, p > .99, and the Stress x Metyrapone, F (1,67) = 0.03, p > .85, Stress x AF-DX 116, F (1,67) = 1.87, p > .17, Metyrapone x AF-DX 116, F (1,67) = 1.91, p > .17, and Stress x Metyrapone x AF-DX 116, F (1,67) = 0.19, p > .66, interactions were not significant. Comparison of the control groups A planned comparison indicated that the experimental unstressed controls (0.20 0.04 Newtons) exhibited a greater startle response to the 90 dB auditory stim uli than the completely unstressed controls (0.11 0.01 Newtons), t (16) = 2.39, p < .05. However, the two groups did not differ in terms of their startle response to the 100 dB, t (18) = 1.14, and 110 dB, t (18) = 0.42, auditory stimuli ( ps > .05). Radial-Arm Water Maze Acquisition (see Figure 19) The within-subjects analysis of arm entry errors from the 12 acquisition trials revealed a main effect of trial, F (11,770) = 43.07, p < .001, indicating that the rats adequately learned the spatial memory task and made less arm entry errors as the trials progressed. The Trial x Stress, F (11,770) = 1.05, p > .39, Trial x Metyrapone, F (11,770) = 0.56, p > .86, and Trial x AF-DX 116, F (11,770) = 1.79, p > .05, interactions were not significan t. The Trial x Stress x Metyrapone, F (11,770) = 0.86, p > .58, Trial x Stress x AF-DX 116, F (11,770) = 0.75, p > .69, Trial x Metyrapone x AF-DX 116, F (11,770) = 1.58, p > .10, and Trial x Stress x Metyrapone x AF-DX 116, F (11,770) = 1.10, p > .35, interactions were not significant. The between-subjects analysis revealed no main effects of stress, F (1,70) = 0.09, p > .76 or metyrapone, F (1,70) = 1.91, p > .17. However, there was a trend effect for

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83 AF-DX 116, F(1,70) = 3.61, p = .06, suggesting that rats treated with AF-DX 116 (1.18 0.07 errors) tended to make more errors during acquisition than rats treated with vehicle (0.99 0.07 errors). The Stress x Metyrapone, F(1,70) = 0.02, p > .89, Stress x AF-DX 116, F(1,70) = 0.67, p > .41, Metyrapone x AF-DX 116, F(1,70) = 1.26, p > .26, and Stress x Metyrapone x AF-DX 116, F(1,70) = 0.2, p > .89, interactions were not significant. 2-Trial Blocks B1B2B3B4B5B6Arm Entry Errors 012345 Metyrapone-AF-DX 116-Stress Metyrapone-AF-DX 116-No Stress Metyrapone-Vehicle-Stress Metyrapone-Vehicle-No Stress Vehicle-AF-DX 116-Stress Vehicle-AF-DX 116-No Stress Vehicle-Vehicle-Stress Vehicle-Vehicle-No Stress Acquisition Curve in the Radial-Arm Water Maze Figure 19. Acquisition curve (+ SE) in the radial-arm water maze. All rats adequately learned the task, as evidenced by fewer arm entry errors as the trials progressed. One-hour memory (see Figure 20). The analysis of arm entry errors on the 1-hr memory test trial revealed no main effects of stress, F(1,70) = 2.55, p > .12, metyrapone, F(1,70) = 2.55, p > .12, or AF-DX 116, F(1,70) = 0.28, p > .59. There was a significant Stress x Metyrapone interaction, F(1,70) = 4.53, p < .05. Post hoc analyses indicated that the metyrapone-treated, stressed rats (0.45 0.09 errors) made more errors than the vehicle-treated, stressed rats (0.10 0.10 errors) and the unstressed rats that were

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84 administered metyrapone (0.10 0.09 errors) or vehicle (0.15 0.09 errors) (ps < .05). There was also a significant Stress x AF-DX 116 interaction, F(1,70) = 4.53, p < .05. Independent of being stressed (0.15 0.09 errors) or not (0.20 0.09 errors), the rats treated with AF-DX 116 made a comparable number of arm entry errors. However, when the vehicle-treated rats were stressed (0.40 0.10%), they made more arm entry errors than the unstressed, vehicle-treated rats (0.05 0.09%) (ps <. 05). Arm Entry Errors 0.00.20.40.60.81.01.2 AF-DX 116 Vehicle No StressMetyraponeVehicleVehicleMetyrapone Stress *Arm Entry Errors on the One-Hour Memory TestTrial in the Radial-Arm Water Maze Figure 20. Arm entry errors (+ SE) on the 1-hr memory test trial in the radial-arm water maze. Stressed rats treated with metyrapone made more arm entry errors than all other groups. p < .05 compared to all other group combinations. The Metyrapone x AF-DX 116 interaction was significant, F(1,70) = 10.18, p < .01. Post hoc analyses revealed that rats treated with AF-DX 116 made a comparable number of arm entry errors, regardless of whether they had been administered metyrapone (0.10 0.09 errors) or its vehicle (0.25 0.09 errors). On the contrary, rats injected with the vehicle for AF-DX 116 made more errors when they were administered

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85 metyrapone (0.45 0.09 errors) than when they were treated with the metyrapone vehicle (0.00 0.00 errors) (ps < .05). The Stress x Metyrapone x AF-DX 116 interaction was not significant, F(1,70) = 2.55, p > .12. Twenty-four hour memory (see Figure 21). The analysis of arm entry errors on the long-term memory test trial revealed no main effect of stress, F(1,64) = 3.04, p > .08. There was a trend effect of metyrapone, F(1,64) = 3.65, p = .06, suggesting that rats treated with metyrapone (0.68 0.16 errors) made more errors than rats treated with vehicle (0.23 0.17 errors). There was no main effect of AF-DX 116, F(1,64) = 1.39, p > .24. Arm Entry Errors 0.00.51.01.52.02.53.0 AF-DX 116 Vehicle No StressMetyraponeVehicleVehicleMetyrapone Stress *Arm Entry Errors on the Twenty-Four Hour Memory Test Trial in the Radial-Arm Water Maze Figure 21. Arm entry errors (+ SE) on the 24-hr memory test trial in the radial-arm water maze. Stressed rats treated with metyrapone made more arm entry errors than all other groups. p < .05 compared to other group combinations. The Stress x Metyrapone interaction was significant, F(1,64) = 6.82, p < .05. Post hoc analyses revealed that the metyrapone-treated, stressed rats (1.20 0.22 errors) made

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86 more errors than the vehicletreated, stressed rats (0.13 0.25 errors) and the unstressed rats that were administered metyrapone (0.17 0.24 errors) or vehicle (0.33 0.24 errors) ( ps < .05). The Stress x AF-DX 116, F (1,64) = 2.00, p > .16, Metyrapone x AF-DX 116, F (1,64) = 1.24, p > .27, and Stress x Metyrapone x AF-DX 116, F (1,64) = 1.82, p > .18, interactions were not significant. Comparison of the control groups A mixed-model ANOVA was utilized to compare arm entry errors on the acquisition tria ls between the control groups, with trials serving as the within-subjects variable. The an alysis revealed a main effect of trial, F (11,198) = 11.67, p < .001, indicating that all of the rats made fewer arm entry errors as the trials progressed. The Trial x Gr oup interaction was not significant, F (11,198) = 0.55, p > .86, nor was the between-subjects effect of group, F (1,18) = 0.56, p > .46. Independent samples t -tests indicated that the cont rol groups made a comparable amount of arm entry errors on the 1-hr, t (18) = -1.00, and 24-hr memory test trials, t (16) = 1.11 (all p s > .05). Fear Conditioning Contextual fear memo ry (see Figure 22) The amount of time spent freezing during the 5-min contextual fear memory test was converted into a pe rcent of total time score. The analysis of these scores revealed a main effect of stress, F (1,70) = 4.34, p < .05, indicating that the stressed rats ( 68.09 4.55 %) froze more than the unstressed rats (54.89 4.41 %). There were no main effects of metyrapone, F (1,70) = 3.26, p > .07, or AF-DX 116, F (1,70) = 1.09, p > .30. There was a signific ant Stress x Metyrapone interaction, F (1,70) = 6.49, p < .05. While the rats treated wi th vehicle exhibited similar

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87 levels of freezing independent of being stressed (65.73 6.62 %) or not (68.67 6.24%), those rats treated with metyrapone froze more if they were stressed (70.44 6.24 %) than if they were not stressed (41.10 6.24 %) (ps < .05). The Stress x AF-DX 116, F(1,70) = 0.18, p > .67, Metyrapone x AF-DX 116, F(1,70) = 1.50, p > .22, and Stress x Metyrapone x AF-DX 116, F(1,70) = 0.01, p > .91, interactions were not significant. Freezing % 020406080100 AF-DX 116 Vehicle StressNo StressMetyraponeVehicleVehicleMetyrapone **Freezing during the Contextual Fear Memory Test Figure 22. Freezing (+ SE) during the contextual fear memory test. p < .05 compared to respective unstressed rats treated with metyrapone. Context test fecal boli. The analysis of fecal boli deposited during the context test revealed no main effects of stress, F(1,69) = 0.01, p > .93, metyrapone, F(1,69) = 0.01, p > .93, or AF-DX 116, F(1,69) = 0.00, p > .96. The Stress x Metyrapone, F(1,69) = 0.40, p > .52, Stress x AF-DX 116, F(1,69) = 2.34, p > .13, Metyrapone x AF-DX 116, F(1,69) = 0.34, p > .56, and Stress x Metyrapone x AF-DX 116, F(1,69) = 0.53, p > .46, interactions were not significant.

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88 Cued fear memory (see Figures 23 & 24). The amount of time spent freezing during the 6-min cue fear memory test (no tone, tone) was converted into a percent of total time score. For the within-subjects analysis, there was a main effect of tone, F(1,67) = 147.51, p < .001, indicating that rats froze more in the presence of the tone (61.65 3.72 %) than when the tone was not presented (23.93 2.45 %). The Cue x Stress interaction was not significant, F(1,67) = 0.45, p > .50. There was a significant Cue x Metyrapone interaction, F(1,67) = 7.43, p < .01. When there was no tone presented, rats exhibited similar levels of freezing, independent of being administered metyrapone (24.22 3.31 %) or vehicle (23.63 3.61 %). However, those rats treated with metyrapone (53.48 5.02 %) froze less when the tone was presented than rats treated with vehicle (69.83 5.49 %) (ps < .05). Freezing % 01020304050 AF-DX 116 Vehicle StressNo StressMetyraponeVehicleVehicleMetyrapone Freezing during the Cue Memory Test without thePresence of the Tone Figure 23. Freezing (+ SE) during the cue memory test without the presence of the tone.

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89 The Cue x AF-DX 116 interaction was also significant, F (1,67) = 4.50, p < .05. When the tone was presented, rats exhibited similar levels of freezing, independent of being administered AF-DX 116 (59.73 5.02 %) or vehicle (63.57 5.49 %). However, those rats treated with AF-DX 116 (28.59 3.31 %) froze more when there was no tone presented than rats treated with vehicle (19.26 3.61 %) ( p s < .05). The Cue x Stress x Metyrapone, F (1,67) = 1.14, p > .29, Cue x Stress x AF-DX 116, F (1,67) = 0.09, p > .76, Cue x Metyrapone x AF-DX 116, F (1,67) = 0.21, p > .64, and Cue x Stress x Metyrapone x AF-DX 116, F (1,67) = 2.59, p > .11, interactions were not significant. For the between-subjects analysis, ther e was a main effect of stress, F (1,67) = 5.23, p < .05, indicating that the stressed rats (49.05 4.00 %) froze more than the unstressed rats (36.53 3.75 %). There we re no main effects of metyrapone, F (1,67) = 2.07, p > .15, or AF-DX 116, F (1,67) = 0.25, p > .61. The Stress x Metyrapone interaction was at a trend level, F (1,67) = 3.60, p = .06. While the rats treated with vehicle exhibited similar levels of freezing independent of being stressed (47.79 6.04%) or not (45.66 5.37 %), those rats treated with metyrapone tended to freeze more if they were stressed (50.31 5.23 %) than if they were not stressed (27.39 5.23 %) ( ps < .05). The Stress x AF-DX 116, F (1,67) = 0.41, p > .52, Metyrapone x AF-DX 116, F (1,67) = 0.26, p > .61, and Stress x Metyrapone x AF-DX 116, F (1,67) = 0.44, p > .51, interactions were not significant.

