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A modifed version of the defensive burying paradigm exposes group differences in behavior, vasopressin… Maynard, Kyle 2013

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   A MODIFED VERSION OF THE DEFENSIVE BURYING PARADIGM EXPOSES GROUP DIFFERENCES IN BEHAVIOR, VASOPRESSIN AND c-FOS EXPRESSION IN TESTOSTERONE-REPLACED RATS   by   Kyle Maynard  B.Sc., Thompson Rivers University, 2009     A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF   MASTER OF SCIENCE   in   THE FACULTY OF GRADUATE STUDIES (Neuroscience)     THE UNIVERSITY OF BRITISH COLUMBIA (Vancouver)    February 2013    © Kyle Maynard 2013  ii  ABSTRACT  Testosterone has been shown to have marked effects on different types of coping behaviors. The current study was done to test the capacity by which testosterone alters the amount of defensive burying in the defensive burying paradigm. Twenty high and twenty low testosterone-replaced (T), castrated rats were used to determine the effects of testosterone using a modified version of the defensive burying paradigm. Rats were exposed to either a remotely triggered mousetrap (snap), or neutral trap (yoke), creating four groups; high-T snap, high-T yoke, low-T snap and low-T yoke. Under habituation conditions, both high- and low-T replacement groups show a characteristic behavioral profile that indicated they habituated. As a function of testosterone replacement, animals showed significantly less burying but more rearing with high-T compared to low-T replacement (p = 0.004 and p = 0.02, respectively). Under aversive trap conditions, testosterone showed a general effect to decrease burying behavior. Testosterone affected rearing behavior that was highest within high-T groups and with direct exposure to the snap. There were limited neurochemical effects of testosterone other than a significant increase in vasopressin mRNA expression in high-T compared to low-T animals in both the medial amygdala and posterior bed nucleus of the stria terminalis. In addition, animals that were exposed to either test condition showed higher c-Fos activation in the septum compared to transport controls.  These results show that high testosterone replacement can reduce signs of neophobia, alter the magnitude of adaptable behaviors and promote proactive exploration under aversive conditions.     iii  PREFACE  The experiments conducted in this study were to determine the effects of testosterone on behavior and could not have been done without the use of animals. The University of British Columbia Animal Care Committee approved all experimental protocols (Certificate number: 3543-09).                iv  TABLE OF CONTENTS  ABSTRACT .................................................................................................................. ii PREFACE ......................................................................................................................................... ii TABLE OF CONTENTS .................................................................................................. iv LIST OF TABLES .......................................................................................................... vi LIST OF FIGURES ....................................................................................................... vii LIST OF ILLUSTRATIONS............................................................................................ viii ACKNOWLEDGEMENTS .............................................................................................. ix 1. Introduction......................................................................................................... 1 1.1 Coping Behavior ........................................................................................................... 1 1.2 Defensive Burying ........................................................................................................ 3 1.3 Anatomy and Neurochemistry of Defensive Behavior .................................................... 5 1.4 Summary ..................................................................................................................... 8 2. Materials and Methods ......................................................................................... 10 2.1 Animals...................................................................................................................... 10 2.2 General Treatment Schedule ...................................................................................... 10 2.3 Analysis ..................................................................................................................... 12 3. Animals With High and Low Testosterone Replacement are Analyzed for Differences in Behavior During 3 Days Of Habituation ..................................................................... 16 3.1 Introduction ............................................................................................................... 16 3.2 Results ....................................................................................................................... 17 3.3 Discussion .................................................................................................................. 18 4. Differential Effects of High and Low Testosterone Replacement on Coping and Behavior under Aversive Conditions ........................................................................................ 23 4.1 Introduction ............................................................................................................... 