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Title:
Influence of temporary inactivation of the prefrontal cortex of hippocampus during stress on the subsequent expression of anxiety and memory
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Book
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English
Creator:
Halonen, Joshua D
Publisher:
University of South Florida
Place of Publication:
Tampa, Fla
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Subjects / Keywords:
PTSD
Fear conditioning
Novel object recognition
Flashbulb memory
Muscimol
Dissertations, Academic -- Psychology -- Masters -- USF   ( lcsh )
Genre:
non-fiction   ( marcgt )

Notes

Abstract:
ABSTRACT: The neural pathways underlying the symptoms of Post Traumatic Stress Disorder (PTSD) have not been fully elucidated. Intrusive memories, persistent anxiety and other cognitive deficits have been attributed to maladaptive or otherwise aberrant processing in specific brain regions, including the hippocampus, amygdala and prefrontal cortex. Our laboratory has developed an animal model of PTSD which results in the enhancement of memory for a place associated with exposure to a predator, anxiety-like behavior, increased startle and impaired memory in a non-aversive memory task. To better understand how the interaction of the hippocampus and prefrontal cortex contribute to the different symptoms of the disorder, we investigated the transient inactivation of each structure during an intense stressor. Our results show that long-term contextual fear associations involve activity in both the hippocampus and the prefrontal cortex, but only the prefrontal cortex is involved in cued fear memories as well.
Thesis:
Thesis (M.A.)--University of South Florida, 2009.
Bibliography:
Includes bibliographical references.
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Mode of access: World Wide Web.
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Statement of Responsibility:
by Joshua D. Halonen.
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Title from PDF of title page.
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Document formatted into pages; contains 50 pages.

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aleph - 002029091
oclc - 436869684
usfldc doi - E14-SFE0002880
usfldc handle - e14.2880
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ABSTRACT: The neural pathways underlying the symptoms of Post Traumatic Stress Disorder (PTSD) have not been fully elucidated. Intrusive memories, persistent anxiety and other cognitive deficits have been attributed to maladaptive or otherwise aberrant processing in specific brain regions, including the hippocampus, amygdala and prefrontal cortex. Our laboratory has developed an animal model of PTSD which results in the enhancement of memory for a place associated with exposure to a predator, anxiety-like behavior, increased startle and impaired memory in a non-aversive memory task. To better understand how the interaction of the hippocampus and prefrontal cortex contribute to the different symptoms of the disorder, we investigated the transient inactivation of each structure during an intense stressor. Our results show that long-term contextual fear associations involve activity in both the hippocampus and the prefrontal cortex, but only the prefrontal cortex is involved in cued fear memories as well.
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Influence of Temporary Inactivation of the Prefrontal Cortex or Hippocampus during Stress on the Subsequent Expression of Anxiety and Memory by Joshua D. Halonen A thesis submitted in partial fulfillment of the requirements for the degree of Mas ter of Arts Department of Psychology College of Arts and Sciences University of South Florida Major Professor: David Diamond, Ph.D. Cheryl Kirstein, Ph.D. Keith Pennypacker, Ph.D. Jonathan Rottenberg, Ph.D. Date of Approval: March 4, 2009 Keyword s: PTSD, fear conditioning, novel object recognition, flashbulb memory muscimol Copyright 2009, Joshua D. Halonen

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In loving memory of Eugene Halonen

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Acknowledgements I would first like to thank my ma jor professor, Dr. David Diamond, for affording me the opportunity to conduct this research and providing his guidance and expertise. I would also like to thank my committee members, Dr. Keith Pennypacker, Dr. Jonathan Rottenberg, and Dr. Cheryl Kirstein for their understanding and helpful input from my initial proposal to the final manuscript. I am also grateful for the assistance of my fellow laboratory assistants, Dr. Collin Park, Dr. Phillip Zoladz, and Shyam Seetharamen. You have each challenged me and helped me become a better researcher, and I am thankful to have you as my colleagues. I would also like to thank all of my past professors and instructors for inspiring my love of science. Finally, I would like to thank my friends and family who have helped me through my entire academic career thus far

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i Table of Contents List of Tables iii List of Figures iv Abstract v Chapter One: Background Multiple Memory Theory 1 Emotion and Memory Post Traumatic Stress Diso rder 2 Memory Enhancement 3 Memory Impairment 4 Animal Models of PTSD 6 Neuroanatomy 10 Hippocampus 10 Prefrontal Cortex 11 Hypothes es of the Present Experiments 12 Chapter Two: Experiments Does In activation of the Hippocampus or Prefrontal Cortex affect Memory and Anxiety like Behaviors induced by Predator Exposure? Methods Design 1 5 Animals 1 5 Surgery 1 6 Infusions 1 7 Histology 1 7 Stress Procedure 1 8 Behavioral Apparatus Fear A ssociations 1 9 Elevated Plus Maze 2 0 Startle Response 2 1 Object Recognition 2 1 Statistical Analysis 2 2 Results Fear Conditioning PFC Contextual 2 3

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ii PFC Cued 2 3 Hippocampal Context 24 Hippocampal Cue 24 Elevated Plus Maze PFC Percen t Time Spent in the Open Arms 26 PFC Percent Time Spent in the Closed Arms 27 Hippocampa l Time Spent in the Open Arms 28 Hippocampal Percent Time Spent in the Closed Arms 28 Startle Respons e 28 Novel Object Reco gnition Prefrontal Cortex 28 Hippocampus 29 Fecal Boli 3 2 Discussion Major Findings and Significance Prefrontal Cortex 3 4 Hippocampus 3 5 Limitations 3 6 Gener al Discussion and Conclusions 3 8 References 40

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iii List of Figures Figure 1. Schematic Diag ram of the Elevated Plus Maze 2 1 Figure 2. Schematic Diagra m of the Open Field Apparatus 2 2 Figure 3. Effects of Cat and Immobiliza tion with vmPFC Inactivation 2 6 on Contextual Freezing Figure 4. Ef fects of Cat and Immobilization wi th vmPFC Inactivation 2 6 on Cued Fear Conditioning Figure 5. Effects of Cat and Immobilization with CA1 Inactivation 2 7 on Contextual Freezing Figure 6. Effects of Cat and Immobilization wit h CA1 Inact ivation on 2 8 Cued Fear Conditioning Figure 7. Effects of Cat and Immobiliz ation with vmPFC Inactivat ion 2 9 on Elevated Plus Maze Figure 8. Effects of Cat and Immobil ization with CA1 Inactivation 30 on Elevated Plus Maze Figure 9. Effe cts of Cat and Immobiliz ation with vmPFC Inactivation 3 1 on Novel Object Recognition Figure 10. Effects of Cat and Immobili zation with CA1 Inactivation 3 2 on Novel Object Recognition Figure 11. Effects of Cat and Immobiliz ation with vmPFC Inactivat ion 3 3 on Total Boli Figure 12. Effects of Cat and Immobiliz ation with vmPFC Inactivation 3 4 on Total Boli

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iv Inactivation of the Prefrontal Cortex or Hippocampus Differentially Affects Predator Induced Fear Memories and Blocks Non Stressful Memo ry Impairments Joshua D. Halonen ABSTRACT The neural pathways underlying the symptoms of Post Traumatic Stress Disorder (PTSD) have not be en fully elucidated. Intrusive memories, persistent anxiety and other cognitive deficits have been attributed to mal adaptive or otherwise aberrant processing in specific brain regions, including the h ippocampus, a mygdala and p refrontal cortex. O ur laboratory has developed an animal model of PTSD which result s in the enha ncement of memory for a place associated with expo sure to a predator, anxiety like behavior increased startle and impaired me mory in a non aversive memory task. T o better unders t and how the interaction of the hippocampus and p refrontal c ortex contribute to the different symptoms of the disorder, we inve stigated the transient inactivation of each structure duri ng a n intense stressor Our results show that long term contextual f ear associations involve activity in both the hippocampus and the prefrontal cortex, but only the prefrontal c ortex is involved i n cued fear memories as well.