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90 Freezing % 020406080100 AF-DX 116 Vehicle StressNo StressMetyraponeVehicleVehicleMetyrapone Freezing during the Cue Memory Test with thePresence of the Tone Figure 24. Freezing (+ SE) during the cue memory test with the presence of the tone. Cue test fecal boli. The analysis of fecal boli deposited during the cue test revealed no main effects of stress, F(1,70) = 1.62, p > .20, metyrapone, F(1,70) = 0.82, p > .36, or AF-DX 116, F(1,70) = 0.29, p > .59. The Metyrapone x AF-DX 116 interaction was significant, F(1,70) = 7.70, p < .01. Post hoc analyses revealed that rats treated with metyrapone and AF-DX 116 (1.70 0.40 boli) defecated less than rats treated with metyrapone (3.05 0.40 boli) or AF-DX 116 alone (3.20 0.40 boli) (ps < .05). The Stress x Metyrapone, F(1,70) = 2.40, p > .12, Stress x AF-DX 116, F(1,70) = 0.28, p > .59, and Stress x Metyrapone x AF-DX 116, F(1,70) = 0.03, p > .86, interactions were not significant. Comparison of the control groups. Independent samples t-tests were used to compare the control groups freezing and defecation during the context test. The groups did not differ in the amount of freezing they displayed during the context test, t(18) =

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91 -0.14, p > .89. They also did not differ in the am ount of fecal boli that was deposited in the apparatus during the test, t (18) = 0.11, p > .91. A mixed-model ANOVA was employed to compare the control groups freezing during the cue test, with cue (no tone, tone) serving as the within-subjects factor. This analysis revealed a main effect of cue, F (1,18) = 47.55, p < .001, indicating that the rats froze more when the tone was presented (69.93 6.44 %) than when it was not presented (31.48 6.19 %). However, the Cue x Group interaction was not significant, F (1,18) = 2.42, p > .13, and there was no main effect of group, F (1,18) = 0.46, p > .50. An independent samples t -test also indicated that the groups did not differ in terms of the amount of fecal boli deposited in the apparatus during the cue test, t (18) = 0.00, p = 1.00. Final Days Heart Rate and Blood Pressure Heart rates (see Figure 25) The analysis of HRs from the final day of testing revealed a main effect of stress, F (1,64) = 12.88, p < .01, indicating that the stressed rats (386.13 5.48 bpm) had lower HRs than th e unstressed rats (413.50 5.31 bpm). There was also a main effect of metyrapone, F (1,64) = 11.21, p < .01, indicating that rats treated with metyrapone (387.05 5.15 bpm) had lower HRs than rats treated with vehicle (412.58 5.62 bpm). Moreover, rats treate d with AF-DX 116 (386.62 5.29 bpm) had lower HRs than rats treated with vehicle (413.00 5.50 bpm), F (1,64) = 11.96, p < .01. There was also a significant Me tyrapone x AF-DX 116 interaction, F (1,64) = 4.04, p < .05. Specifically, rats that were treated w ith metyrapone (392.57 7.19 bpm), AF-DX 116 (391.72 7.57 bpm), or both drugs (381.52 7.38 bpm) had lower HRs than rats that had been administered both vehicles (433.44 8.32 bpm) ( ps < .05). The Stress

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92 x Metyrapone, F(1,64) = 3.06, p > .08, Stress x AF-DX 116, F(1,64) = 1.47, p > .23, and Stress x Metyrapone x AF-DX 116, F(1,64) = 1.37, p > .24, interactions were not significant. Planned comparisons indicated that the stressed rats treated with both vehicles (404.00 4.16 bpm) exhibited lower HRs than the unstressed rats treated with both vehicles (462.88 10.22 bpm), t(13) = -5.06, p < .001. Heart Rate (bpm) 0350400450500 AF-DX 116 Vehicle StressNo StressMetyraponeVehicleMetyraponeVehicle *Heart Rates during the Final Day of Behavioral Testing Figure 25. Heart rates (+ SE) during the final day of behavioral testing. Unstressed rats treated with both vehicles showed the greatest heart rates. p < .001 compared to all other groups. The stressed rats treated with metyrapone (385.40 12.36 bpm), t(16) = -4.50, AF-DX 116 (380.44 6.56 bpm), t(15) = -6.45, or both drugs (374.67 5.62 bpm), t(15) = -7.18, displayed lesser HRs than the unstressed rats treated with both vehicles (ps < .01). The unstressed rats treated with metyrapone (399.73 13.03 bpm), t(16) = -3.54, AF-DX 116 (403.00 13.84 bpm), t(15) = -3.29, or both drugs

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93 (388.38 10.47 bpm), t (16) = -4.79, exhibited lesser HRs than the unstressed rats treated with both vehicles ( ps < .01). Systolic blood pressure (see Figure 26) There was a main effect of stress, F (1,63) = 4.37, p < .05, indicating that the stressed rats (135.60 2.44 mm Hg) exhibited greater systolic BP than the unstressed rats (128.32 2.48 mm Hg). There were no main effects of metyrapone, F (1,63) = 0.12, p > .73, or AF-DX 116, F (1,63) = 1.03, p > .31. There was a significant Stress x Metyrapone interaction, F (1,63) = 9.57, p < .01. Independent of being stressed (130.80 3.33 mm Hg) or not (134.30 3.24 mm Hg), rats that were treated with metyrapone tended to exhibit similar systolic BP. However, stressed rats that were treated with vehicle (140.39 3.57 mm Hg) displayed gr eater systolic BP than unstressed rats that were treate d with vehicle (122.35 3.75 mm Hg) ( ps < .05). The Stress x AF-DX 116, F (1,63) = 0.33, p > .56, Metyrapone x AF-DX 116, F (1,63) = 0.81, p > .37, and Stress x Metyrapone x AF-DX 116, F (1,63) = 0.04, p > .85, interactions were not significant. A planned comparison indicated that the st ressed rats treated with both vehicles (136.38 2.57 mm Hg) exhibited gr eater systolic BP than the unstressed rats treated with both vehicles ( 119.69 5.11 mm Hg), t (12) = 2.92, p < .01.

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94 Systolic Blood Pressure (mm Hg) 0110120130140150 AF-DX 116 Vehicle StressNo StressMetyraponeVehicleMetyraponeVehicle **Systolic Blood Pressure during the Final Day ofBehavioral Testing Figure 26. Systolic blood pressure (+ SE) during the final day of behavioral testing. Stressed rats, in general, exhibited elevated systolic blood pressure. However, this effect was most pronounced in the stressed rats treated with the vehicle for metyrapone. p < .05 compared unstressed rats treated with both vehicles. The stressed rats treated with AF-DX 116 (144.40 5.51 mm Hg) displayed greater systolic BP than the unstressed rats treated with both vehicles, t(15) = 3.14, p < .05. The stressed rats treated with metyrapone, t(15) = 1.26, or both drugs, t(14) = 2.10, did not differ from the unstressed rats treated with both vehicles (ps > .05). The unstressed rats treated with metyrapone, t(15) = 1.80, AF-DX 116, t(13) = 0.87, or both drugs, t(15) = 2.13, did not differ from the unstressed rats treated with both vehicles (ps > .05). Diastolic blood pressure (see Figure 27). There was a main effect of stress, F(1,65) = 21.45, p < .001, indicating that the stressed rats (95.51 1.46 mm Hg) had greater diastolic BP than the unstressed rats (85.71 1.53 mm Hg). There were no main

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95 effects of metyrapone, F(1,65) = 1.42, p > .23, or AF-DX 116, F(1,65) = 0.22, p > .64. The Stress x Metyrapone, F(1,65) = 1.91, p > .17, Stress x AF-DX 116, F(1,65) = 1.63, p > .20, Metyrapone x AF-DX 116, F(1,65) = 0.74, p > .39, and Stress x Metyrapone x AF-DX 116, F(1,65) = 0.04, p > .84, interactions were not significant. Planned comparisons indicated that the stressed rats treated with both vehicles (96.67 3.82 mm Hg) exhibited greater diastolic BP than the unstressed rats treated with both vehicles (86.24 2.80 bpm), t(13) = 2.15, p = .05. Diastolic Blood Pressure (mm Hg) 08090100 AF-DX 116 Vehicle StressNo StressMetyraponeVehicleMetyraponeVehicle **Diastolic Blood Pressure during the Final Day ofBehavioral Testing Figure 27. Diastolic blood pressure (+ SE) during the final day of behavioral testing. Stressed rats, in general, exhibited elevated diastolic blood pressure. However, this effect was most pronounced in the stressed rats treated with the vehicle for metyrapone. p < .05 compared to unstressed rats treated with both vehicles. The stressed rats treated with AF-DX 116 (99.80 2.09 mm Hg) displayed greater diastolic BP than the unstressed rats treated with both vehicles, t(15) = 3.97, p < .05. The stressed rats treated with metyrapone, t(15) = 1.70, or both drugs, t(15) =

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96 2.01, did not differ from the unstressed rats treated with both vehicle (ps > .05). The unstressed rats treated with metyrapone, t(15) = 0.40, AF-DX 116, t(14) = 0.44, or both drugs, t(15) = 0.04, did not differ from the unstressed rats treated with both vehicles (ps > .05). Correlation between Cardiovascular Activity and Anxiety Average Diastolic BP (mm Hg) during the Stress Sessions 708090100110120% Time in the Open Arms of the EPM 020406080 r2 = 0.19Relationship between Average Diastolic Blood Pressure &Percent Time Spent in the Open Arms of the Elevated Plus Maze Figure 28. Relationship between average diastolic blood pressure during the stress sessions and percent time spent in the open arms of the elevated plus maze. Greater diastolic blood pressure during the stress sessions was associated with less time spent in the open arms of the elevated plus maze during behavioral testing. Relationship between cardiovascular activity during the stress sessions and anxiety-like behavior on the elevated plus maze (see Figure 28). Since stress produced a robust increase in rats diastolic BP during the stress sessions and on the last day of behavioral testing, an analysis was run to examine the correlation between rats average diastolic BP during the stress sessions and their behavior on the EPM (specifically, the

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97 percent time spent in the open arms). The analysis of this data revealed a significant negative relationship between these two variables, r(70) = -0.43, p < .001, indicating that as the rats diastolic BP (during the stress sessions) increased, the amount of time that they spent in the open arms of the EPM (weeks later) decreased. Correlation between Corticosterone Levels and Average Startle Response Corticosterone Levels (g/dL) during Stress Session One 0102030405060Average Startle Response (Newtons) 0.00.51.01.52.02.53.0 r2 = 0.14Relationship between Corticosterone Levels duringStress Session One & Average Startle Response Figure 29. Relationship between corticosterone levels during stress session one and average startle response. Greater corticosterone levels during stress session one was associated with greater startle responses during behavioral testing. Relationship between corticosterone levels during stress session one and average startle responses (see Figure 29). Since stress produced a robust increase in rats CORT levels during the stress sessions, an analysis was run to examine the correlation between rats CORT levels during stress session one and their average startle response (the effect was witnessed for each level of auditory stimulus, so rats startle responses from the 90, 100, and 110 dB noise bursts were averaged to facilitate the discussion and graphical

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98 representation of the data). The analysis of this data revealed a significant positive relationship between these two variables, r (68)= 0.37, p < .01, indicating that as the rats CORT levels (during stress se ssion one) increased, their av erage startle response (weeks later) also increased. Final Days Corticosterone Levels (see Figure 30) The within-subjects analysis revealed a main effect of time, F (2,54) = 86.76, p < .001. Post hoc analyses indicated that the rats exhibited greater CORT levels 20 min after the onset of stress (22.82 0.85 g/dL) than they did at baseline (4.68 0.69 g/dL). These levels were lower 1 hr after the cessation of stress (12.54 1.47 g/dL), but they were still el evated relative to baseline levels ( ps < .05). The Time x Metyrapone interact ion was also significant, F (2,54) = 5.28, p < .01. While the metyrapone-treated rats showed a reduction in CORT levels 1 hr after the cessation of stress (Stress: 23.20 1.17 g/dL; Return -to-Baseline: 9.59 2.03 g/dL), the CORT levels in the vehicle-treated rats remained elevated (Stress: 22.44 1.23 g/dL; Returnto-Baseline: 15.49 2.14 g/dL) ( ps < .05). The Time x Stress, F (2,54) = 1.31, p > .27, and Time x Stress x Metyrapone, F (2,54) = 0.75, p > .47, interactions were not significant. The between-subjects analysis reve aled no main effects of stress, F (1,27) = 2.28, p > .14, or metyrapone, F (1,27) = 0.36, p > .55. However, the Stress x Metyrapone interaction was significant, F (1,27) = 6.40, p < .05. Rats that were treated with vehicle exhibited comparable CORT levels, independent of whether they were stressed (13.05 1.56 g/dL) or not (14.47 1.28 g/dL). However, rats that were treated with

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99 metyrapone exhibited greater CORT levels when they had been stressed (15.74 1.36 g/dL) than when they had not been stressed (10.12 1.36 g/dL) (ps < .05). 0 min20 min40 min60 min80 minCorticosterone (g/dL) 051015202530 Vehicle-Vehicle-Stress Vehicle-Vehicle-No Stress Metyrapone-Vehicle-Stress Metyrapone-Vehicle-No Stress (Baseline)(Stress)(Return-to-Baseline) Restraint Stress Corticosterone Levels during the Final Day of Behavioral Testing Figure 30. Corticosterone levels ( SE) during the final day of behavioral testing in Experiment 1. All rats showed a significant increase in corticosterone levels following restraint stress. However, only the metyrapone-treated rats showed a return-to-baseline of corticosterone levels 1 hr later. The corticosterone levels of those rats treated with vehicle remained elevated. Weight (see Table 1) The within-subjects analysis revealed a main effect of time, F(2,138) = 2999.26, p < .001, indicating that all of the rats gained a significant amount of weight over the course of the experiment. The Time x Stress, F(2,138) = 2.32, p > .10, Time x Metyrapone, F(2,138) = 0.14, p > .77, and Time x AF-DX 116, F(2,138) = 1.20, p > .30, interactions were not significant.