23  v  4.2 Results ....................................................................................................................... 24 4.3 Discussion .................................................................................................................. 33 5. Differential Effects of High and Low Testosterone Replacement on Vasopressin mRNA Expression and Cellular Activation of limbic Regions ................................................. 37 5.1 Introduction ............................................................................................................... 37 5.2 Results ....................................................................................................................... 38 5.3 Discussion .................................................................................................................. 39 6. Differential Effects of High and Low Testosterone Replacement on Hypothalamic- Pituitary-Adrenal (HPA) Axis Activation ..................................................................... 46 6.1 Introduction ............................................................................................................... 46 6.2 Results ....................................................................................................................... 47 6.3 Discussion .................................................................................................................. 47 7. General Discussion ................................................................................................ 52 LITERATURE CITED .................................................................................................... 57               vi  LIST OF TABLES  Table 1  Post hoc analysis of 2-way ANOVA comparing main effects of habituation .............. 20                         vii  LIST OF FIGURES  Figure 1 Mean ± SEM Testosterone results ........................................................................... 19  Figure 2 The mean duration of burying behavior ± SEM ........................................................ 25  Figure 3 The mean duration of freezing behavior ± SEM ....................................................... 26  Figure 4 Average grooming duration ± SEM. ......................................................................... 28  Figure 5 Average rearing behavior ± SEM after exposure to an aversive snap. ...................... 29  Figure 6 Average percent time spend in the trap zone ± SEM. ............................................... 30  Figure 7 Average stretch-attend frequency ± SEM.. .............................................................. 31  Figure 8 Mean ± SEM frequency of line crossess ................................................................... 32  Figure 9 Average reactivity score ± SEM. .............................................................................. 34  Figure 10 Average c-Fos MeA cell count ± SEM. .................................................................... 40  Figure 11 Average c-Fos pBST cell count ± SEM. .................................................................... 41  Figure 12 Average c-Fos LS.v ± SEM ...................................................................................... 42  Figure 13 Average optical density of AVP mRNA ± SEM. ....................................................... 43  Figure 14 Average AVP pBST cell count ± SEM. ..................................................................... 44  Figure 15 Average c-Fos PVN ± SEM ..................................................................................... 42  Figure 16 Average AVP mRNA of the PVN ± SEM. ................................................................. 43  Figure 17 Average Corticosterone ± SEM. ............................................................................. 50   viii  LIST OF ILLUSTRATIONS  Illustration 1 Schematic showing general shift in behavior for both T-replaced groups under habituation conditions ........................................................................................ 53                        ix  ACKNOWLEDGEMENTS   During the last three years I have received support and encouragement from a great number of individuals. My supervisor, Dr. Viau, with his knowledge and guidance has been inspirational as I continue my path in science. I would also like to thank the members my committee Dr. Weinberg and Dr. Gorzalka for their patience and support. During data collection and writing, Nirupa, Leyla, Adam, Megan and Brenda have spent countless hours teaching me various techniques, listening to me talk about my research, and giving me daily advice. Without their help, none of my experiments would have worked and this thesis would not have been possible. I would also like to thank my classmates who where there supporting me and generously sharing their time and ideas. I have learned much through our discussions. Finally, thanks to Dawn and Cindy for giving me the push I needed to get this finished. Without their maternal guidance, I would have never made it this far. I have been exposed to a great number of experiences, and gained a substantial amount of knowledge over the last three years and my success is only possible because of all these awesome people.        1  1. Introduction  Some individuals are predisposed to develop posttraumatic stress disorder or major depression when exposed to psychological stress.  Part of this susceptibility to psychopathology may be the result of failing to effectively cope with a stressor (Gross and Hen, 2004; de Kloet et al, 2005; Walker et al, 2008).  