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1 Chapter One: Background Multiple Memory Systems The idea that brain structures network with one another during learning has lead to a better understanding of the multiple systems that mediate the formation of memories (McDonald & White, 1993; McDonald & White, 19 95; McDonald, Devan, & Hong, 2004; Sutherland, McDonald, Hill, & Rudy, 1989; Kim, Lee, Han, & Packard, 2001; Packard & Cahill, 2001; Packard & Teather, 1998; Packard, Hirsh, & White, 1989; Poldrack & Packard, 2003) Although there is debate among researc hers ab out the distinctions between which brain structures are involved in particular memory fu nctions, multiple memory theory is accepted as the nature of memory processes (Meeter, Veldkamp, & Jin, 2009 ; Weber et al., 2005) Neural networks orchestrate distinct types of skill learning, declarative neutral and emotional memories in paral lel. Individual structures process information and communicate with other structures to form a memory and influence b ehavior to later stimuli. Strong emotional experiences have powerful eff ects on the formation of memory, often facilitating durable memories. P eople who experience acute trauma and respond with intense fear, helplessne ss or horror and then relive the trau ma through intrusive flashback memories are prone to be diagnosed with PTSD Not everyone exposed to trauma develops PTSD, but in those individuals that develop the disorder it seems the abnormally durable memory of a particular event comes at the cost o f concentration and

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2 memory for trauma neutral information (Moores et al. 2008 ; Gil, Calev, Greenberg, Kugelmass, & Lerer, 1990) The following sections will briefly outl ine PTSD and the paradox between emotional en hancement of traumatic memory and impairment of post trauma working memor y, followed by a summary of the brain structures implicated in these phenomena. Post Traumatic Stress Disorder One of the diagnostic crit eria of PTSD is experiencing a traumatic event (McN ally, 2003; Moores et al., 2008 ) To be considered “traumatic,” the event must pose a n actual or a t least a perceived threat to the individual’s physical well being and cause a sense of loss of control. Individuals diagnosed with PTSD experience trouble concentrating and functioning in their daily lives These symptoms are exacerbated by reminders of the trauma which trigger intr usive memories (Bryant, 2003; Reynolds & Brewin, 1999) Accordingly, individuals with PTSD make great efforts to avoid stimuli that remind them of their trauma. While the type (rape, combat, natural disaster, etc.) and characteristics (duration, intensity, or stage of life) of the trauma (Heim & Nemeroff, 2001; Stam, 2007) play a role in w hether or not an individual will develop PTSD, only about 25% of individuals who are exposed to trauma develop the disorder (Yehuda, 2001) This s upports the hypothesis that there is some fundamental difference between individuals that do and individuals that do not de velop the disorder. One criticism of the reports of cognitive impairments in PTSD patients is that often these investigations report high comorbid ity with major depressive disorder (MDD) and a history of substance abuse in PTSD groups, making it difficult to attribute the

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3 cognitive deficits to a single cause. Cognition can be negatively affected by both MDD (Veiel, 1997) and substance abuse (Goldman, 1999; Goldman, Brown, Christiansen, & Smith, 1991) In order to c ontrol for these factors Neylan et al. ( 2004) excluded individuals with MDD or substance abuse an d found no differences between PTSD patients and controls on measures of cognitive functioning, including assessme nts of attention. T hese findings suggest that deficits in cognitive functioning may not be a characteristic of PTSD; rather, they could be a predisposition to mental illness in general Enhancement The sights, sounds, or even smells related to trauma can evoke a powerful memory In patients with PTSD reminders of the traumatic event can lead to reliving the initial experience commonly refer red to as a “flashback” In order to conceptualize these flashbacks, researchers have put forth the notion that negative emotion is associated wi t h information learned around the time of a trauma (Ehlers & Clark, 2000 ) Thus when presented with a sensory cue associated with negative emot ions people with PTSD have anxiogenic intrusive memori es specific to the trauma (Diamond, Campbell, Doan, & Park, 1999; Diamond, Park, & Woodson, 2004; Ehlers, Hackmann, & Michael, 2004; Ehlers & Steil, 1994) These intrusive memories can lead people with PTSD to avoid reminders of the t rauma because of the intense anxiety they cause (Burste in, 1985) and negatively affect their daily life functioning, as mentioned earlier. Understanding how memory is enhanced by emotion has long been of interest to psychologists. One type of this enhancement has been lab eled a “flashbulb memory” (Berntsen & Rubin, 2006; Brown & Kulik, 1977; Conway et al., 1994; Diamond, Campbel l, P ark, Halonen, & Zoladz, 2007 ) In this form of memory, strong emotion

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4 enhances the salience of the sensory information of an experience resulting in a powerful form of learning (Christianson, 1992; Richter Levin & Akirav, 2003; Diamond et al., 1999; Diamond et al., 2004; Ehlers & Clark, 2000 ) Thus, emotion enhances recall compared to learning the same information under non emotional circumstances. People with PTSD show notable differences in bra in activity in the amygdala, hippocampus and the prefrontal cortex as compared to controls (Bremner, 1999) The amygdala has been heavily implicated in PTSD and a vast literature exists describing the role it plays in emotion regulation and fear conditioning. The hippocampus and prefrontal cortex are critical for learning and memory (Goldman Rakic, 1987; Morris, Garrud, Rawlins, & O'Keefe, 1982; O'Keefe & Speakman, 1987) The interactions among these structures during an intensely emotion al event, such as a tr auma, play a role in the enhancement of memory. In summary, the hippocampus and prefrontal cortex in conjunction with the amygdala are likely candidates for the neural processing responsible for forming flashbulb memories. Impairment Many investigators have suggested that i ntrusive mem ories are detrimental to concentration and working memory in PTSD patients (Bremner, Vermetten, Afzal, & Vythilingam, 2004; Chemtob et al., 1999; Halligan, Clark, & Ehlers, 2002; Jelinek et al., 2006; Kivling Boden & Sundbom, 2003; Litz et al., 1996; McFarlane, Weber, & Clark, 1993; Schonfeld & Ehlers, 2006; Sutker, Winstead, Galina, & Allain, 1991; Tapia, Clarys, El Hage, Belzung, & Isingrini, 2007; Vasterling, Brailey, Constans, & Sutker, 1998b; Yehuda et al., 1995) Chemtob et al. (1999 ) examined the ability o f Vietnam veterans with PTSD to attend to a primary digit detection paradigm while c oncurrently