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100 The Time x Stress x Metyrapone interaction wa s significant, F (2,138) = 4.12, p < .05. Post hoc analyses indicated that the unst ressed, metyrapone-treated rats (57.90 2.11 g) gained more weight from stre ss session one to stress session two than the stressed, metyrapone-treated rats (45.90 2.11 g). However, the vehicle-treated rats gained a comparable amount of weight fr om stress session one to stress session two, regardless of whether they were stressed (50.14 2.24 g) or not (51.19 2.17 g). By the start of behavioral testing, th ese differences were not evident. However, they resurfaced by the final day of testing, indicating that testing may have had a greater impact on the stressed, metyrapone-treated rats than the unstressed, metyrapone-treated rats ( p s < .05). The Time x Stress x AF-DX 116 inte raction was also significant, F (2,138) = 7.18, p < .001. According to post hoc analyses, rats th at were stressed and treated with AF-DX 116 gained less weight than all other groups from stress session one to stress session two (46.90 2.11 g), from stress session one to the last day of handling (130.70 4.46 g), and from stress session one to the last day of testing (135.30 4.50 g) ( ps < .05). The Time x Metyrapone x AF-DX 116 interaction was significant, F (2,138) = 9.99, p < .001. Post hoc analyses indicated that while there were no differences between the groups in terms of body wei ght gained from stress sess ion one to stress session two, the metyrapone-treated rats that were al so administered the vehicle for AF-DX 116 gained more weight than all of the other gr oups from stress session one to the last day of handling (148.20 4.46 g) and to the last day of behavioral testing (153.75 4.50 g) ( ps < .05). The Time x Stress x Mety rapone x AF-DX 116 interaction was not significant, F (2,138) = 0.76, p > .47.

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101 Table 1 Average Raw Weights for all Groups in Experiment 1 Raw weights (g SE) _____________________________________________________ Stress Stress Last day Final day Group session 1 session 2 of handling of testing Stress Metyrapone AF-DX 116 275.90 (1.04) 323.10 (2.66) 401.30 (5.86) 401.10 (6.37) Vehicle 278.00 (3.24) 322.60 (3.83) 424.20 (6.35) 423.50 (6.56) Vehicle AF-DX 116 275.80 (2.39) 322.40 (3.65) 411.80 (5.61) 421.20 (6.36) Vehicle 275.45 (3.11) 330.90 (6.05) 418.13 (10.60) 421.50 (10.00) No Stress Metyrapone AF-DX 116 287.50 (3.36) 340.10 (5.64) 425.10 (10.44) 434.80 (10.42) Vehicle 266.10 (2.31) 329.30 (3.08) 416.30 (6.89) 428.10 (6.57) Vehicle AF-DX 116 282.90 (3.27) 333.50 (4.20) 426.40 (8.01) 428.40 (8.50) Vehicle 292.78 (2.60) 344.56 (4.22) 419.33 (6.41) 429.33 (5.46) Completely unstressed controls 300.25 (2.10) 376.60 (7.79) 470.00 (18.09) 478.85 (20.33)

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102 The between-subjects analyses revealed no main effect of stress, F(1,69) = 1.84, p > .18, metyrapone, F(1,69) = 0.17, p > .67, or AF-DX 116, F(1,69) = 2.61, p > .11. The Stress x Metyrapone interaction was significant, F(1,69) = 5.17, p < .05. Post hoc analyses indicated that while the rats that were treated with vehicle gained a comparable amount of weight, regardless of being stressed (112.41 3.75 g) or not (109.08 3.64 g), the metyrapone-treated rats that were stressed (105.68 3.54 g) gained less weight than the metyrapone-treated rats that were not stressed (118.82 3.54 g) (ps < .05). The Stress x AF-DX 116, F(1,69) = 1.02, p > .31, Metyrapone x AF-DX 116, F(1,69) = 3.63, p > .06, and Stress x Metyrapone x AF-DX 116, F(1,69) = 0.96, p > .33, interactions were not significant. SS1 SS2SS1 BehaviorSS1 Final DayWeight Gained (g) 050100150200 Stress No Stress Completely Unstressed Body Weight Gained in Experiment One Figure 31. Body weight gained ( SE) throughout the course of Experiment 1. Both the experimental controls and the stressed rats gained less weight than the completely unstressed controls throughout the course of the experiment.

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103 Comparison of the control groups A mixed-model ANOVA was run on the weights that the rats in the c ontrol groups gained, with time serving as the within-subjects variable. The analysis revealed a significant effect of time, F (2,28) = 114.23, p < .001, indicating that all rats gain ed a significant amount of wei ght over the course of the experiment. The Time x Group in teraction was not significant, F (2,28) = 1.93, p > .16. However, there was a main effect of group, F (1,14) = 4.58, p = .05, indicating that the completely unstressed controls (141.57 11.37 g) gained a more weight than the experimental controls (101.83 14.68 g). Discussion Major Findings and Significance The primary finding of this study is that st ress alone increased rats cardiovascular and hormonal activity at the time of stress a nd led to heightened anxiety, an exaggerated startle response, and a sensiti zed blood pressure response to acute stress 3 weeks later. Although stress alone did not affect learning and memory in the water maze, the rats that had been administered metyrapone in additi on to being stressed exhibited significant memory impairments on the 1-hr and 24-hr me mory test trials. These findings suggest that a reduced amount of CORT at the time of stress may exacerbate its long-term effects on cognition. Despite the fact that metyrapone and AF-DX 116 blunted the stress-induced increases in CORT (Figures 9 and 10) and le d to greater HRs and BP (Figures 3 through 8), respectively, there was little evidence to indicate that these agents exacerbated the effects of stress on rats anxiet y-like behavior. In fact, ther e was actually some indication that the drugs, particul arly metyrapone, actually blocked the stress-induced increase in

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104 startle. Although the present pharmacologica l manipulations were unsuccessful in exacerbating the stress-induced effects on anxi ety, this study does provide evidence for a novel model of PTSD in rats. Specifically, it demonstrates that exposure to unpredictable and uncontrollable stress, in conjunction with a reminder experience, produces behavioral and physiological symptoms in rats that are comparable to those observed in humans with PTSD. In contrast to some animal mode ls of PTSD, the present model used an ethologically relevant stressor, cat exposure, to produce the e ffects. Moreover, it consists of a sequence of events, incl uding a traumatic reminder experience and daily mild stress, which closely resembles the daily life of a human subject with the disorder. Stress-Induced Enhancements of An xiety and Acoustic Startle Response The finding that stress led to a signifi cant reduction of open-arm exploration on the EPM is consistent with the findings of other animal models of PTSD (Adamec & Shallow, 1993; Cohen et al., 2006; Cohen et al., 2003). When rats are placed on the EPM, they face a decision: (1) to enter the open arms and explore novel places (which they naturally do in experimental sett ings) or (2) to stay in the cl osed arms and remain out of harms way. Following stress in the present study, the rats appeared to choose the latter, inhibiting their natura l tendency to explore novel places. However, there was evidence indicating that the stressed rats exhibited less activity (i.e ., fewer ambulations) than the unstressed rats on the plus maze. To contro l for these findings, an ANCOVA was run on the open arms data (i.e., percent time spent in the open arms) with ambulations serving as the covariate. The results revealed that the effects of stress on open-arm exploration remained significant after controlling for the ra ts general activity. Thus, it would appear

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105 that the effects of stress on ope n-arm exploration, at least in the present paradigm, are independent of its effects on general locomotor activity. The finding that stressed rats tended to move less on the EPM is consistent with previous research. Many studies have shown that stress can le ad to a general decrease in activity on the EPM and other be havioral apparatuses, such as the open field (Adamec et al., 1999; Adamec & Shallow, 1993; Bowman, Ferguson, & Luine, 2002; Katz, Roth, & Carroll, 1981; Ottenweller, Natelson, Pitma n, & Drastal, 1989). In fact, when Adamec and Shallow (1993) controlled for the effects of predator stress on general activity, cat exposure no longer influenced open-arm explorat ion in the plus maze. Nevertheless, the finding that stress leads to a reduction in locomotor activity should not negate the conclusion that stressed rats are exceedingl y anxious. For one, their avoidance of the open arms leaves them with less area to travel (i.e., just the closed arms). Thus, one would expect these rats to move less and not incessantly pace back and forth from one closed arm to the other. This argument is suppo rted by the fact that th e stressed rats in the present study traveled less distance per time in the closed arms than the unstressed rats. In general, rats reluctance to enter the open arms can be re lated to the behavior often observed in PTSD patients. These individuals often face the choice of (a) going out into the world and running the risk of experienci ng a panic attack or (b) remaining home and avoiding any threat of traumatic reminders. Th e avoidant behavior th at is exhibited by individuals with PTSD often leads to agoraphobia, a diso rder that is commonly diagnosed in individuals with PTSD (Maes, 2000). The fi ndings of the current study relate nicely to these observations.