Unfortunately, there has been little advancement towards understanding the neurobiological basis or behavioral responses that may contribute to resiliency (Charney, 2004). Therefore, this raises the importance of determining the neural substrates responsible for individual variations in behavioral coping responses to threatening stimuli. Studies using animal models have found that coping behavior is regulated by several hormones and neuropeptides that mediate defensive circuits in the brain (Gross and Hen, 2004; Kabbaj, 2004). This circuitry includes many limbic structures and disruption or manipulation of this circuitry may explain why some individuals develop mental illness (de Kloet et al, 2005). This introduction will review the anatomy and chemistry of defensive circuitries of the limbic system that may be involved in promoting adaptive behavior in response to stress. Ultimately, this information can be used to gain important insight into individual differences in the formation, treatment and vulnerability to psychopathology.  1.1 Coping Behavior The acute stress response is an adaptive process that is utilized to meet the physiological demands of environmental or psychological stressors (de Kloet et al, 2005). Often overlooked are the behavioral consequences of acute stress that work to modify sensory cues that induce the stress response. This behavioral response is termed coping, and its successful adaptation to the stimuli will prevent a prolonged stress response. (Charney, 2004; Koolhaas et al, 2010). Depending on the  2  characteristics of the stimulus, coping can reduce the physiological load on the organism through one of two types of behavioral responses: active or passive (Koolhaas et al, 1999; Steimer and Driscoll 2003).  Although the nomenclature for the two response types are opposing, they can both be considered as successful or adaptive coping strategies (Koolhaas et al, 2010). Active and passive coping behaviors are differentiated by an organism’s routine of interacting with a particular stimulus. Active coping is a proactive attempt to reduce the threats in the environment directly, through behaviors such as active avoidance, offensive aggression, and nest building (Pinel et al, 1990; Koolhaas et al, 1999; Frank et al, 2006; Arakawa, 2007; Koolhaas, 2010). Passive coping is accompanied by withdrawal-like behaviors from threats in the environment, including passive avoidance, freezing, submission, immobility and even migration (Frank et al, 2006; Arakawa, 2007; Koolhaas et al, 2010). These coping responses are acquired through experience during early development. Such behavior increases an animal’s chance of survival by protecting and gathering food, partners and nesting sites (Blanchard et al. 1998). These early life experiences, along with genetic, environmental and pharmacological influences are what give rise to individual differences in innate coping behavior (De Boer and Koolhaas, 2003; Ebner et al, 2005; Frank et al, 2006; Goel and Bale, 2009; Koolhaas et al, 2010). When considering coping behavior, the term “maladaptive” refers to when an animal succumbs to the physiological effects of stress due to the animals innate mode of coping being unable to remove or adapt to the demands of the situation. Since, the underlying mechanisms and neurophysiology of individual differences in coping behavior is not well understood, it is essential to exploit individual differences in coping using animal models to determine the neurological underpinnings of this behavior.     3  1.2 Defensive Burying 1.2.1 Introduction Until 1978, biologists were familiar with three classic defensive behaviors; fight, flight, and freezing. Defensive burying, first noted by Hudson (1950), is the newest defensive strategy to be described. Pinel and Treit (1978) extensively explored this behavior and developed a paradigm in which rats are placed in a bedding-filled chamber with an electrified or non-electrified wire-wrapped prod protruding through one of the walls. They noticed the defensive burying response was a remarkably stereotyped active avoidance tactic, directed toward sand seen only in the presence of the shock- prod (Pinel and Treit 1978; Treit et al, 1980; Pinel et al, 1985). They defined “defensive burying” as spraying bedding material with forward treading motion using the snout and/or forepaws towards an aversive source (Pinel and Treit 1978; Pinel et al, 1985; Lapiz-Bluhm et al, 2008).  These results are not just a laboratory phenomenon since defensive burying has also been observed in the wild (Calhooun, 1962; Silverman, 1978). It was concluded that defensive burying was a conditional defensive response that is now classified to be a novel tool in a rodent’s behavioral toolbox of survival mechanisms. 1.2.2 Defensive Burying as a Behavioral Tool Similar to elevated-plus maze and social interaction tests, defensive burying may also be used as a tool for assessing components of anxiety-like behavior in rodent models (Lapiz-Bluhm, 2008). However, unlike other tests, anxiety-related behaviors examined in this paradigm may be looked upon as a defensive coping strategy (Lapiz-Bluhm, 2008). There are multiple benefits for choosing this particular test. First, burying is specific to the stimulus, which eliminates nonspecific treatment effects that may alter baseline behavior (Lapiz-Bloom et al, 2008).  