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5 viewing either neutral or Vietnam – related distracters and found that PTSD patients’ performance was worse than other groups of combat expos ed or psychopathology patients when trauma related pictures were presented These and other results (Bremner et al., 1995 ) indicate that i ntr us ive memories can interfere with daily functioning in PTSD patients by reducing their ability to pay attention to information like names and other pieces of information. P hysiological studies using event related potentials (ERP) suggest that trauma neutr al information is abnormally processed, such that PTSD augments the way people evaluate the significance of a stimulus and the subsequent executive processes associated with working memory ( McFarlane et al., 1993; Galletly, Clark, McFarlane, & Weber, 2001) The stimulus is then proc essed as a danger signal, rather than a neutral environmental stimulus. Stress can have detrimental effects on hippocampus dependent learning an d memory. In fact numerous studies have reported declarative and working memory impairments, along with deficit s in attention, in PTSD patients (Bremner, Krystal, Southwick, & Charney, 1995; Bremner et al., 1993; Gilbertson, Gurvits, Lasko, Orr, & Pitman, 2001; Golier et al., 2002; Jenkins, Langlais, D elis, & Cohen, 1998; Sachinvala et al., 2000; Moradi, Doost, Taghavi, Yule, & Dalgleish, 1999; Uddo, Vasterling, Brailey, & Sutker, 1993; Vasterling, Br ailey, Constans, & Sutker, 1998 ; Barrett, Gree n, Morris, Giles, & Croft, 1996 ) However, some research ers have found no diff erences between individuals with PTSD and healthy control s or subjects when not using trauma related material to influence cognitive functioning (Barrett, Green, Morris, Giles, & Croft, 1996 ; Crowell, Kieffer, Siders, & Vanderp loeg, 2002; Neylan et al., 2004 ; Zalewski, Thompson, & Gottesman, 1994) This means that not all cognitive capabilities of PTSD

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6 patients are always affected by the disorder and they are able to perform some tasks normally Animal Models of PTSD Although PTSD remains a disorder that is unique to humans, the limitations of human rese arch necessitate a valid animal model of PTSD. Such a model would potentially allow investigations into the factors that contribute to the disorder’s development, as well as, the neurobiological progression of the disorder This would pave the way to stu dy effects of therapeutic agents on the treatment of the disorder. Stressors such as electric shock, immobilization (i.e., restraint stress), underwater trauma, and predator stress have been used to produce behavioral effects in rodents that are comparabl e to those observed in humans with PTSD. However, the fact that many of these models do not reliably generate the range of symptoms displayed in PTSD p atients warrants a set of criteria that all animal models of PTSD should meet before they are accepted b y the scientific community. According to Yehuda & Antelman ( 1993) animal models of PTSD should attempt to inc orporate five key aspects: 1) Very brief stressors should be capable of inducing the biological and behavioral sequelae of PTSD. 2) The stressor should be capable of producing the PTSD like sequelae in a dose dependent manner because the disorder is produ ced by a threshold “dose” of stress in humans 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 biological and behavioral alterations that have the potential for enhanced or reduced responsiveness to different aspects of the environment. 5) Variability in response to a stressor should be

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7 present either as a function of experience (e.g., prior history and post stress adaptations), genetics, or an interaction of t he two ( Yehuda & Antelman, 1993 p. 480 482) Once an animal model satisfies these criteria, the behavioral and biological consequences of trauma can be further elucidated. To model PTSD, most investigators expose rodents to some form of stress and then assess the effects of that stress on physiology and behavior. The investigators typically compare the entire stressed groups to control animals. Cohen, Zohar, & Matar ( 2003) argued that some animals appear to be more vulnerable to the stress than others, which supports the fifth criteri on in Yehuda & Antelman’s (1993 ) manuscript. Given this argument, Cohen et al. (2003 ) examined the differen tial response of rats to intense stress by exposing 150 rats individually to a cat for a period of 10 minutes and then examined their behavior on the elevated plus maze one week later. As a group, the stressed rats exhibited greater levels of anxiety on t he elevated plus maze, relative to controls. Interestingly, within the stressed group of rats, some did not show elevated levels of anxiety and freely explored the open arms of the maze. Therefore, the investigators used cutoff behavioral criteria to div ide the stressed rats into well adapted (WA) or maladapted (MA) rats, based on time spent in the closed arms and entries into the open arms. Physiological analyses indicated that the MA rats exhibited greater levels of adrenal hormones shortly after the s tress compared to WA rats. The MA rats also displayed greater sympathetic nervous system tone and lower vagal tone base d on lower heart rate variability, showing higher high frequency and lower low frequency component of their heart rates Cohen and coll eagues have replicated and extended this work by reporting similar effects on other behavioral measures, such a s the acoustic

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8 startle response; as well as the use of a different stressor ( underwater trauma ) to manifest similar results (Cohen, Zohar, Matar, Kaplan, & Geva, 2005; Cohen et al., 2004) Collectively, these findin gs support the notion 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 indivi dual reacts the same way to stress and that trau ma can come in different forms The study of varying types, intensities, and durations of stress have also provided valuable insight into the physiological and behavioral changes in rodents. Chronic restraint stress (6 hrs/d ay for 21 days) leads to the remodeling of hippocampal dendrites (Magarinos, McEwen, Flugge, & Fuchs, 1996; Magarinos & McEwen, 1995) and impairments of hippocampus dep endent, spatial memory (Conrad, Galea, Kuroda, & McEwen, 1996; Luine, 1994; Luine, Villegas, Martinez, & McEwen, 1994) Other investigators have studied the effects of a small number of stress sessions or a single stress session with periodic reminders of the “trauma” on long term behavior in rodents. (Pynoos, Ri tzmann, Steinberg, Goenjian, & Prisecaru, 1996) exposed mice to footshock (2 mA for 10 seconds) and then assessed their behavioral response 1, 21, or 42 days later. Some of the stressed mice were reminded weekly throughout the experiment by placing them back in the apparatus where they received the shock. Only mice that were given reminders of the shock exhibited increased anxiety on the elevated plus maze 1, 21, and 42 days after being shocked, but they did not demonstrate an exaggerated startle respons e until six weeks post stress. Servatius and colleagues (Servatius, Ottenweller, Bergen, Soldan, & Natelson, 1994; Servatius, Ottenweller, & Natelso n, 1995; Adamec & Shallow, 1993 ) also observed a delayed sensitization of startle following exposure to

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9 repeated restraint a nd tailshock s tress. T hese findings have been inconsistent, stress may induce a delayed sensitization of rats’ startle response, but the timeline for this effect is unclear. An investigation by Adamec & Shallow (1993 ) found heightened anxiety like behavior in rats following a single five min ute exposure to a cat a s indicated by a r eduction in the ratio of time spent in the open to closed arms on the elevated plus maze up to three weeks later. This effect is theoretically based on NMDA receptor dependent plasticity in the amygdala which is responsible for the las ting effects of cat exposure on anxiety (Adamec, Muir, Grimes, & Pearcey, 2007) Further work by Adamec and colleagues (Adamec, Burton, Shallow, & Budgell, 1999a; Adamec, Burton, Shallow, & Budgell, 1999b; Blundell, Adamec, & Burton, 2005) has supported the argument that these effects are mediated, in part, by NMDA receptor de pendent plasticity. When rats were administered competitive NMDA receptor antagonists 30 minutes prior to cat exposure, it blocked lasting increases in anxiety like behaviors. However, these drugs were incapable of blocking the stress induced increase in anxiety like behaviors if they were administered 30 minutes after cat exposure, suggesting that they had to be present at the time of the stress to be effective. Studies have observed NMDA receptor dependent synaptic plasticity in the amygdala as a resu lt of fear conditioning (Bauer, Schafe, & LeDoux, 2002; Rogan, Staubli, & LeDoux, 1997) and the administration of NMDA receptor antagonists within the ventricular system (Fanselow, Kim, Yipp, & De Oca, 1994; Kim, DeCola, Landeira Fernandez, & Fanselow, 1991 ; Kim, Fanselow, DeCola, & Landeira Fernandez, 1992) or amygdala (Maren, Aharonov, Stote, & Fanselow, 1996) prevents the formation of fear