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106 Although it is not clear if individuals with PTSD exhi bit an exaggerated startle response, the stressed rats in the present study exhibited si gnificantly greater startle during behavioral testing. However, their startle response appeared to be elevated only when they had received vehicle injections during the stress sessions The rats that had been stressed and administered one or both dr ugs did not show this enhancement. It is possible that low CORT at the time of stress prevents long-las ting increases in startle. In support of this argument, there was a positive correlation between CORT levels at the time of stress session one and rats startle responses during behavioral testing. That is to say, greater CORT levels during stress predic ted greater startle responses weeks later. The finding of an enhanced startle response in th e stressed rats is cons istent with previous work showing that a single exposure to a pred ator can lead to enhanced startle one week later (Adamec, 1997). The present study extends this finding, sugges ting that predator stress can have longer-lasting e ffects on this type of behavior. Although the primary hypothesis of the pr esent study was that a blunted CORT response at the time of stress would exacerbate its effects on behavior, it is possible that a blunted CORT response to stress actually amelio rates some of these effects. In support of this argument, Cohen and colleagues (Cohe n, Benjamin, Kaplan, & Kotler, 2000b) found that the administration of ketoconazo le, a steroid synthesis inhibitor, prevented the effects of stress on anxiety-like beha vior in rats. In the presen t study, metyrapone appeared to block the stress-induced increase in startle, but it di d not alleviate the effects of stress on open-arm exploration in the EPM. In addition, metyrapone actually exacerbated the effects of stress on learning and memory in the RAWM. Therefore, a reduced amount of

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107 CORT at the time of trauma may differentially affect, rather than simply exacerbate or ameliorate, rats long-term response to stress. Heightened startle in PTSD patients has b een linked with an elevated state of anxiety at the time of testing, thus raising the possibility th at individuals with PTSD do not have an exaggerated baseline startle response, but rather a greater fear-potentiated startle response. It is also possible that PTSD patients do not exhibit heightened startle at baseline because they are already anxious a nd cannot manifest a larger response to the acoustic stimuli. This is related to work conducted by Cohen and colleagues (Cohen et al., 2000a; Cohen et al., 1998), who failed to fi nd a significant increase in the autonomic activity of PTSD patients when they were e xposed to a recollection of their trauma. The investigators reasoned that si nce the PTSD patients displaye d autonomic dysregulation at rest, they could not manifest further increases in sympathetic tone in response to the traumatic reminders. The same argument could provide an alternative explanation for why not all of the stressed rats displayed an enhanced startle response in the present study. A heightened state of anxiety in these ra ts could have led to a greater overall state of arousal, making a larger re sponse relative to this st ate of arousal unlikely. Spatial Learning & Fear Memory Stress alone did not have any long-term effects on l earning and memory in the RAWM. However, the stressed rats that had been admini stered metyrapone during the stress sessions exhibited si gnificant memory impairments on the 1-hr and 24-hr memory test trials. These findings sugge st that a reduced amount of CO RT at the time of trauma may have long-lasting effects on cognitive processing. Moreover, it is possible that

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108 certain physiological responses at the time of trauma are re sponsible for different longterm effects of stress on behavior. While a reduction in CORT at the time of stress may be more predictive of long-term cognitive de ficits, it may not lead to an exacerbation of the stress-induced effects on a nxiety, given the lack of hei ghtened startle in the same groups of rats. Regardless, this is the first study to suggest that metyrapone, in conjunction with intense stress, can have la sting consequences on spatial learning and memory in rats. Although the stressed rats generally froze more than the unstressed rats on the contextual and cue fear memory tests, the reduction of freezing in the metyraponetreated, unstressed rats was primarily respons ible for this effect. The unstressed rats treated with both vehicles fr oze just as much as the stressed rats treated with both vehicles during both the context and cue tests. Thus, it would not appear that the stress during trauma had a long-term effect on rats fe ar conditioning. Since all of the rats in the present study were stressed dur ing the stress sessions when they were restrained for the measurement of HR/BP and the collection of blood samples, it is possible that the unstressed rats exhibited enhanced fear conditioning. Indeed, research has shown that even a single stressful experience can lead to the enhancement of contextual fear conditioning (Cordero, Venero, Kruyt, & Sandi, 2003). However, since the completely unstressed controls were not significantly diffe rent from the experimental controls, there is little evidence to support this claim. A nother possibility is that the shock amplitude used in the present study was t oo high, especially since the ra ts were most likely stressed during the behavioral testing that occurred prior to fear conditioning training.

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109 Long-Term Stress Effects on the Cardiovascular System Of particular interest is the finding that stress led to a long-term sensitization of rats cardiovascular response to acute stress. Rats that had previously been stressed exhibited a significant elevation of systolic BP and diastolic BP on the final day of behavioral testing. These elev ations were apparent almost an entire month after the second stress session, indicating th at intense stress, in conjunc tion with daily mild stress, can have lasting influences on how the car diovascular system responds to future stressors. Similar work (Bruijnzeel, Stam, Croise t, & Wiegant, 2001) found that a single stressful experience could lead to long-term sensitization of rats cardiovascular stress response. Bruijnzeel et al. (2001) exposed ra ts to a 15-min session of scrambled electric footshocks (ten 6-sec footshocks @ 0.5 mA) a nd then left them undisturbed for 2 weeks. After this period of time, the experimenters exposed rats to a novel environment (Day 14) and a repeated shock prod procedure (D ays 15 and 16). The repeated shock prod procedure consisted of placing an electrified prod into the rats home cages on Day 15 and then reinserting an unelectrified pr od into the home cages on Day 16 and observing their behavior. During Days 14-16, the inve stigators continuously recorded rats cardiovascular response to the stressors. Base line HR and BP did not differ between the pre-shocked and control rats on Days 14-16. However, rats th at were shocked two weeks earlier exhibited significantly greater elevati ons in mean arterial BP than controls in response to the novel stressors. They did not demonstrate a greater HR response to the stressors than the control rats. These results co incide with the current findings in that the

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110 stressed rats demonstrated a significant in crease in BP, but not HR, when exposed to acute stress four weeks afte r the second stress session. Although elevated systolic BP and diastolic BP have both been associated with an increased risk for the development of cardi ovascular disease (Joint National Committee on the Detection, Evaluation, and Treatment of High Blood Pressure, 1997; Sesso et al., 2000), elevations of systolic BP appear to gene rate a greater risk fo r developing this type of ailment (Haider, Larson, Franklin, & Levy, 2003; Izzo, Levy, & Black, 2000). A considerable amount of research on humans has also indicated th at a range of life stressors (e.g., job stress, anxiety disorders, personality characteristics hostility for instance, lack of social support, depression) are strongly associ ated with an increased risk for developing cardiovascular disease (see Rozanski, Blumenthal, & Kaplan, 1999 for a review), and the present study provides evid ence for the long-term effects of stress on cardiovascular reactivity in rats. The stressed rats in this study demonstrated a robust increase in BP during both st ress sessions and during the final day of behavioral testing. Since individuals with PTSD display hei ghtened baseline HR and BP and greater autonomic reactivity to stress, these findings further validate the use of the current paradigm as an animal model of PTSD. They also suggest that the current paradigm may be useful in examining the long-lasting eff ects of stress on the cardiovascular system. Stressed Controls and the Effecti veness of Metyrapone and AF-DX 116 It is important to bear in mind that th e unstressed rats in the present study were exposed to moderate amounts of stress duri ng the stress sessions when they were restrained for HR/BP measurements and the co llection of blood samples. Therefore, the

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111 stressed and unstressed rats in this study were in all actuality exposed to traumatic and mild-to-moderate stress, respectively. Given these circumstances, it is not surprising that the stressed rats did not gain less weight, relative to the unstressed rats, throughout the course of the experiment. There is eviden ce to suggest that as little as 20 min of immobilization stress can reduce the amount of food that a rat ingests for several days thereafter (Valles, Marti, Garcia, & Armario, 2000). In addition, the completely unstressed controls gained more weight th roughout the study than the experimental unstressed controls, providing fu rther support for this argument. Many investigations involving chronic st ress paradigms have reported a smaller amount of weight gain in th e stressed rats, relative to c ontrols (Karst & Joels, 2003; Magarinos & McEwen, 1995; Park et al., 2001; Sandi, Merino, Cordero, Touyarot, & Venero, 2001). However, a majority of studies that have used a small number of stress exposures (Servatius et al., 1994) or even just a single st ress exposure (Adamec et al., 1999; Adamec & Shallow, 1993; Blundell et al., 2005; Cohen et al., 2003) to model PTSD in rodents have not reported, or ha ve reported a quick recovery in, such differences. In addition, Gerges et al. (2004) exposed rats to unstable housing conditions (similar to those employed in the presen t study) for 4-6 weeks and found no weight differences between the stressed and unstresse d rats at the end of the study. This finding provides support for the weight effects in the current study th at is, it is likely that the unstable housing conditions were not stressful enough to sustain the smaller weight gain in the traumatized rats, relative to those rats that were still mildly stressed during the stress sessions.

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112 Based on the results of this study, the ab ility of metyrapone and/or AF-DX 116 to exacerbate rats long-term response to stre ss should not be dismissed. Again, the unstressed rats were still mode rately stressed during the st ress sessions. This piece of information may help explain why some of th e unstressed rats that were treated with pharmacological agents appeared to exhibit gr eater levels of anxiety on the EPM than the unstressed rats treated with both vehicles. It is possible that these agents could intensify the lasting effects of a shorte r and milder stressor, one that does not produce long-lasting effects in rats that have been stressed but not administered the dr ugs, on rats behavior. Why the Pharmacological Manipulations were, for the Most Part, Ineffective There are several reas ons why metyrapone and AF-DX 116 could have been ineffective in exacerbating the effects of st ress on rats behavior. For one, the stress procedure alone in this study le d to a tremendous increase in rats anxiety-like behaviors. On the EPM, rats that had been stressed and treated with both vehicles spent <10% of the trial in the open arms. With this low of a baseline in th e stress condition, it is not surprising that the pharmacological agents could not exacerbate the effect. The floor effect created by the stressed rats treate d with both vehicles rendered a greater enhancement of anxiety-like behavior on the EPM very unlikely. The primary reason for using metyrapone in the present study was to prevent the increase in CORT levels in the stressed ra ts. However, as indicated above, metyrapone only blunted the CORT levels in stressed rats, as th ese levels were still elevated relative to unstressed controls. It is possible that a greater inhibiti on of the stress-induced increase in CORT is necessary to intensify the effects that stress ha s on rats long-term behavior.

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113 Thus, the use of a higher dose of mety rapone, or perhaps even a different pharmacological agent such as dexamethasone (which leads to gr eater suppression of CORT levels), would provide a better model of the low CORT levels that have been observed in traumatized individuals who late r developed PTSD. Metyrapone also led to an unexpected increase in CORT levels in the unstressed rats. There is evidence to suggest that metyrapone actually delays the return of CORT to baseline levels (Rotllant & Armario, 2005). Thus, it is likely that the un stressed rats treated with metyrapone were stressed when they were brought to the lab (b efore they were injected with metyrapone), and the drug subsequently delayed these rats CORT levels from returning to an absolute baseline level. Transportation to the lab a nd the injection procedure are both stressful events to the rats, and since these rats had been acclimated to the HR/BP machine the day before the first stress session, it is also po ssible that the lab reac tivated this memory, leading to an even greater stress response in the rats. Another reason why metyrapone may not ha ve provided greater effects of stress on rats long-term behavior is because of the alternative effects that it has on rats physiology. For instance, this agent blunts the stress-i nduced increase in CORT by inhibiting the final step of CO RT synthesis that is, the conversion of 11-deoxycorticosterone to corticosterone. In doing so, metyrapone prevents the negative feedback of CORT onto areas of the brai n (e.g., hippocampus, hypothalamus, pituitary) that house GRs and regulate pr oduction of the hormone. Without this negative feedback, the hypothalamus is not informed of the CORT status in the body and therefore keeps transmitting CRH to the pituitary, which rel eases ACTH, and so on down the cycle until

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114 11-deoxycorticosterone is produced more a nd more. Ultimately, this means that metyrapone blocks the production of CORT but leads to an increas ed release of the precursors for the hormones synthesis, (CRH, ACTH, and 11-deoxycorticosterone) at the same time. These are not the kind of physio logical conditions that have been observed in traumatized humans shortly after enduring a traumatic experience. Thus, the release of these additional neuromodulators during th e stress sessions could have very well influenced the long-term effect s of stress on rats behavior. In addition to these claims, there is evidence that metyrapone itself can act as a pharmacological stressor and induce long-term dysregulation of the HPA axis. Rotllant, Ons, Carrasco, & Armario (2002) examined the effects of metyrapone (50, 100, and 200 mg/kg, s.c.) on baseline ACTH levels, plasma glucose levels, and fos-like immunoreactivity (FLI) throughout the brain in rats. The higher doses of metyrapone (100 and 200 mg/kg) led to si gnificant elevations of ACTH and plasma glucose. While only the higher doses of metyrapone (100 and 200 mg/kg) led to FLI in brain regions such as the hypothalamic paraventricular nucleu s, bed nucleus of the stria terminalis, and the lateral septum, even the lower dose of metyrapone (50 mg/kg) was capable of inducing significant FLI in the central am ygdala (ACe) and paraventricular thalamic nucleus. The expression of c-fos in the ACe has been linked with stressful experiences and the induction of defense behaviors in rats (Beckett, Duxon, Aspley, & Marsden, 1997; Moller, Bing, & Heilig, 1994) suggesting that even at this dose, metyrapone may potentiate the stress response in rodents. Further work by Rotllant and colleagues (Rotllant & Armario, 2005) also de monstrated that a single dose of metyrapone

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115 (200 mg/kg, s.c.) could induce long-term dysre gulation of the HPA axis. Specifically, rats that were treated with this dose of metyra pone exhibited increased basal levels of CRH mRNA in the hypothalamic paraventricular nuc leus and a reduction of GR mRNA in the dentate gyrus and CA1 region of the hippocampu s 8 days after the drugs administration. These rats also displayed an exaggerated hormonal response to immobilization stress at this time point. Therefore, it would appear that just a single inject ion of metyrapone could have lasting effects on the HPA axis and the endocrinological response to stress. In the present study, there was also evidence to suggest that metyrapone may reduce baseline HR and BP in rats and perh aps even blunt the stre ss-induced increase in cardiovascular activity. To my knowledge, this is the first evidence of a metyraponeinduced reduction of HR/BP in rats. Neverthele ss, van den Buuse and colleagues (van den Buuse, van Acker, Fluttert, & de Kloet, 2002) found that adrenalectomized rats exhibited a reduced HR/BP response to nove lty stress and that administering CORT could reverse these effects. There has also been some work in humans indicating that metyrapone prevents stress-induced increases in HR and BP. Broadley et al. (2005) found that individuals who rece ived 1500 mg of metyrapone prio r to mental stress did not display significant increases in HR and BP, as exhibited by placebo-treated controls. Preventing the stress-induced increase in cardiovascular activity would be counterproductive for the purposes of the pr esent study, which provides another reason why metyrapone may not be the best pharm acological agent to model the low CORT levels during stress that have been observ ed in individuals w ho eventually develop PTSD.