For example, any response elicited by a rat in the elevated plus-maze could be a general response to the entire paradigm since there is neither a habituation trial nor any specific stimulus source. Defensive burying, however,  4  includes a habituation stage making the test specific to the stimuli. Second, in addition to the standard set of common behaviors (burying and latency to bury) there are several others available (e.g. grooming, rearing, freezing and latency to approach) which provide an opportunity to gain a more thorough understanding of animal behavior. Finally, instead of using the common shock prod or predator odor (Dielenberg and McGregor, 2001) that induce very robust effects on behavior, a mild stimulus source can be used such as an air-puff or a remotely-triggered mousetrap. Studies have shown that the moderate nature of these latter methods prevent ceiling effects in behavior and can therefore exploit individual differences in coping behavior (Pinel et al, 1994; Linfoot et al, 2009).  The flexibility and specificity of the stimulus, along with all the behavioral measures, make the defensive burying paradigm a very powerful behavioral test of the animal’s defensive repertoire. Following its discovery, several experiments have identified factors that may influence the level of defensive burying activity in a laboratory setting. These factors include chamber size (Pinel et al, 1980), stimulus intensity (Treit et al, 1980), type of stimulus (Pinel et al, 1979), age, sex (Pinel et al, 1990; Arakawa, 2007), time of day, metabolism (De Boer and Koolhaas, 2003), bedding type and amount (Pinel and Treit, 1979; De Boer et al, 1989; Bondi et al, 2007), maternal care (Menard et al, 2007), and even the complexity of the object (Pinel et al, 1979).  Collectively, these studies emphasize the sensitivity of defensive burying paradigm. Defensive burying remains a useful tool for studying stimulus-directed behavior and permits the detection of individual differences in coping strategies.  Detecting variations in behavior also facilitates investigation of its underlying anatomical and neurochemical correlates, which are currently not well understood.     5  1.3 Anatomy and Neurochemistry of Defensive Behavior 1.3.1 Defensive Anatomy Group differences in coping and stress related behavior have been found to correlate with functional manipulations of the limbic system. Lesion studies using rats suggest that multiple subregions of the septum are involved in the burying response (Pesold and Treit, 1992). When anterior and posterior electrolytic lesions were compared, Gray et al, (1980) found that posterior septal lesions abolish defensive behaviors, whereas anterior lesions have no effects. These results were similar to excitotoxic lesions targeting the cell bodies of the posterior septum, confirming the behavioral effects were limited to the destruction of septal nuclei rather than input or output connections (Singewald et al, 2011). The posterior septum can be further subdivided into medial and lateral subdivisions, which differ in major connections, cytoarchitecture, and neurochemistry (Caffe et al, 1987; Canteras et al, 1995; Menard and Treit, 1996; Dong and Swanson, 2004).  Despite these functional and anatomical subdivisions, both medial and lateral lesions decrease burying behavior (Menard and Treit, 1996).  These results suggest that the posterior septum is an important mediator of the defensive burying response. Numerous inputs from the limbic system converge on the septum. Several tract-tracing studies provide evidence that the medial amygdala (MeA), the posterior bed nucleus of the stria terminalis (pBST) and the hippocampus, project directly and indirectly to the septum (Caffe et al, 1987; Risold and Swanson, 1997; Dong and Swanson, 2004). It has been postulated that any of these limbic regions may be in a position to modulate defensive behavior since they are capable of relaying sensory information to the septum. For example, the MeA receives input from several cortical and thalamic projections and a direct connection from the olfactory bulb (Samuelsen and Meredith, 2009). The MeA serves to grade incoming sensory information as emotionally relevant or not, and to transmit this processed information to elicit the most appropriate behavioral response  6  (Canteras et al, 1995; Caffe et al, 1987; Dong and Swanson, 2004). The MeA influences the septum indirectly via the pBST, which projects directly to the septum.  Defensive behavior regulated by the septum may involve the premamilliary and anterior hypothalamic nuclei, which have been shown to be involved in freezing and aggressive postures in the rat (Dong and Swanson, 2004; Motta et al, 2009). In summary, the septum plays an important role in defensive behavior by integrating converging information arising from the MeA and pBST, in addition to other limbic-related structures. 1.3.2 Androgen Receptors and Testosterone The limbic system in males appears to be the most responsive to circulating levels of testosterone. This may be due to the high concentration of androgen receptors in the pBST and MeA (de Vries et al, 1983; 1984). Using in situ hybridization, it was demonstrated that neurons of the pBST and MeA express high levels of androgen receptor mRNA (Dong and Swanson, 2004). This suggests that changes in testosterone may influence defensive behaviors by acting at the level of the MeA and pBST. Furthermore, Linfoot et al (2009) identified a positive correlation between the number of androgen receptor expressing cells in the MeA and pBST and burying behavior. Whether this relation reflects an effect of testosterone to increase defensive burying activity remains to be determined. Testosterone can influence mood, promote different forms of coping and can dampen the physiological responses to stress in both humans and animals (Albert et al, 1990; Frye and Seliga, 2001; Veenema et al, 2010).  