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10 memories in rodents These findings support the idea that stress induces N MDA receptor dependent plasticity probably within the amygdala, results in heightened anxiety like and fear behavior. Our laboratory has recently developed a novel form of modeling PTSD in rats using the combination of predator exposure and restraint, in conjunction with daily social instability (Zoladz, Conrad, Fleshner, & Diamond, 2008) In this model two stress sess ions with social instability were su fficient to produce enhanced anxiety like behavior on the ele vated plus maze, increased startle and an impaired ability to recognize a familiar object. Animals that went through the trauma like ex periences also exhibited differences in their hypothalamic pituitary axis and cardiac function s compared to controls T he following experiments were designed to manip ulate rats’ information processing in specific brain regions to exacerbate or mitigate the expression of PTSD like symptoms. These experiments will also extend our model by manipulating the number and type of stressors to investigate the effects on contextual and cued fear memory, and other behavioral consequences, that a single cat a nd immobilization session have on rats Neuroanatomy Hippocampus Hippocampal divisions are based primarily on the cellular org anization and neuroanatomical features of each region conserved across mammals. The p erforant pathway is fibers from the entorhinal cortex that terminate in the dentate gyrus and CA3 regions. Schaffer collaterals, which are axons from the CA3 pyramidal c ells, project to CA1 pyramidal cells. Neurons in the CA1 project to entorhinal cells, which relay to the

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11 cortex. According to some reports, the CA1 region of the hippocampus play s an integrative role in memor y because it receives input from various modal ities and outputs to the cortex (Akirav, Sandi, & Richter Levin, 2001; Artola et al., 2006; Cao, Chen, Xu, & Xu, 2004; Kim, Foy, & Thompson, 1996) The capacity of the hippo campus to receive and integrate information fr om different senses allows the hippocampus to generate a coherent representation of the context through the associat ions made between the information according to Shapiro & Eichenbaum ( 1999) Thus, the hippocampus is important for acquiring new declarative memories (Bunsey & Eichenbaum, 1996; Eichenbaum, 2004) which can be either emotional or neutral in nature. In la boratory animals, damage to the hippocampus seven days before contextual learning (Selden, Ev eritt, Jarrard, & Robbins, 1991 ) or musca rinic cholinergic receptor antagonism of the hippocampus fifteen minutes prior to the learning (Anagnostaras, Maren, & Fanselow, 1999; Selden, Ev eritt, Jarrard, & Robbins, 1991 ) impair performance on con textual fear conditioning. Prefrontal Cortex The prefrontal cort ex is particularly impor tant for flexible behavioral reactions (Aston Jones, Rajkowski, & Cohen, 2000) and has demonstrated involvement in sus tained attention in rodents (Granon, Hardouin, Courtier, & Poucet, 1998) The prefrontal cortex monitor s incoming environmen tal information and initiate s appropriate behavior based on the circumstances at any given time (Dalley, Cardinal, & Robbins, 2004 ) Trauma related memories in abused women result in overactive prefrontal cortex (Bremner et al., 2005) Extinction of fear con ditioning in rodents has also been shown to

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12 depend on the prefrontal cortex (Herry & Garcia, 2002; Morgan & LeDoux, 1999; Quirk, Garcia, & Gonzalez Lima, 2006; Sotres Bayon, Cain, & LeDoux, 2006) These effects have been attributed to a failure of the prefrontal cortex to suppress attention to trauma related stimuli subsequently allows over excitability of the amygdala and results in resilient extinction resistant memories (Gilboa et al., 2004) The ventromedial area of the prefrontal cortex, including the prelimbic and infralimbic sections have strategic connections involved in the extinction of fear behaviors in rodents (Amat et al., 2005 ; Da lley, Cardinal, & Robbins, 2004; Ishikawa & Nakamura, 2003 ; Vertes, 2004) Direct neural pathways with the hippocampus and the amygdala, along with lower brainstem areas that are involved in the regulation of neurotransmitter syste ms such as the ventral tegmental area, dorsal raphe nucleus and locus c o eruleus support the role for the prefrontal cortex in the regulation of behavior (Burette, Jay, & Laroche, 1997; Herman, Prewitt, & Cullinan, 199 6; Irle & Markowitsch, 1982; Ishikawa & Nakamura, 2003 ; Shu, Wu, Bao, & Leonard, 2003) Thus, the prefrontal cortex likely plays an integral role in the formation of memories and behavioral reactions to environmental stimuli. Hypotheses In order to gain insight into the multiple components of memory researchers have utilized the GABA agonist muscimol to suppress neural activity in specific structures. Understanding the functional interactions among the prefrontal cortex and hippocampus during a traumati c experience may lead to effective diagnostic and treatment strategies for PTSD and other anxiety disorders.

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13 The present experiments were designed to selectively and transiently suppress activity in the ventromedial portion of the prefrontal cortex or CA1 region of the hippocampus before conditioning rats in a single stress model of flashbulb memories. This model generates a durable fear association to a context and produces high levels of anxiety along with deficits in non aversive memory of intact rats t hat receive inescapable intense predator stress. To test the extent of interactions between the hippocampus and prefrontal cortex in the formation of long term fear memories, anxiety like behaviors startle and general non emotional memories, mus cimol was used to inactivate the neural activity of these structures at the time of the inescapable cat exposure My first hypothesis was that suppression of the prefrontal cortex at th e time of emotional learning would allow more amygdalar activation than during vehicle treatment rendering a more emotional memory This should have facilitate d a more durable and salient fear memory and increased anxiety, rendering a more fearful animal in the presentation of the context and cue s associated with a strong stressor a nd a more anxious animal in general. M y second hypothesis was that suppressing the hippocampus before the stress would weaken the formation of contextual fear. However, these animals should have exhibit ed heightened anxiety like behaviors, but maintain ed equivalent cognitive capacity as compared to vehicle stress animals. All vehicle treated stressed animals were hypothesized to show heightened fear, anxiety, and poorer object recognition memory as compared to non stressed controls. The general hypothesi s was that in both humans and rats the hippocampus and prefrontal cortex interact to mediate memory and anxiety. During a traumatic experience

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14 each structure provides unique processing, such that the inactivati o n of each individual area during trauma woul d influence different aspects of memory and anxiety.

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15 Chapter Two : Experiments Methods Design Hippocampal and pre frontal manipulations were conducted in 2 experiments based on the targeted brain structure. Each of the experiments utilized a 2x 2 factorial design with artificial cerebral spinal fluid (aCSF ; Harvard Laboratories ) used as v ehicle or m uscimol and immobilization with cat exposure (Cat) or homecage (No Cat) as the levels. Animals A to tal of 78 male Sprague Dawley rats (Charles River) weighing 225 250g on arrival were acclimated to the vivarium and cage changes for at least 7 days before any experimental manipulat ions are conducted. Rats were housed 2 per cage (standard Plexiglas – 46 x 25 x 21 cm) until s urgery, after which they were singly housed Tap water and rat chow were avai lable ad libitum The animal housing room was maintained at 20 1 C with a humidity range of 60 3%, and a 12hr light cycle (on at 0700 hr). All procedures wer e approved by the Institutional Animal Care and Use Committee at the University of South Florida. Surgery On the day of surgery, rats were brought to the laboratory where all surgical procedu res were performed under aseptic conditions Rats were deeply anesthetized using isoflurane. Their heads were shaved and placed level on a stereotaxic device.