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116 I had also hypothesized that the admini stration of AF-DX 116 would lead to an exacerbation of the stress-induced increase in rats HR and BP. However, despite the main effects of AF-DX 116 for HR and BP, th e drug did not produce a greater increase in HR and BP than that which was produced by the stress alone. This finding may explain why AF-DX 116 did not exacerbate rats response to stress. Limitations of the Present Study There were some limitations to the present study that must be considered. First, as mentioned above, the control rats in this experiment were not completely unstressed, as I would have hoped. However, given that HR/BP and CORT levels needed to be obtained from the controls to compare with the stressed rats, this situation seemed unavoidable. Perhaps separate groups of rats that did not undergo behavioral testing and were simply used for means of obtaining HR /BP and CORT levels could have been run, but this would have made it impossible to co rrelate the rats physiological measures to their behavior weeks later. Mo reover, if the behaviorally-tes ted, unstressed rats were not exposed to the HR/BP and CORT measurements their experiences during the experiment would have been even more different from the stressed rats, making it difficult to infer the cause of any significant effects. It is also possible that th e rats in the present study we re exposed to an excessive amount of behavioral testing. This could ha ve led to one behavioral task having an influence on subsequent tasks. Theoreticall y, a valid animal model of PTSD should be able to show that stress has an effect on seve ral measures of behavi or in the same rats. Certainly, this seems to be the case in huma ns with the disorder, and my primary goal is

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117 to create a model that can be applied to the human species. One could potentially run separate groups of rats for each behavioral tes t; however, this is simply an inefficient way to test the present hypotheses and, quite fr ankly, would take a considerable amount of time and money to accomplish. Nevertheless, when interpreting the results of the present study, especially those from the tasks that took place near the end of behavioral testing, it is important to keep these caveats in mind.

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118 Chapter Three: Experiment Two Does Stress Produce Dynamic Brain Changes that Increase Rats Long-Term Sensitivity to Yohimbine? The second experiment was designed to examine the effects of stress on rats long-term sensitivity to yohimbine, an 2 adrenergic receptor antagonist. This drug enhances neuronal firing in the LC, increases levels of NE, and tends to induce panic attacks in PTSD patients. Ra ts in the second experiment were again subjected to two stress sessions. However, given that HR/B P and CORT data were collected during the stress sessions in the first experiment, th ese assessments were avoided in the second experiment to prevent the c ontrol rats from being unnecessa rily stressed. The behavioral testing paradigm was also slig htly altered. Three weeks after the second stress session, all rats sensitivity to yohimbine was examined by injecting them with yohimbine or vehicle prior to tests of general locomotor activity, anxiety, and startle. Si nce the stress alone produced the most robust effects in Experiment One, the only groups to be compared in Experiment Two were the stress and no stress groups. Methods Rats Adult male Sprague-Dawley rats (225-250 g upon delivery) obtained from Charles River laboratories were used for th e present experiment. The rats were housed two to a cage (standard Plexiglas 46 x 25 x 21 cm), maintained on a 12-hr light-dark

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119 cycle (lights on at 7:00 a.m.), and had access to food and water ad libitum. Upon arrival, all rats were afforded a habituation period of one week to acclimate to the housing room and cage changes before any experimental ma nipulations took place. All procedures were approved by the Institutional Animal Care a nd Use Committee at the University of South Florida. Design The present study employed a 2 x 2 factor ial design. The two manipulated factors were stress (stress, no stress) and yohimbine (yohimbine, vehicle). The sample sizes for each cell of the factorial are listed in Table 2. Table 2 Samples Sizes for all Groups in Experiment 2 Condition Sample size Stress Yohimbine (1 mg/kg) 10 Vehicle 9 No Stress Yohimbine (1 mg/kg) 12 Vehicle 8 Stress Manipulations After the one-week acclimati on period, all rats were tr ansported to the lab. Each rat was tail-marked with a permanent black marker and weighed. Then, the rats were

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120 randomly assigned to the stress or no st ress group. Rats in the stress group were restrained in plastic DecapiCones and placed in a perforated wedge-shaped Plexiglas enclosure. After 15 min, the st ressed rats, restrained in the plastic DecapiCones and resting in the perforated, wedge-shaped Plexig las enclosure, were placed in a metal cage with an adult female cat for 45 min in a room located adjacent to the rat housing rooms. Canned cat food was placed on top of the Pl exiglas enclosure to direct cat activity towards the rats. After 45 min had elapsed, th e rats were returned to the laboratory and then transported back to the housing room. Af ter being weighed, the unstressed rats spent 1 hr (yoked to the stress procedure for the stressed rats) in their home cages in the laboratory, after which they were al so returned to the housing room. Stress Sessions All of the manipulations during the firs t stress session were performed during the rats light cycle, betw een 0700 and 1500 hrs. As in Experime nt One, all rats that were stressed in the initial session were exposed to a second stress session 10 days later. The second stress session took place during the ra ts dark cycle. During the second stress session, all rats were transpor ted to the laboratory. The sa me manipulations that were performed prior to the first stress session were performed again. All manipulations during the second stress session took place between 1900 and 0200 hrs. Randomized Housing After the initial stress se ssion, the stressed rats were exposed to unstable housing conditions until the commencement of behavioral testing. All stressed rats were still housed two per cage, but every day, the cage mate of each rat was changed. The

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121 randomization of the stressed rats occurred within groups, so each rat was exposed to every other rat within its own group between 3 and 4 times prio r to behavioral testing. No rat had the same cage mate on two c onsecutive days, and the randomization manipulations always took place between 0800 and 1200 hrs. Handling and Weight Before behavioral testing began, all rats were handled for three consecutive days, as per the methods in Experiment One. All rats were weighed prior to the first stress session, prior to the second stress sess ion and on the last day of handling. Behavioral Testing Three weeks after the second stress se ssion, all rats were transported to the laboratory and received i.p. injections of yohimb ine (1 mg/kg) or vehicle. These doses of yohimbine were chosen because pilot work in our lab revealed that they produced a threshold level of anxiety-related behaviors in control animals. The drug was dissolved in distilled H 2 O, brought to the appropriate volume with 0.9% saline, and then administered at a volume of 1 ml/kg. All behavioral tes ting took place during the light cycle between 0700 and 1500 hrs. Behavioral Apparatus Open field (see Figure 32) Thirty minutes after receiving injections of yohimbine or vehicle, all rats underwent open field testi ng, a test of general locomotor activity. Rats were placed in a large, tr anslucent, plastic box (Hamilton-Kinder, San Diego, CA 40 x 47 x 70 cm) with an open top in a lighta nd sound-attenuated room for 10 min. Rats behavior was monitored by infrared photob eams connected to a computer program

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122 (Motor Monitor) that analyzed the behavior. The program allowed for assessment of the rats total distance traveled in each area of the open field (center and perimeter), total time spent in each area of the open field, rearing, ambulations, and entries into each area of the open field. Figure 32. Schematic diagram of the open field apparatus. Elevated plus maze (EPM). After all rats had been tested in the open field (approximately 45-60 min later), they were subjected to the EPM assessment. Testing conformed to the protocol used in Experiment One. Startle response. After all rats have been tested on the EPM (approximately 45-60 min later), they were tested for their startle response. The rats were tested in same fashion as in Experiment One. Blood samples. Twenty-four hrs after behavioral testing, all rats were transported to the laboratory and received i.p. injections of yohimbine (1 mg/kg) or vehicle. Thirty minutes later, three blood samples were collected from the rats at three separate time

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123 points (0, 20, and 80 min). These three sample s represented baseline, stress, and returnto-baseline samples, respectively, and the sa me procedures that were employed on the final day of Experiment One were employed here (with the exception of recording rats HR/BP). The procedure was employed in Experiment Two to assess the rats endocrinological response to yohimbine. Statistical Analysis Some data were analyzed through use of between-subjects analyses of variance (ANOVAs); mixed-model ANOVAs were employe d when repeated measures variables were a part of the behavioral assessment being examined. In other cases, the behaviors were expressed as a percent change from th e vehicle-injected animals to emphasize the stress-yohimbine interactions. Since it wa s hypothesized that yohimbine would produce an exaggerated behavioral response in th e stressed rats, planned comparisons were conducted between the stressed, yohimbine-in jected rats and the unstressed, yohimbineinjected rats in these instances. Alpha was set at .05 for all analyses, and post-hoc comparisons were made through use of Bonferroni-corrected t -tests. Data points that were greater than three standard deviation units beyond the exclusiv e mean were considered outliers and removed from data analysis. Less than 1% of the data were ou tliers. All data are expressed as means SEM. Open field In all cases, the data were expr essed as a percent change from the vehicle-injected animals to emphasize the stress-yohimbine interactions. Planned

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124 comparisons were conducted between the stressed, yohimbine-injected rats and the unstressed, yohimbine injected rats. Elevated plus maze (EPM) Some of the dependent measures acquired from the EPM were subjected to two-way ANOVAs, with Stress and Yohimbine serving as the between-subjects factors. In other cases, th e behaviors were expr essed as a percent change from the vehicle-injected animals to emphasize the stress-yoh imbine interactions. In these instances, planned comparisons were conducted between the stressed, yohimbine-injected rats and the unstressed, yohimbine injected rats. Startle response. For each rat, there we re eight startle responses at each of three auditory intensities. These eight responses we re averaged to create one data point per intensity per rat. For the reasons emphasized above, the data were analyzed using three separate two-way ANOVAs, with Stress and Y ohimbine serving as the between-subjects factors at each auditory stimulus intensity. Corticosterone. A mixed-model ANOVA was empl oyed to analyze the CORT samples collected 24 hrs after behavi oral testing. Stress and Yohimbine (vehicle, 1 mg/kg) served as the between-subjects factor s, and time points (baseline, stress, returnto-baseline) served as th e within-subjects factor. Weight. Although each group of rats was ordered for the same weight range, an independent-samples t -test indicated that there were weight differences between the stressed and unstressed rats, t (37) = 7.60, p < .001. Nevertheless, both groups reached adult weight by the time of stress session one (see Table 3). In order to examine group differences in weight over the course of th e experiment, the amount of weight that rats

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125 gained between stress session one and each subsequent time point (i.e., stress session two and last day of handling) was analyzed using a mixed-model ANOVA. Since rats were injected with yohimbine on the day of behavioral testing, there was only one between-subjects factor for this analysis, which was stress. Time was the within-subjects factor (first stress session to second stress session, second stress session to last day of handling). Results Open Field Ambulations (see Figure 33). An independent samples t-test indicated that there was no difference between the stressed and unstressed rats treated with vehicle in terms of ambulations in the open field, t(18) = 0.40, p > .69. Stress-Yoh% Change from Respective Vehicle -20-15-10-505101520 No Stress-Yoh*Yohimbine Suppressed Ambulations in Stressed Rats, butEnhanced this Measure in Unstressed Rats Figure 33. Percent change of ambulations (+ SE) in the open field in yohimbine-treated rats. Yohimbine suppressed the number of ambulations made by stressed rats, but enhanced this measure in the unstressed rats. p < .001 compared to the unstressed rats treated with yohimbine.