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16 After the skull was exposed, the topographical coordinates for the landmarks of bregma and lambda were recorded for targeting purposes. All targets are in reference to the skull surface of bregma in m illimeters and insertions wer e made with 26 gauge, stainless steel, guide cannula e (Plastics One Inc., Roanoke, VA). Target coordinates for the vmPFC were +2.7 anterior posterior (AP), 0.5 medial lateral (ML), and 5.0 dorsal ventral (DV). The target of the hippocampus was the dorsal CA1 region, and the coordinates used were 3.8 AP, 3.0 L, 2.8 DV. These target coordinates were based on the Paxinos & Watson rat brain atlas and pilot data. Bilateral g uide ca nnula e were held in place by dental cement and anchored to the skull with four skull screws. Removable stylets projecting 1mm from the tip of the guide cannula were inserted and held in place with a screw on dust cap (Plastics One Inc., Roanoke, VA) to kee p the cannula patent. Infusions All animals were given one week to recuperate from surgery before data collection. All infusion and behavioral procedures were performed between 0900 1500 hours. For three consecutive days, in order to acclimate the animal s to the infusion procedure, animals were brought into the laboratory and given at least 30 minutes to adjust to the surroundings. On the first day, the dust cap was removed and a mock injection tube placed on the cannula e pedestal to familiarize the rats to the sensation of the tube on their head. The second and third day consisted of the removal the dust cap and stylet, and gently placing the injectors (Plastics One) in the guide cannula e A Harvard Apparat us pump (Holliston, MA) was connected to 25l syringe injectors (Hamilton) b y plastic tubing (Plastics One) and infused aCSF at a rate of 0.1l/min for 3

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17 minutes. After the infusion, the pump was turned off and the fluid given 1 minute to diffuse before the dummy cannula e were replaced and dust cap s crewed back on the top of the pedestal. On th e third day, aCSF or muscimol were administered. Histology A total of 70 rats completed the battery of tests. Upon completing the behavioral tasks all animals were euthani zed with an overdose of k etamine and x ylaxine, cresyl violet was infused into the cannula e at a rate of 0.1l/min for 5 minutes to allow visual inspection of cannula e placement The brains were extracted and flash frozen in 2 methylbutane and the tissue was stored at 80C until it was sliced in co ronal sections in 40m s ections on a Cryostat held at 16C and mounted on microscope slides. After histological analysis of 30 representative samples four animals were eliminated for placement outside the target area. Behavior Stress Procedure Approximately 15 minutes after the rats were infused with aCSF or muscimol, they were placed in a dark fear conditioning chamber (25.5 x 30 x 29 cm; Coulbourn Instruments; Allentown, PA) that consists of two aluminum sides, an aluminum ceiling, and a Plexiglas front and back covered with black plastic. The cham ber served as the cont ext associated with a c at The floor consists of 18 stainless steel rods, spaced 1.25 cm apart. Exposure to the chamber for three minutes terminated with presentation of a single 30 second, 74 dB 2500 Hz tone, which served as the au ditory cue. Animals in the c at groups were immediately immobilized using a plastic DecapiCone (Braintree Scientific; Braintree, MA) and then placed in a pie shaped

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18 Plexiglas enclosure (Braintree Scientific; Braintree, MA; 20 x 20 x 8 cm). This container was placed inside a cage containing a female cat to keep the cat in close proximity to the rats. T o direct the cat’s activity to the container, a small amount of wet cat food was smeared on top of the container. Rats then remained with the cat for one ho ur. This procedure has been developed to produce a fear memory in rats to a context that is, otherwise, innocuous. A nimals in the no c at groups were placed back in their home c ages for one hour Fear Association Three weeks lat er, each rat was brought ba ck to the laboratory and allowed 30 minutes to acclimate to the environment. After acclimation rats were placed in the same fear conditioning chamber as the one in which they were placed just before receiving the stress treatment 3 weeks earlier. The fr eezing behavior of each rat was monitored by computer for five minutes. Approximately 45 60 minutes after the contextual memory test, rats were individually placed in the light side (25 x 22.5 x 33 cm) of a shuttle box (Coulbourn Instrumen ts; Allentown, P A) that consisted of two aluminum sides, an aluminum ceiling, and a Plexiglas front and back, with the shuttle door in the closed position. A house light was turned on, and a metal plate (21.5 x 21.5 cm) placed on the floor to eliminate the sensation of t he stainless steel rods beneath their paws. These conditions reduce the similarities between the conditioning chamber context and the auditory cue testing chamber. The rats remained in the light side of the shuttle box for a total of six minutes. Our pa radigm consisted of no tone present for the first three min utes and introducing a tone (74 dB; 2500 Hz) for the last three min utes This paradigm provides a measure for a novel context and a more direct measure of the cue memory. Therefore, rats’ freezin g behavio r in response to the tone was considered as a

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19 measure of their memory for the tone cat association, independent of the context. The amount of time freezing and feca l boli were recorded and considered as an index of fear. Freezing was measured by a 24 cell infrared activity monitor (Coulbourn Instruments; Allentown, PA), mounted on the top of the fear conditioning chamber, which uses the emitted infrared body heat image (1300 nm) from the animal to detect relative changes in movement. Freezing wa s defined as perio ds of inactivity for more than three seconds, except for movement required for respiration. A Microsoft Excel spreadsheet with a macro designed to analyze freezing behavior was used to calculate the total number of seconds spent freezing by each animal at 30 sec epochs divided by the total amo unt of time in the chamber, providing a percentage of time freezing for each animal. The use of automated parameters have been employed by labora tories elsewhere (Lee & Kim, 1998 ) and have been shown to significantly correlate with time sampling observer methods often employed to assess freezing behavior (Kim, DeCola, Lande ira Fernand ez, & Fanselow, 1991 ) Elevated Plus Maze (EPM; see Figure 1) Twenty four hours after the context and cue tests, all rats were brought to another room of the laboratory and subjected to the EPM assessment. The EPM (Hamilton Kinder; San Diego, CA) i s an apparatus that has be en used extensively to study anxiety like behavior in rodents (Korte & De Boer, 2003) It consists of 2 open (10.80 x 51.17 cm) and 2 closed arms (10.80 x 51.17 cm) that intersect e ach other to form the shape of a plus sign. The intersection area is 10.80 by 10.80 cm, and the walls of the closed arms are 40.01 cm high. The more time rats spe nd in the closed arms is considered to be indicative of anxiety like behavior In other wor ds, time spent in the open arms is considered risk taking behavior, as it theoretically places

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20 the rat in open view to predators and susceptible to danger. Each rat was placed on the EPM for 10 minutes, and its behavior monitored by 48 infrared photobeams connected to a computer program (Motor Monitor) that analyzes the behavior. The program enables the experimenter to assess the rats’ total movement, distance traveled in each area of the maze, distance traveled overall, and time spent in each area of the maze. The primary measurement of concern is the percentage of time that each rat spends in the open arms, as compared to t he closed arms. The EPM was wiped down with 25% ethanol solution after removal of fecal boli between sessions to reduce odor betwee n testing Figure 1 Schematic Diagram of the Elevated Plus Maze Startle Response Approximately one hour after the EPM assessme nt, all rats were subjected to test s of startle reflex. To measure the startle, each rat was pla ced inside a restraint box that is inside of a larger startle monitor cabinet (Hamilton Kinder; San C L O S E D A R M S ARMS OPEN 51.17 CM INTERSECTION (10.80 CM X 10.80 CM)