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126 The number of ambulations for all yohimbine-treated rats was converted into a percent change from vehicle score. A planned comparison was conducted on this data to compare the stressed, yohimbine-treated rats with the unstressed, yohimbine-treated rats. While yohimbine decreased the number of ambulations that stressed rats (-10.06 4.06%) made in the open field, it led to an increase of this measure in the unstressed rats (13.86 4.16%), t(19) = -4.10, p < .001. Rearing (see Figure 34). An independent samples t-test indicated that there was no difference between the stressed and unstressed rats treated with vehicle in terms of rearing in the open field, t(15) = 0.51, p > .62. Stress-Yoh % Change from Respective Vehicle -60-50-40-30-20-100 No Stress-Yoh*Yohimbine led to a Greater Suppression of Rearingin the Stressed Rats Figure 34. Percent change of rearing episodes (+ SE) in the open field in yohimbine-treated rats. Yohimbine led to a greater suppression of rearing in the stressed rats. p < .05 compared to the unstressed rats treated with yohimbine. The number of rearings for all yohimbine-treated rats was converted into a percent change from vehicle score. A planned comparison was conducted on this data to

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127 compare the stressed, yohimbine-treated rats with the unstressed, yohimbine-treated rats. The stressed rats treated with yohimbine (-50.25 6.37%) displayed a greater suppression of rearing in the open field than the unstressed rats treated with yohimbine (-32.70 4.53%), t(20) = -2.30, p < .05. Time spent in the perimeter (see Figure 35). An independent samples t-test indicated that there was no difference between the stressed and unstressed rats treated with vehicle in terms of the time that they spent in the perimeter of the open field, t(15) = 1.38, p > .18. Stress-Yoh% Change from Respective Vehicle -10-50510152025 No Stress-Yoh*Yohimbine Increased the Time that Stressed Rats Spent in the Perimeter, but Decreased this Measure in Unstressed Rats Figure 35. Percent change in time spent in the perimeter (+ SE) of the open field in yohimbine-treated rats. Yohimbine increased the amount of time that the stressed rats spent in the perimeter of the open field, but decreased this measure in the unstressed rats. p < .01 compared to the unstressed rats treated with yohimbine. The time spent in the perimeter for all yohimbine-treated rats was converted into a percent change from vehicle score. A planned comparison was conducted on this data to

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128 compare the stressed, yohimbine-treated rats with the unstressed, yohimbine-treated rats. While yohimbine increased the amount of time that the stressed rats (16.06 4.38%) spent in the perimeter of the open field, it led to a decrease of this measure in the unstressed rats (-3.42 3.23%), t(20) = 3.65, p < .01. Time spent in the center (see Figure 36). An independent samples t-test indicated that there was no difference between the stressed and unstressed rats treated with vehicle in terms of the time that they spent in the center of the open field, t(15) = 1.39, p > .18. Stress-Yoh% Change from Respective Vehicle -60-40-200204060 No Stress-Yoh*Yohimbine Suppressed the Time that Stressed Rats Spent in theCenter, but Increased this Measure in Unstressed Rats Figure 36. Percent change in time spent in the center (+ SE) of the open field in yohimbine-treated rats. Yohimbine suppressed the amount of time that the stressed rats spent in the center of the open field, but increased this measure in the unstressed rats. p < .01 compared to the unstressed rats treated with yohimbine. The time spent in the center for all yohimbine-treated rats was converted into a percent change from vehicle score. A planned comparison was conducted on this data to compare the stressed, yohimbine-treated rats with the unstressed, yohimbine-treated rats.

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129 While yohimbine decreased the amount of time that the stressed rats (-39.54 11.06%) spent in the center of the open field, it led to an increase of this measure in the unstressed rats (23.81 15.64%), t(19) = -3.25, p < .01. Distance in the open field (see Figure 37). An independent samples t-test indicated that there was no difference between the stressed and unstressed rats treated with vehicle in terms of the distance traveled in the open field, t(13) = 1.43, p > .17. Stress-Yoh% Change from Respective Vehicle -15-10-505101520 No Stress-Yoh*Yohimbine Suppressed the Distance that Stressed Rats Traveled,but Increased this Measure in Unstressed Rats Figure 37. Percent change in distance traveled (+ SE) in the open field in yohimbine-treated rats. Yohimbine suppressed the distance that the stressed rats traveled in the open field, but increased this measure in the unstressed rats. p < .01 compared to the unstressed rats treated with yohimbine. The distance traveled in the open field for all yohimbine-treated rats was converted into a percent change from vehicle score. A planned comparison was conducted on this data to compare the stressed, yohimbine-treated rats with the unstressed, yohimbine-treated rats. While yohimbine decreased the distance traveled by

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130 the stressed rats (-8.28 2.03 %), it increased the distance traveled by the unstressed rats (10.82 4.59 %), t(18) = -3.53, p < .01. Fecal boli. The analysis of fecal boli deposited in the open field revealed no main effect of stress, F(1,34) = 0.58, p > .45, or yohimbine, F(1,34) = 1.82, p > .18, and the Stress x Yohimbine interaction was not significant, F(1,34) = 0.43, p > .51. Elevated Plus Maze Ambulations (see Figure 38). An independent samples t-test indicated that the stressed rats treated with vehicle (307.88 22.08 ambulations) made fewer ambulations on the EPM than the unstressed rats treated with vehicle (425.50 31.45 ambulations), t(14) = 3.06, p < .01. Stress-Yoh% Change from Respective Vehicle -30-20-1001020 No Stress-Yoh*Yohimbine Suppressed the Ambulations Made by StressedRats, but Slightly Increased this Measure in Unstressed Rats Figure 38. Percent change in ambulations (+ SE) on the elevated plus maze in yohimbine-treated rats. Yohimbine suppressed the number of ambulations made by the stressed rats, but slightly increased this measure in the unstressed rats. p = .05 compared to the unstressed rats treated with yohimbine.

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131 The number of ambulations for all yohimb ine-treated rats wa s converted into a percent change from vehicle score. A pla nned comparison was conducted on this data to compare the stressed, yohimbine-treated rats with the unstressed, yohimbine-treated rats. While yohimbine decreased the number of ambulations made by the stressed rats (-23.18 8.01 %), it increased the number of ambulations made by the unstressed rats (0.90 8.49 %), t (20) = -2.03, p = .05. Percent time spent in the open arms (see Figure 39) Time spent in the open arms of the EPM was converted into a percent of to tal time score. The analysis of these scores revealed a main effect of stress, F (1,33) = 52.98, p < .001, indicating that the stressed rats (2.94 3.71 %) spent less percent tim e in the open arms than the unstressed rats (39.99 3.49 %). There was also no main effect of yohimbine, F (1,33) = 2.12, p > .15, and the Stress x Yohimbine inte raction was not significant, F (1,33) = 1.82, p > .18. Percent time spent in the open ar ms, controlling for ambulations Since the stressed rats treated with vehicle made fewe r ambulations than the unstressed rats treated with vehicle, one could argue that the stre ssed rats spent less time in the open arms because they moved less. Therefore, a two-way ANCOVA was used to examine group differences in the percent of time spent in the open arms, with ambulations on the EPM serving as the covariate. The analysis revealed a main effect of stress, F (1,33) = 12.92, p < .001. There was no main effect of yohimbine, F (1,33) = 3.91, p > .05, and the Stress x Yohimbine interaction was not significant, F (1,33) = 0.49, p > .49.

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132 Stress % Time 0102030405060 Vehicle Yohimbine No Stress**Percent Time Spent in the Open Arms of the Elevated Plus Maz e Figure 39. Percent time spent in the open arms (+ SE) of the elevated plus maze in Experiment 2. Stressed rats spent less time in the open arms than the unstressed rats. p < .001 compared to respective unstressed groups. Percent time spent in the closed arms (see Figure 40). Time spent in the closed arms of the EPM was converted into a percent of total time score. The analysis of these scores revealed a main effect of stress, F(1,33) = 69.76, p < .001, indicating that the stressed rats (93.22 3.84 %) spent more percent time in the closed arms than the unstressed rats (49.23 3.61 %). There was no main effect of yohimbine, F(1,33) = 1.01, p > .32, and the Stress x Yohimbine interaction was not significant, F(1,33) = 1.17, p > .28.

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133 Stress% Time 020406080100 Vehicle Yohimbine No Stress**Percent Time Spent in the Closed Arms of the Elevated Plus Maz e Figure 40. Percent time spent in the closed arms (+ SE) of the elevated plus maze in Experiment 2. Stressed rats spent more time in the closed arms than the unstressed rats. p < .001 compared to respective unstressed groups. Movement per unit time in the closed arms (see Figure 41). The distance that each rat traveled in the closed arms was divided by the amount of time it spent in the closed arms to produce a distance/time score. This score represented the distance that each rat traveled in the closed arms per the amount of time that it spent in the closed arms. The analysis of this data revealed a main effect of stress, F(1,32) = 37.97, p < .001, indicating that the stressed rats (7.46 0.57 cm/sec) traveled less distance/time in the closed arms than the unstressed rats (12.38 0.56 cm/sec). There was no main effect of yohimbine, F(1,32) = 3.03, p > .09, and the Stress and Yohimbine interaction was not significant, F(1,32) = 0.73, p > .40.

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134 Stress Distance Traveled per Time (cm/sec) 0246810121416 Vehicle Yohimbine No Stress**Distance/Time Traveled in the Closed Arms of theElevated Plus Maze Figure 41. Distance/time traveled in the closed arms (+ SE) of the elevated plus maze in Experiment 2. Stressed rats traveled less distance/time in the closed arms than the unstressed rats. p < .001 compared to respective unstressed groups. Distance on the elevated plus maze (see Figure 42). An independent samples t-test indicated that the stressed rats treated with vehicle (5461.11 430.84 cm) traveled less distance on the EPM than the unstressed rats treated with vehicle (7110.75 355.84 cm), t(15) = 2.91, p < .05. The distance scores for all yohimbine-treated rats were converted into percent change from vehicle scores. A planned comparison was conducted on this data to compare the stressed, yohimbine-treated rats with the unstressed, yohimbine-treated rats. The stressed rats that were treated with yohimbine (-26.01 6.74 %) exhibited a greater suppression of distance traveled on the EPM than the unstressed rats that were treated with yohimbine (-7.60 4.05 %), t(20) = -2.43, p < .05.

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135 Stress-Yoh % Change from Respective Vehicle -35-30-25-20-15-10-50 No Stress-Yoh*Yohimbine Led to a Greater Suppression of Distance Traveled in Stressed Rats Figure 42. Percent change in distance traveled (+ SE) on the elevated plus maze in yohimbine-treated rats. Yohimbine led to a greater suppression of distance traveled on the elevated plus maze in the stressed rats. p < .05 compared to the unstressed rats treated yohimbine. Fecal boli. The analysis of fecal boli deposited on the EPM revealed a main effect of stress, F(1,32) = 9.60, p < .01, indicating that the stressed rats (0.72 0.15 boli) defecated more on the EPM than the unstressed rats (0.07 0.07 boli). There was no main effect of yohimbine, F(1,32) = 1.24, p > .27, and the Stress x Yohimbine interaction was not significant, F(1,32) = 0.18, p > .67. Startle Response 90 dB auditory stimuli (see Figure 43). The analysis of startle responses to the 90 dB auditory stimuli revealed a main effect of stress, F(1,32) = 39.76, p < .001, indicating that the stressed rats (0.31 0.02 Newtons) exhibited greater startle than the unstressed rats (0.16 0.02 Newtons). There was no main effect of yohimbine, F(1,32) =

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136 0.89, p > .35, and the Stress x Yohimbine interaction was not significant, F(1,32) = 0.72, p > .40. StressStartle Response (Newtons) 0.00.10.20.30.4 Vehicle Yohimbine No Stress**Startle Responses for the 90 dB Noise Bursts Figure 43. Startle responses (+ SE) for the 90 dB noise bursts in Experiment 2. Stressed rats exhibited greater startle responses than the unstressed rats. p < .001 compared to respective unstressed groups. 100 dB auditory stimuli (see Figure 44). The analysis of startle responses to the 100 dB auditory stimuli revealed a main effect of stress, F(1,33) = 32.43, p < .001, indicating that the stressed rats (1.76 0.12 Newtons) exhibited greater startle responses than the unstressed rats (0.82 0.12 Newtons). There was no main effect of yohimbine, F(1,33) = 0.01, p > .92, and the Stress x Yohimbine interaction was not significant, F(1,33) = 0.07, p > .78.