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21 Diego, CA; 35.56 x 27.62 x 49.53 cm). Within the recording chamber, the rat sits on a sensory transducer, which records startle reflexes. The startle tria l began with a 5 minute 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 bu rsts at 100 dB, etc.), and the time between each noise burst varied in a pseudorandom fashion between 25 and 55 seconds. Upon the commencement of the first noise burst, the startle apparatus provides an uninterrupted background noise of 57 dB. Each start le reflex was recorded in Newtons, and the compl ete session lasted 16 minutes. Figure 2 Schematic Diagram of the Open Field Apparatus Object Recognition Twenty four hours after the startle test, animals were returned to the lab oratory After acclimation animals were placed individually in to an Open Field (see Figure 2). This field is a large, black walled, plastic box (Hamilton Kinder, San Diego, CA – 40 x 47 x 70 cm). It has an open top, and is in a light and

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22 sound attenuated room for 5 minutes. The rat s ’ behavior was monitored by a video feed to a computer program (Any Maze; St oelting) and analyzed The program allows for assessment of the rats’ total distance traveled in each area of the open field (center and perimeter), t otal time spent in each area of the open field, rearing, and entries into each quadrant of th e open field. This exposure serve d as habituation to the apparatus and provide d an assessment of general behavior Another day after the five min ute habituation t rial, the animals were again brought back to the laboratory and re exposed to the open field; but this time 2 identical objects were placed diagonally opposite to one another for the rat to explore for a total of 5 minutes (training phase). Three hours la ter, rats were replaced into the open field, a replica of one of the original objects and a completely novel object were placed in the same orientation as the training procedure for 5 minutes (testing phase). The amount of time spent with the novel object was then recorded by Any Maze monitoring the head of the rat in relation to th e objects in the field and served as an index of memory for the original familiar object. Video files were also made for experimenter coding. Rats normally spend more time wit h the novel object than with the familiar This paradigm is interpreted as a f orm of non stressful memory. Statistical Analysis Most of the data were analyzed through use of appropriate types of two way analyses of v ariance (ANOVA ) A priori p lanned co mparisons wer e tested with two tailed Student’s t tests, between Cat and No Cat aCSF and muscimol treated groups in each behavioral test of the experiments. Alpha was set at 0 .05 for all analyses.

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23 Results Fear Conditioning (see figures 3 6) The 3 week fear conditioning retention tests (contextual fear conditioning, cue based fear conditio ning) wer e analyzed separately. Contextual fear conditio ning freezing percentages, excluding the first 30 seconds and last minute of the 5 minute tests, w e re compared u sing a two way ANOVA for each brain target, with Stress (cat or n o c at) and Inactivation (aCSF or muscimol) serving as t he between subjects variables. Cued fear conditioning was analyzed using the percent time spent freezing during the 3 minute tone prese ntation. Analysis of the vmPFC group’s contextual fear response at three weeks revealed a significant overall effect with F (3,31) = 7.80, p < 0 .01. A significant main effect of Stress was found ( F (1,31 ) = 15.21, p < .01) with the stress procedure produci ng higher levels of freezing (M = 17.08, SEM = 2.02) compared to animals not receiving the stress procedure (M = 6.58, SEM = 1.18). Inactivation also produced a significant main effect ( F (1,31) = 7.15, p < 0 .05), animals having aCSF infused into the vmPFC prior to conditioning expressed higher levels of freezing (M = 15.43, SEM = 1 .86) than animals infused with m uscimol (M = 8.23, SEM = 1.95). The interaction between Stress and Inactivation was not significant with F (1,31) = 3.61, p = 0 .07. However, plan ned comparison two tailed t tests indicated that Stress rats with the vmPFC inactivated prior to conditioning froze (M = 10.92, SEM = 2.38) significantly (p < 0 .05) less than Stress – aCSF animals (M = 23.25, SEM = 4.92); in addition, these animals froze s ignificantly (p < 0 .01) more than Unstressed – aCSF animals (M = 7.62, SEM = 1.60). Analysis of t he cued fear response of vmPFC targeted animals indicated no significant differences overall with F (3,31) = 1.03, p = 0 .39.

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24 Effects of Cat + Immobilization and vmPFC Inactivation on Contextual Fear Memory % Time Immobile 0 10 20 30 40 No Cat Cat aCSF No Cat Cat Muscimol p < .05 Fi gure 3 Inactivation of the vmPFC with muscimol before conditioning blocked the stress induced increase in freezing to the context 3 weeks later. (* p < 0 .05) Effects of Cat + Immobilization and vmPFC Inactivation on Cue Fear Memory % Time Immobile 0 10 20 30 40 aCSF Muscimol No Cat Cat No Cat Cat Figure 4 There was no effect of stress or in activation on the cu ed freezing.

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25 Analysis of varia nce for the CA1 targeted groups’ contextual fear revealed an overall significant effect with F (3,28) = 4.11, p < 0 .05. There was a significant main effect of both Stress ( F (1,28) = 4.46, p < 0 .05) and Inactivation ( F (1,28) = 5.95, p < 0 .05). In similar fashion to the vmPFC group, the stressed animals froze more (M = 18.88, SEM = 3.12) than unstressed (M = 8.86, SEM = 3.58), and aCSF treated animals expressed more fea r (M = 19.66, SEM = 3.22) than m uscimol treated animals (M = 8.08, SEM = 3.49). The Stress by Inactivation interaction was also not significant ( F (1,28) = 1.34, p = 0 .26). A similar pattern to the vmPFC group emerged in the CA1 group when analyzed using planned comparison t tests. Muscimol infused prior to the s tress procedure (M = 10.35, SEM = 3.99) significantly reduced (p < 0 .03) freezing compared to aCSF (M = 27.41, SEM = 5.91), which was also significantly greater than (p 0 .05) aCSF unstressed animals (M = 11.90, SEM = 4.31). The cued fear response in CA1 targeted animals did show significant overall differences ( F (3,31) = 3.65, p < 0 .05); with no significant main effect of Inactivation ( F (1, 31 ) = .93, p = 0 .34) or the Stress by Inactivation interaction ( F (1,31) = 1.39, p = 0 .25). However, a significant main effect was observed in the Stress manipulation with F (1,31) = 8. 10, p < 0 .01; where the Stress procedure resulted in animals freezing more to the cue (M = 21.487, SEM = 3.11) than unstressed animals (M = 7.80, SEM = 3.67). Planned comparison t tests revealed the Stressed aCSF animals froze significantly more (M = 29.6 5, SEM = 7.10) than both Unstressed aCSF and – m uscimol (M = 7.29, SEM = 2.75 and M = 8.31 SEM = 2.26; p < 0 .05). However, Stressed m uscimol animals (M = 16.34, SEM = 3.21) were not significantly different from their Stressed aCSF counterparts in percent time freezing to the tone (p = 0 .096).