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137 StressStartle Response (Newtons) 0.00.51.01.52.02.5 Vehicle Yohimbine No Stress**Startle Responses for the 100 dB Noise Bursts Figure 44. Startle responses (+ SE) for the 100 dB noise bursts in Experiment 2. Stressed rats exhibited greater startle responses than the unstressed rats. p < .001 compared to respective unstressed groups. 110 dB auditory stimuli (see Figure 45). The analysis of startle responses to the 110 dB auditory stimuli revealed a main effect of stress, F(1,33) = 11.98, p < .001, indicating that the stressed rats (3.07 0.19 Newtons) exhibited greater startle responses than the unstressed rats (2.15 0.19 Newtons). There was no main effect of yohimbine, F(1,33) = 2.72, p > .10, and the Stress x Yohimbine interaction was not significant, F(1,33) = 0.29, p > .59.

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138 StressStartle Response (Newtons) 01234 Vehicle Yohimbine No Stress**Startle Responses for the 110 dB Noise Bursts Figure 45. Startle responses (+ SE) for the 110 dB noise bursts in Experiment 2. Stressed rats exhibited greater startle responses than the unstressed rats. p < .001 compared to respective unstressed groups. Fecal boli. The analysis of fecal boli deposited in the startle apparatus revealed no main effects of stress, F(1,35) = 0.24, p > .62, or yohimbine, F(1,35) = 0.87, p > .35, and the Stress x Yohimbine interaction was not significant, F(1,35) = 0.39, p > .53. Final Days Corticosterone Levels (see Figure 46) The within-subjects analysis revealed a main effect of time, F(2,46) = 71.18, p < .001. Post hoc analyses indicated that rats CORT levels were greater after 20 min of restraint stress (31.59 1.02 g/dL) than at baseline (9.17 1.16 g/dL). These levels significantly declined 60 min later (16.71 1.95 g/dL), but were still elevated, relative to baseline levels (ps < .05). The Time x Stress interaction was significant, F(2,46) = 10.65, p < .001. The stressed rats exhibited greater CORT levels (baseline: 12.35 1.29 g/dL; stress: 38.35 1.14 g/dL) at baseline and after 20 min of restraint stress

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139 than the unstressed rats (baseline: 5.99 1.93 g/dL; stress: 24.82 1.70 g/dL). However, the CORT levels of stressed (14.70 2.17 g/dL) and unstressed rats (18.72 3.24 g/dL) did not differ 60 min later (ps < .05). The Time x Yohimbine interaction was significant, F(2,46) = 3.82, p < .05. Post hoc analyses indicated that yohimbine led to greater CORT levels at baseline and after 20 min of restraint stress. While the CORT levels of vehicle-treated rats (6.34 2.47 g/dL) returned to baseline levels 60 min later, those of yohimbine-treated animals (27.08 3.01 g/dL) remained elevated (ps < .05). The Time x Stress x Yohimbine interaction was not significant, F(2,46) = 1.03, p > .36. 0 min20 min40 min60 min80 minCorticosterone (g/dL) 0102030405060 Stress-Vehicle Stress-Yohimbine No Stress-Vehicle No Stress Yohimbine (Baseline)(Stress)(Return-to-Baseline) Restraint StressCorticosterone Levels during the Final Day of Behavioral Testing Figure 46. Corticosterone levels ( SE) during the final day of behavioral testing in Experiment 2. Stressed rats showed greater baseline levels and stress-induced elevations of corticosterone. Yohimbine also led to greater baseline levels and stress-induced elevations of corticosterone. Stressed rats treated with yohimbine demonstrated the greatest stress-induced elevation of serum corticosterone.

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140 The between-subjects analysis revealed a main effect of stress, F (1,23) = 8.29, p < .01, indicating that the stressed rats (21.80 1.02 g/dL) had greater levels of CORT than the unstressed rats (16.51 1.53 g/dL). Th ere was also a main effect of yohimbine, F (1,23) = 82.72, p < .001. Post hoc analyses indicated th at the rats treated with yohimbine (27.51 1.42 g/dL) had greater CORT levels than the rats treated with vehicle (10.80 1.17 g/dL). The Stress x Yohimbin e interaction was not significant, F (1,23) = 0.00, p > .95. Weight (see Table 3 & Figure 47) Table 3 Average Raw Weights for the Stre ssed Groups in Experiment 2 Raw weights (g SE ) ________________________________________________ Stress Stress Last day Group session 1 session 2 of handling Stress 300.63 (2.76) 345.61 (4.03) 430.00 (7.20) No Stress 273.98 (2.19) 344.63 (4.89) 429.48 (7.72) The within-subjects analysis revealed a main effect of time, F (1,37) = 650.51, p < .001, indicating that all of the rats gain ed a significant amount of weight over the course of the experiment. The Time x Stress interaction was not significant, F (1,37) = 0.05, p > .94. The between-subjects analysis revealed a main effect of stress, F (1,37) = 18.91, p < .001, indicating that the st ressed rats (87.17 4.27 g) gained less weight than the unstressed rats (113.08 4.16 g).

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141 SS1 SS2SS1 BehaviorWeight Gained (g) 020406080100120140160180 Stress No Stress **Body Weight Gained in Experiment Two Figure 47. Body weight gained ( SE) throughout the course of Experiment 2. Stressed rats gained less weight. p < .001 compared to unstressed rats. Discussion Major Findings and Significance The most important finding of the current study is that the stressed rats exhibited an exaggerated response to yohimbine 3 weeks after the second stress session. This finding was predominantly evident for the first behavioral task examined (i.e., the open field) and provides support for the idea that the present stress paradigm has lasting effects on the noradrenergic system. The present study also replicated the stress effects found in Experiment One for the EPM and startle assessments. Specifically, the stressed rats exhibited greater levels of anxiety on the EPM and an exaggerated startle response, relative to the unstressed rats.

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142 Enhanced Sensitivity to Yohimbine in the Open Field The stressed rats displayed an enhan ced sensitivity to yohimbine treatment, as measured by general locomotor activity in the op en field. First, the stressed rats showed a greater suppression of rearing in the open field after yohimbine treatment, relative to the unstressed rats. In addition, yohimbine reduced the total distance that the stressed rats traveled in the open field, while it increased this measure in the unstressed rats. This finding is comparable to the differential eff ects that yohimbine had on the time that rats spent in the perimeter and center areas of the open field. The stressed rats that had been administered yohimbine spent more time in the perimeter and less time in the center of the open field, relative to vehicl e-injected controls. This was in stark contrast to the unstressed rats, which spent less time in the perimeter and more time in the center of the open field after being treated with yohimb ine. Upon placement in a novel environment, rats tend to avoid exploring the center area and spend more time in the perimeter. In fact, most of the rats in the present study spen t approximately 70-80% of the trial in the perimeter of the open field. This type of behavi or theoretically minimizes the rats risk of being exposed to a predator-dominated, open ar ea. When rats are anxious, this type of avoidant behavior is even more prevalent (Beck & Luine, 2002). Si nce the stressed rats displayed greater avoidance of the center ar ea after being treated with yohimbine, it can be reasoned that the drug produced an anxi ogenic response in these rats, a response that was not observed in the unstressed rats.

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143 Elevated Plus Maze and the Magnitude of the Stress Effect As in Experiment One, the stressed rats in the present study spent less time in the open arms of the EPM than the unstressed rats The magnitude of this effect was again enough to generate a floor effect, which c ould not be further exacerbated by yohimbine treatment. The stressed rats that were treated with vehicle spent <5% of the trial in the open arms of the EPM. Clearly, it would very di fficult, if not impossible, to intensify this effect. At any rate, it is impor tant to emphasize the fact that these findings replicated the findings of Experiment One. Replication is always an important part of developing new paradigms and, in this case, provides further corroboration for the vali dity of the present stress paradigm. Although yohimbine did not exacerbate the am ount of time that the stressed rats spent in the open arms of the EPM, it did lead to a greater su ppression of distance traveled on the plus maze in the stressed rats In addition, the stressed rats that were treated with yohimbine made fewer ambulations than vehicle-injected controls, while the unstressed rats treated with yohimbine made more ambulations than vehicle-injected controls. Collectively, these findings indi cate that yohimbine induced a greater suppression of locomotor activity in the stre ssed rats, relative to the unstressed rats. Startle Response Yohimbine did not lead to an exacerbation of startle in the stressed rats. This may be due to the fact that stress alone resulted in a significant en hancement of rats startle, or it could be the case that the effects of yohimb ine on startle are simp ly linear in nature, regardless of whether or not the rats are stressed. Furthe rmore, yohimbine alone did not

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144 augment rats startle responses. This finding lie s in contrast to previous work that has reported yohimbine-induced enhancements of the acoustic startle res ponse in rats (Kehne & Davis, 1985) and humans (Morgan, Sout hwick, Grillon, Davis, Krystal, & Charney, 1993). The inconsistency between previous work and the findings presented here is most likely attributable to the different doses of yohimbine that were employed in each case. The dose of yohimbine (1 mg/kg) employed in th e present study is a very low, threshold dose that does not have signi ficant effects on anxiety in uns tressed rats (as evidenced by the data presented here). This low of a dose was employed because I was testing for a hyperresponsivity to the drug in the stressed rats; if the drug alone had too great of effects on controls, it would be difficult to detect an exaggerated res ponse to the agent in stressed rats. Weight as an Index of Stress The effects of stress on body weight were much clearer in the present study than in Experiment One, given that the control rats were not stresse d by HR/BP measurement and the collection of blood samples during th e stress sessions. As shown in Figure 47, the stressed rats gained less wei ght from the first stress sessi on to the second stress session and this effect was maintained to the commencement of behavioral testing. It would appear from the graph that it is the first stress session that leads to a reduction in weight gain, and this initial effect on the rats is maintained throughout the course of the experiment by the second stress session and randomized housing. It is clear from this study that the stress paradigm does lead to a significant reduction in weight gain, a

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145 finding that is consistent with other research on chronic stress and supports the use of this paradigm as a model of PTSD in rats. Effects of Stress and Yohimbine on Adrenal Activity The stressed rats exhibited a greater stress-induced increase in CORT 3 weeks after the second stress session. This finding is consistent with the re sults of Experiment One, where the stressed rats exhibited greater increases in systolic BP and diastolic BP after acute restraint stress on th e final day of behavioral test ing. It is possible that the stressed rats in Experiment One did not de monstrate the same relative increase in CORT because the control rats themselves had been exposed to a moderate amount of stress throughout the course of the study. Indeed, the control rats from Experiment One (21.09 2.19 g/dL) appeared to exhibit s lightly higher stress-i nduced increases in CORT on the final day of behavi oral testing than the contro l rats from Experiment Two (17.37 1.34 g/dL). Ultimately, the finding of a greater stress-induced increase in CORT levels in the stressed rats is consiste nt with research in humans that have been diagnosed with PTSD. As mentioned above, these individuals often display significantly greater hormonal responses to acu te stress than do control subj ects. Elzinga et al. (2003) found that individuals with a buse-related PTSD exhibited gr eater cortisol levels than control subjects after exposure to a trauma-relate d script. The important point here is that the control subjects had also experienced severe childhood abuse, but they did not develop PTSD as a result of the trauma. Si nce stressed rats in the present study were immobilized during the stress sess ions, it is possible that these rats demonstrated greater

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146 increases in CORT after acute restraint stress 3 weeks later because it reminded them of the trauma that they had previously endured. Yohimbine led to elevated baseline levels of CORT in both stressed and unstressed rats 30 min after its administration. This finding is consistent with previous work indicating that yohimbine increases HPA activity and anxiety-lik e behavior in both humans and rodents (Grunhaus, Tiongco, Ze lnik, Flegel, Hollingsworth, & Smith, 1989; McDougle, Price, Heninger, Krystal, & Charney, 1995; Myers, Banihashemi, & Rinaman, 2005; Vythilingam et al., 2000). In addition, the stressed rats that had been treated with yohimbine appeared to show a greater stress-indu ced increase in CORT than the unstressed rats that had been treated with yohimbine. This is the first study to show such a response in stressed rats. Additiona lly, research on humans with PTSD has only shown an exaggerated autonomic response to yohimbine (Southwick et al., 1993), not an exaggerated hormonal response. Thus, the fi ndings of the present study demand further investigation of the physiologi cal response to yohimbine in individuals diagnosed with PTSD. Limitations of the Present Study As in Experiment One, there are some lim itations and/or caveats to the findings of the present study that must be considered. Si nce all of the rats were tested on multiple behavioral assessments, it is again possible that some of the tasks interacted with one another. That is to say, one behavioral task could have affected some groups of rats differently than the way it affected others. Given that most of the evidence for an

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147 exaggerated response to yohimbine was found for the open field assessment, which was the first task examined, this argument is very convincing. In addition, it is possible that the effect s of stress were so robust, that they prevented the detection of yohimbine effects in the stressed rats. This was particularly the case for the EPM, where stress alone led to a considerable suppression of open-arm exploration. Given that the stressed rats that were tr eated with vehicle spent approximately 2.66% of the trial in the open arms, it is not surprising that yohimbine did not exacerbate this effect. In sum, it is likely that this behavioral as sessment did not retain the sensitivity to detect a heightened sensitivity to yohi mbine in the stressed rats.