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26 Effects of Cat + Immobilization and CA1 Inactivation on Contextual Fear Memory % Time Immobile 0 10 20 30 40 aCSF Muscimol No Cat Cat No Cat Cat Figure 5 Muscimol inactivation of the hippocampus before conditioning blocked the stress induced increase in freezing to the context 3 weeks later. (* p < 0 .05)

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27 Effects of Cat + Immobilization and CA1 Inactivation on Cue Fear Memory % Time Immobile 0 10 20 30 40 aCSF Muscimol No Cat Cat No Cat Cat Figure 6. Stressed animals infused with aCSF into the hippocampus exhibited heightened cued fear conditioning after 3 weeks as compared to non stressed rats. Animals infused with muscimol were no t significantly different than any group. EPM ( see figures 8 & 9) The data for the first five minute s of the ten minute test analyzed separately for each brain region using two two way ANOVAs, comparing the percent time each group spent in the open arms or closed arms. For the vmPFC group a significa nt overall difference for percent time spent in the open arms was found with F (3,30) = 5.22, p < 0 .01. There was a significant main effect of Stress ( F (1,30 ) = 10.86, p < 0 .01) with the stress procedure resulting in less percent time (M = 10.52, SEM = 3.9 2) in the open arms as compared to animals not put through the procedure (M = 27.77, SEM = 3.48). However, there was no significant main effect of Inactivation ( F (1,30) = 2.13, p = 0 .16) or Stress x Inactivation interaction ( F (1,30) = 2.56, p = 0 .12) for the percent time spent in open arms. The analysis of the percent time spent in the closed

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28 arms for the vmPFC group indicated a significant overall effect with F (3, 30) = 6.23 p < 0 .01. A s ignificant main effect for Stress ( F (1,30) = 18.49 p < 0 .01) was found; however, no significant main effect of Inactivation ( F (1, 30) = 0.02, p = 0 .90 ) or Stress x Inactivation interaction ( F (1,30) = 0.003, p = 0.96) were found. Both the Stress aCSF (M = 82.35, SEM = 6.77 ) and Stress Muscimol (M = 82.87, SEM = 7.24 ) t reated groups spent significantly greater percent time i n the closed arms than the U nstressed aCSF (M = 53.79, SEM = 5.77 ) and Unstresse d muscimol (M = 54.98, SEM = 6.38 ). The CA1 groups showed no significant overall differences in percent time spent in t he open ( F (3,30) = 1.60, p = 0 .21) or closed arms ( F (3,30) = 0.53, p = 0.68 ). Effects of Cat + Immobilization and Inactivation of vmPFC on Elevated Plus Maze % Time in Arms 0 20 40 60 80 100 Open Arms Closed Arms aCSF Muscimol No Cat Cat No Cat Cat * Figure 7. Inactivation of the vmPFC had no effect on stress effects on time spent in open or closed arms of the EPM. (* indicates p < 0 .05 comp aring closed arms or open arms between stressed and unstressed rats)

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29 % Time in Arms 0 20 40 60 80 100 Open Arms Closed Arms aCSF Muscimol Effects of Cat + Immobilization and Inactivation of CA1 on Elevated Plus Maze No Cat Cat No Cat Cat Figure 8. There was no overall effect of stress or inactivation on EPM behavior in the hippocampal manipulation. Startle Response (not shown) For each rat, eight startle responses at each of three auditory intensities were averaged to create one data point per auditory i ntensity per rat. The data were analyzed using a mixed model ANOVA, with Stress and Inactivation serving as the between subjects varia bles and I ntensity (90, 100, and 110 dB) serving as the within subjects variable. For each brain structure, only significant differences were found between startle intensities; with the vmPFC ( F (2,66) = 85.30, p < 0 .01) and CA1 ( F (2,60) = 86.54, p < 0 .01) groups both expressing more startle as the intensities increased. Object Recognition (see figures 9 & 10) The object recognition data were analyzed using two way ANOVAs with the same between subjects variables as before, with time spent with the novel and familiar objects as the within subjects variables. For

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30 the vmPFC group an overall within subjects trend was revealed for object preference (F(1,28) = 2.58, p = 0 .12), with the Novel object (M = 18.79, SEM = 2.63) being investigated more than the Famil iar object (M = 13.68, SEM = 1.53). Planned comparison repeated measure t tests were also used to investigate the effects of inactivating the brain structures during the stress procedure on this non stressful memory. These tests indicated that each of th e Unstressed aCSF, Unstressed muscimol, and Stressed muscimol groups spent significantly (p < 0 .05) more time with the Novel object (M = 19.78, SEM =2.05; M = 17.9, SEM = 1.93; M = 21.3, SEM = 5.38, respectively) than the Familiar object (M = 13.33, SEM = 1.16; M = 13.73, SEM = 0.83; M = 10.26, SEM = 1.78). However, the aCSF Stress animals showed no difference (p = 0 .43) between the time spent with the Novel (M = 13.67, SEM = 1.01) and Familiar (M = 13.33, SEM = 1.09) objects.

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31 Effect of Cat + Immobilization and Inactivation of vmPFC on Novel Object Recognition 0 5 10 15 20 25 30 Novel Familiar aCSF Muscimol Seconds Spent with Object * No Cat Cat No Cat Cat Figure 9. Only the vehi cle treated stress animals did not investigate the novel object more than the familiar object. (* p < 0 .05) The hippocampal manipulations showed a significant within subjects difference for object preference as indicated by F (1,2 6) = 7.25, p < 0 .05, with more time being spent with the Novel object (M = 18.40, SEM = 2.65) than the Familiar object (10.92, SEM = .99). The planned comparisons for these groups showed similar patterns to that of the vmPFC results. The only group not t o show a significant preference for the Novel object (M = 17.8, SEM = 4.40) over the Familiar (M = 11.59, SEM = 1.48) was the aCSF Stress group.

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32 Effect of Cat + Immobilization and Inactivation of CA1 on Novel Object Recognition Seconds Spent with Object 0 5 10 15 20 25 30 Novel Familiar aCSF Muscimol * No Cat Cat No Cat Cat Figure 10. Only the vehicle treated stress animals did not investigate the no vel object more than the familiar object. (* p < 0 .05) Fecal Boli (see figures 11 & 12) The total number of fecal boli ea ch animal produc ed for contextual and cued fear conditioning, EPM, Startle, Open Field, Object Training, and Object Recognition behav ioral tests was averaged and analyzed using a 2 x 2 ANOVA. For the vmPFC group a significant overall difference was found ( F (3,34) = 4.91, p < 0 .01). There was a significant main effect of Stress with F (1,34) = 11.33, p < 0 .01, where the Stress groups (M = 10.75, SEM = 1.30) produc ed more boli than the No Stress groups (M = 5.02, SEM = 1.13). Planned comparison analysis revealed that the Stress aCSF group defecated more than both of the No Stress groups; however the Stress muscimol group was no differ en t statistically from the Stress aCSF group.

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33 Effects of Cat + Immobilization and Inactivation of vmPFC on Total Boli Boli 0 5 10 15 20 aCSF Muscimol No Cat Cat No Cat Cat Figure 11. The stressed animals receiving vehicle infusions defecated more than the unstressed groups, but stressed animals receiving muscimol were no t significantly different th an any other groups. (* p < .05) Analysis of the hippocampal manipulations revealed a main effect of Stress ( F (1,28) = 4.87, p < 0 .05), with the Stress groups (M = 13.40, SEM = 1.87) defecating more than the No Stress groups (M = 7.31, SEM = 2.03). Planne d comparison t tests indicated the Stress aCSF (M = 14.56, SEM = 2.57) produc ed more boli than either of the No Stress groups (a CSF, M = 6.78, SEM = 2.57; and m uscimol, M = 7.83, SEM = 3.15).

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34 Effects of Cat + Immobilization and Inactivation of CA1 on Total Boli Boli 0 5 10 15 20 aCSF Muscimol No Cat Cat No Cat Cat Figure 12. The stressed anima ls receiving vehicle infusions defecated more than the unstressed groups, but stressed animals receiving muscimol were no t significantly different than any other groups. (* p < 0 .05)

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35 C ha p ter T hr ee : Discussion Prefrontal Cortex The most intriguing findi ng of these exper iments was counter to the hypothesis, infusion of muscimol into the prefrontal cortex resulted in lower freezing to the contextual fear conditioning tests. While this finding is cont rary to the hypothesis derived from the literature that the inactivation of t he prefrontal cortex would result in a mo re active amygdala and produce a more robust fear memory, there is a similar finding in humans. Combat veterans who received brain damage to the prefrontal cortex had significantly less occurrence of PTSD than those with damage to any other brain region, except the amygdala ( Koenigs, et al., 2008 ). The results of the present study are comparable to this finding in humans. The prefrontal circuitry involved has been theorized to rely on the percept of control, such that in rats the infralimbic and prelimbic cortex orchestrate inhibitory influence over the dorsal raphe nucleus (DRN). The DRN provides the majority of 5 hydroxytrypta mine signaling to the rest of the brain. It has been demonstrated that the vmPFC is important for controlling the DRN and that the construct of control mitigates these effects in rats (Christianson, Thompson, Watkins, & Mai er, 2008; Amat, Paul, Watkins, & Maier, 2008; Baratta, Lucero, Amat, Watkins, & Maier, 2008; Baratta et al., 2007; Maier, Amat, Baratta, Paul, & Watkins, 2006; Amat, Paul, Zarza, Watkins, & Maier, 2006; Amat et al., 2005) This design specifically set ou t to reduce the perception of control the animal had Then, why would inhibi ting the

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36 prefrontal cortex reduce the memory of the context and cue associated with immobilization and predator exposure? H uman imaging studies have provided evidence that the vm PFC is more likely responsible for attention (Geday & Gjedde, 2009) If thi s is the case in rodents then one possible explanation for the present finding is the animals were unable to attend to the context and the cue when the information would have normally been processed, and were then subsequently unable to recognize them as predictors of an aversive stimulus. The regulation of emotional behavior by the PFC is two fold. That is, while electrical activation of the prelimbic areas stimulates the parasympathetic nervous system, infr alimbic activity is associated with sympathetic nervous system stimulation (Powell, Watson, & Maxwell, 1994) Thus, the obtained results could be due to more consistently ventral placement of the cannula, resulting in a more blunted emotional response at the time of st ress. The stressed animals in the vmPFC group displayed more anxiety like behaviors on the elevated plus maze. This demonstrates the single stress paradigm is sufficient to be anxiogenic, much like our laboratory ’s model of PTSD. However, the inactivation of the vmPFC di d not facilitate any further anx iogenesis. In fact, the reduced amount of time spent in the closed arms in both vehicle and muscimol unstressed rats supports t he idea that these animals were less anxious than the stressed animals. Hippocampus The hippocampal inactivation in stressed animals resulted in a blockade of the stress induced contextua l fear memory, while sparing the cued fear memory. These results supported the hypothesis that hippocampus modulates the contextual, but not

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37 auditory cue information processed in close temporal proxi mity to fear learning i n rats. Thus, this experiment illustrates the importance of the hippocampus in relation to the ti ming of contextual fear memory formation. These findings also provide validity of thi s stress procedure, per Yehuda & Antelman’s (1993) crite ria, because the single “dose” of stress facilitated fear learning The lack of an effect on the EPM is possibly due to an anxiolytic effect of the cannulation via cellular remodeling after the surgery (Dringenberg, Levine, & Menard, 2008) found one second of electrical stimulation of the dorsal but not the ventral hippocampus before behavioral testing r educed the amount of time rats spent in the open arms of an EPM. Furthermore, (McEown & Treit, 2009) showed transient inactivation of the ventral and the dorsal hippocampus during acquisition of a defensive burying task (an anxiogenic type of fear conditioning) reduced anxiety like responses when re ten tion was tested in the burying apparatus However, only transient inactivation of the dorsal hippocampus after acquisition resulted in anxiolytic effects on the 24 hr te st. These reports indicate the hippocampus is involved in anxiety like behaviors i n rats and that the dorsal hippocampus is particularly important for contextual memory of aversive events. Limitations The lack of effects on the startle behavior could have arisen from the fact that these animals underwent stereotaxic surgery, which u sually results in the puncture of the tympanic membrane (Kaplan, Allan, & Wolf, 1983) It is also possible that two stress sessions are necessary to generate the hyperarousal behaviors we observe in our PTSD model. There is also indication from research in humans that startle is context dependent.

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38 In a series of experiments Morgan and colleagues (Grillon, Morgan, So uthwick, Davis, & Charney, 1996 ; Morgan, III, Grillon, Southwick, Davis, & Charney, 1995; Morgan, I II et al., 1995) examined the startle response in PTSD patients and found PTSD patients exhibited greater startle throughout both baseline and threat conditions. However, (Grillon, Morgan, So uthwick, Davis, & Charney, 1996 ) examine d the baseline startle response of Vietnam veterans with PTSD in a familiar environment and found no differences between startle responses of Vietnam veterans with or without PTSD and healthy control subjects. Other studies (Grillon & Morgan, III, 1999; Grillon, Morgan, III, Davis, & Southwick, 1998; Pole, Neylan, Best, Orr, & Marmar, 2003) have found manipulations of the experimental context or the presentation of explicit threat cues consistently leads to enhanced st artle responses in PTSD symptoms indicating the exaggerated startle responses reported in PTSD patients is context dependent, and not necessarily a stable trait of these individu als. Thus, testing startle in the fear provoking context could have facilitated enhanced startle expression. There are methodological limitations of this investigation. One li mitation is the fact that only one type of GABA agonist was utilized to inactiv ate the brain regions of interest. It is possible that the single administration of this powerful drug was sufficient to induce a general anxiolytic effect in treated animals. Future investigations could determine whether inactivation of the prefrontal c ortex or hippocampus using different pharmacological agents, such as lidocaine or tetrodotoxin result in similar findings. Another limitation arises from the 90 degree angle used i n cannulae placement of these investigations. This could account for the l ack of cued fear conditioning in the prefrontal cortex manipulations from cortical damage from cannula e placement. The fact that all of

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39 the behavioral tests were done on the same animals; that is separate groups were not used to look at each behavior coul d have influence the results. However, the most intriguing findings were found in the first and last behavioral tests indicating that at least the non aversive memory effects are robust. General Conclusions Overall, these studies have shown that the hippocampus and the prefrontal cortex are necessary to form long term contextual fear memories. Furthermore, these investigations call into question the current theories of how multiple brain regions interact to form traumatic memories. If the prefrontal cortex “goes offline” during a traumatic experience and allows the amygdala to form a more emotional memory than usual, then the stressed rats with muscimol inactivation and the veterans with prefrontal damage (Koenigs et al., 2008) would both have had more robust fear and anxiety symptoms than has been reported. This serendipit ous finding call s into question the curren t theoretical framework that many researchers are using.

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