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148 Chapter Four: General Discussion and Conclusions Mechanisms Mediating the Long-Term Effect s of Stress on Physiology and Behavior Amygdala Plasticity A considerable amount of work by Adamec and colleagues has suggested that NMDA-dependent plasticity in the amygdala is responsible for the long-term effects of predator stress on anxiety-like be havior in rats. Indeed, when the investigators treated rats with competitive NMDA receptor antagonists 30 min prior to cat exposure, the rats did not show heightened levels of anxiety or startle one week later (Adamec et al., 1999). Given the role of NMDA receptors in learning and memory, it would appear that these pharmacological agents prevent the memory of the traumatic experience from being adequately formed. Intuitively, this makes sense, since it is the powerful traumatic memories that ultimately lead to the diso rder in humans. Adamec, Strasser, Blundell, Burton, & McKay (2006) provided further s upport for this argument when they found that the effects of predator stress on anxiety-like behaviors is dependent upon protein synthesis in the amygdala. Since memory cons olidation has been shown to be dependent on protein synthesis (Lamprecht & LeDoux, 2004) this finding provides support for the idea that it is the memory of the cat exposure that leads to long-lasting effects on behavior. Further work by Adamec and colleague s has examined the neural circuitry involved in the effects of predator stress on rats behavior. Adamec, Blundell, and Collins

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149 (2001) have shown that predat or stress induces the potenti ation of several important pathways to and from the amygdala. For inst ance, the right ventra l angular bundle (VAB) input to the basolateral amygda la (BLA) has been linked with the anxiogenic effects of predator stress on behavior in the EPM (Ada mec et al., 2001). This pathway is of an excitatory nature and extends from the hippocampus to the BLA. It supports NMDAdependent plasticity and plays an important role in contextual fear conditioning (Maren, De Oca, & Fanselow, 1994; Maren & Fansel ow, 1995). Another pathway that has been implicated in the effects of predator stress on anxiety-like behavior in rats is that which extends from the ACe to the lateral column of the periaqueductal gray (PAG) (Adamec et al., 2001). The ACe and PAG have both been as sociated with rodent defense and anxietylike behaviors, and predator stress enhances activity in both areas. Theoretically, NMDAmediated plasticity within these pathways plays a major role in the lasting effects of predator stress on anxiety-lik e behaviors in rodents. Serotonin Some PTSD patients have been effectivel y treated with a family of drugs known as the selective serotonin reuptake inhibitors (SSRIs), a finding that has drawn attention to the role of 5-hydroxytryptamine (5-HT; sero tonin) in the progres sion of the disorder. Spivak and colleagues (Spivak, Vered, Gra ff, Blum, Mester, & Weizman, 1999) found significantly lower platelet-poor plasma concen trations of 5-HT in PTSD patients, while Fichtner and colleagues (Arora, Fichtner, OConnor, & Crayton, 1993; Fichtner, Arora, OConnor, and Crayton, 1994) detected a lower nu mber of platelet 5-HT transporters in these individuals. Increased serotonergic activity is essential for a successful stress

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150 response, and the findings of reduced 5-HT le vels in PTSD patients suggest that 5-HT dysregulation may play a role in the pathophysiology of the disorder. The 5-HT 1A receptors heavily innervate areas of the hippocampus (Richer, Hen, & Blier, 2002) and play a larg e role in mediating the st ress response (Lopez, Liberzon, Vazquez, Young, & Watson, 1999). In addition, activation of the 5-HT 2 receptors, which primarily occupy cortical areas, leads to a si gnificant increase in anxiety (McKittrick, Blanchard, Blanchard, McEwen, & Sakei, 1995). Investigators have observed elevated 5-HT 1A and 5-HT 2A receptor densities in the hippocampus and pref rontal cortex (PFC), respectively, of fear-sens itized rats (Kalynchuk et al., 2001). Harvey and colleagues (Harvey et al., 2003) examined the effects of the TDS stress model on rats physiology and found that it led to an increase in 5-HT 1A receptor density and a reduction in ligand affinity for this receptor 7 days poststress. These investigators also found an increase in ligand affinity for the 5-HT 2A receptor in the PFC and a marginal increase in its density within the same area. Research has indicated that the link betw een 5-HT and the stress response may involve the HPA axis (Cassano & DMello, 2001 ). For instance, activation of the 5-HT 2A receptors leads to HPA axis activation and a large increase in serum CORT levels (Heimrick-Leucke & Evans, 2002). In additi on, CORT has been shown to downregulate the expression of 5-HT 1A receptors, while adrenalectomy results in an upregulation of these receptors (Neumaier, Sexton, Hamblin, & Beck, 2000). Accordingly, Harvey et al. (2003) found significantly lower baseline CORT leve ls in stressed rats 7 days poststress. These rats also displayed a significant increase in 5-HT 1A receptor density, as mentioned

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151 above. These findings support the idea that reduc ed baseline levels of CORT, as a result of stress, leads to the modulation of 5-HT 1A receptor density in the hippocampus. Noradrenergic Mechanisms As mentioned above, individuals with PT SD tend to exhibit greater sympathetic tone at baseline. Research has found that these individuals ha ve higher resting levels of EPI and NE and that they produce greater el evations of these ag ents in response to traumatic reminders. Previous work has also found that individuals with PTSD react adversely to the administration of yohimbine, an 2 adrenergic receptor antagonist. Specifically, these individuals experience fl ashbacks and panic attacks after taking the drug, due to its effects on th e noradrenergic system (Southw ick et al., 1999b; Southwick et al., 1993). Investigators have observed an alogous results in stressed primates. Specifically, Rosenblum and colleagues (R osenblum, Coplan, Friedman, Bassoff, Gorman, & Andrews, 1994) found that macaque s raised in a stressful environment displayed hyperresponsivity to yohimbine as adults. These investigators attributed the hyperresponsivity to an exaggerated suppres sion of locomotor activity in the stressed monkeys. Such a finding is comparable to the present set of experiments, where yohimbine produced a greater suppression of ge neral activity, as ev idenced by a greater suppression of rearing and total distance tr aveled in the open field and a greater suppression of ambulations and total distance travel ed on the EPM, in the stressed rats. Relevance of the Present Findings to Understanding PTSD in Humans The present set of experiments provide s a novel approach to modeling PTSD in rats, and a number of characteri stics of this stress paradigm relate well to the symptoms

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152 and general environment endured by humans di agnosed with the diso rder. For instance, the present stress paradigm utilizes psychological stress of an ethologically relevant nature to produce its effects. Many other anim al models (as described in the introduction) have failed to use purely psychological stress to produce behavioral responses characteristically observed in human PT SD patients. Instead, these models have employed physical stressors, such as electric shock or swim stress, to produce behavioral abnormalities in common with PTSD. Clearly, the use of an ethologically relevant stressor, such as predator stress, would be preferable as a valid animal model of PTSD. Additionally, the pres ent paradigm uses a stress-restress paradigm that can be related to the experiences of humans with PTSD. These i ndividuals are exposed to an intense traumatic event, which is etched in to the neural circuitr y of their brain and produces the lasting consequen ces that have been descri bed thus far. Individuals diagnosed with PTSD endure flashbacks and unavoidable memories of the initial trauma throughout most of their lives. The present m odel uses a second stressful experience to force rats to relive the initia l event, a situation that is co mparable to the experiences of human subjects with PTSD. This characteristic of the model, along with the presence of daily mild stress, is analogous to the stress that PTSD patients endure on a daily basis. Behaviorally, the stressed rats in th e present experiments exhibited robust enhancements of anxiety, startle, and a hype rresponsivity to yohi mbine long after the initial trauma. The stressed rats showed a si gnificant reduction in open-arm exploration in the EPM, a finding that is indicative of heightened anxiety in rats and that can be related to the anxiety often observed in humans with PTSD. For instance, individuals diagnosed

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153 with PTSD tend to avoid public places because they fear ha ving panic attacks. Consequentially, they become antisocial and, sometimes, even agoraphobic. Thus, the behavior of the stressed rats in the present st udies can be related to the behavior that is often observed in PTSD patients. The stressed rats also exhibited an exa ggerated startle respon se during behavioral testing. Although the literature is not clear on whether or not base line startle is elevated in PTSD patients, the individuals diagnosed w ith the disorder frequently report having heightened startle. Indeed, this symptom is often described as one of the most annoying characteristics of the disorder (Morgan, 1997). Lastly, the stressed rats in the present study exhibited an exag gerated response to yohimbine. Park et al. (2001) found similar result s in rats that had been exposed to a cat for 5 weeks. The present experiments pr ovide the novel observa tion that only two exposures to a predator, along with daily unstable housing conditions, can produce analogous results. In addition, both of these experiments (Park et al, 2001 and the present study) appeared to find that yohimbine led to a greater suppression of locomotor activity in the stressed rats, which is consistent w ith the findings of Rosenblum et al. (1994), who found that yohimbine led to a greater suppression of locomotion in primates that had been raised in a stressful environment. The fi nding of hyperresponsivity to yohimbine in the stressed rats supports human research on PTSD and again provides further validation of the present stress paradigm as an animal model of the disorder. Nevertheless, it must be noted that the present paradigm does not produce a condition in rats that is co mpletely analogous to the span of behavioral symptoms

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154 observed in human subjects with PTSD. It ma y be very difficult, if not impossible, to model every symptom that is observed in humans with the disorder. Even so, the current model is a step towards understanding how the behavioral sy mptoms that are common to PTSD (e.g., heightened anxiet y, exaggerated startle, hyperr esponsivity to yohimbine) are produced and what can be done to reverse thes e manifestations. It is clear that a valid animal model of PTSD would not only al low investigators to understand how the disorder progresses and better characterize its underlying neurobiolog ical alterations, but also how to treat the disorder and potentially, with time, how to alle viate the disorder or even prevent it before it starts The present experiments are only a small step to hopefully affording researchers these opportunities in the future.

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162 Cohen, H., Benjamin, J., Geva, A. B., Matar, M. A., Kaplan, Z., & Kotler, M. (2000a). Autonomic dysregulation in panic disorder and in post-traumatic stress disorder: Application of power spectrum analysis of heart rate variability at rest and in response to recollection of trauma or panic attacks. Psychiatry Research 96, 1-13. Cohen, H., Benjamin, J., Kaplan, Z., & Kotle r, M. (2000b). Admini stration of high-dose ketoconazole, an inhibitor of steroid synthesis, prevent posttraumatic anxiety in an animal model. European Neuropsychopharmacology, 10, 429-435. Cohen, H., Kotler, M., Matar, M. A., Kapla n, Z., Loewenthal, U., Miodownik, H., et al. (1998). Analysis of heart rate variability in posttraumatic stress disorder patients in response to a trauma-related reminder. Biological Psychiatry, 44, 1054-1059. Cohen, H., Kotler, M., Matar, M. A., Kapl an, Z., Miodownik, H., & Cassuto, Y. (1997). Power spectral analysis of heart variability in posttraumatic stress disorder patients. Biological Psychiatry 41, 627-629. Cohen, H., Zohar, J., Gidron, Y., Matar, M. A., Belkind, D., Loewenthal, U., et al. (2006). Blunted HPA axis response to st ress influences susceptibility to posttraumatic stress response in rats. Biological Psychiatry 59 1208-1218. Cohen, H., Zohar, J., & Matar, M. (2003). The relevance of differential response to trauma in an animal model of posttraumatic stress disorder. Biological Psychiatry 53, 463-473. Cohen, H., Zohar, J., Matar, M. A., Kaplan, Z., & Geva, A. B. (2005). Unsupervised fussy clustering analysis suppor ts behavioral cutoff criteria in an animal model of posttraumatic stress disorder. Biological Psychiatry 58, 640-650.

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