Several experiments show that testosterone has a strong ability to promote active coping responses.  Testosterone administration in female mice increases the active coping response in the tail suspension test and increases burying in the marble-burying test (Goel and Bale, 2009). In the resident-intruder paradigm, testosterone administration promotes active behavior (Albert et al, 1990) in residents provided with high levels of testosterone (Ebner et al,  7  2005). Lastly, it was also shown that testosterone is positively correlated with dominance and anxiolytic behavior in humans (Delhez et al, 2003; Kessler, 2003). Since these findings support testosterone’s ability to promote proactive behavior, it is hypothesized that testosterone could increase active coping in the defensive burying paradigm. Previous studies examining testosterone and defensive burying activity in rats have shown that testosterone exerts an inhibitory effect on burying (Fernandez-Guasti and Martinez-Mota, 2005; Gutierrez-Garcia et al, 2009). However, the methods employed in these studies raise several concerns. First, the studies used testosterone injections as a mode of hormone delivery. This mode of steroid delivery can result in doses in excess of normal physiological levels (Strom et al, 2008). At high levels androgens have been shown to possess analgesic properties (Frye and Seliga, 2001).  This could be problematic as burying behavior was provoked by shock prod exposure. Since the shock prod has a component of pain (Pinel et al, 1980) it is possible that the decrease in burying could be due to testosterone’s pain- masking properties. Therefore, the methods used in these studies may actually confound the nature of testosterone’s actions on coping behavior in the defensive burying paradigm. 1.3.3 Vasopressin Testosterone can regulate several forms of behavior in addition to coping, such as aggression and reproductive behaviors. If testosterone has so many effects in the brain, then how can testosterone specifically affect defensive behavior? Co-localization studies indicate that virtually all arginine vasopressin (AVP) producing cells in the MeA and pBST express the androgen receptor (Van Leeuwen et al, 1984; Zhou et al, 1994; Watters et al, 1998; de Vries and Miller, 1998; Kalsbeek et al, 2002; Veenema et al, 2010).  Castration without testosterone replacement abolishes AVP mRNA and protein levels only in the pBST and MeA (Caffe et al, 1987; de Vries and Miller, 1998). Therefore, the  8  effects of circulating testosterone via AVP-related circuits of the pBST and MeA may be important for context-dependent changes in defensive behavior in response to stressful stimuli. Indeed it has been shown that AVP has an influence on burying, as well as active coping behavior. AVP protein levels increase in the septum with the active swimming response in the forced swim test (Ebner et al, 1999) and increased aggression in the resident intruder paradigm (Ebner et al, 2005).  Furthermore, in AVP 1A receptor (V1a) knockout mice, the active burying responses to marbles were attenuated (Egashira, 2007).  Central administration of the V1a receptor antagonist JNJ-17308616 in mice also decreases burying behavior, indicating a role for both AVP and the V1a receptor to promote active coping (Bleickardt et al, 2008). Finally, individual differences in coping behavior have been shown to correlate positively with individual differences in AVP expression in the pBST and MeA, however causality remains to be elucidated (Linfoot et al, 2009). Nonetheless, these results provide evidence that changes in limbic AVP may be a possible mechanism for testosterone’s central influences on defensive coping.  1.4 Summary The present study examined the ability of high and low testosterone replacement levels to modulate defensive burying behavior in rats exposed to an acute mild stimulus. Using a mousetrap as the source of a loud audible snap, behavioral profiles were compared between animals with high or low testosterone and whether or not they are directly exposed to the mousetrap. In addition to burying behavior, several behaviors were measured to determine a full behavioral profile for each group. Vasopressin mRNA, Fos expression, testosterone, and corticosteroid levels were also measured to establish a framework for differences between testosterone-replaced animals. Since AVP is highly dependent on male gonadal status, we expect AVP expression to be higher with increased circulating testosterone. Through testosterone-dependent influences on the  9  limbic system, we also expect to see increased coping behavior and Fos protein patterns (a marker for cellular activation) unique to these regions depending on the level of testosterone replacement. The following sets of experiments are described in four parts: A habituation trial in which rats are exposed to a neutral environment containing a mousetrap; an aversive test where animals are exposed to a remotely triggered mousetrap; analysis of AVP and Fos in the limbic system and in the paraventricular nucleus of the hypothalamus, and assessment of corticosterone in circulation.                   10  2. Materials and Methods 2.1 Animals Forty Long-Evans rats (Charles River Canada, St. Constant, Quebec) were used. Animals were pair- housed under controlled temperature and lighting conditions (lights on at 0700 hours). Acclimatization and testing were restricted to the light phase of the cycle (0900 to 1400 h). Food and water was available ad libitum. The University of British Columbia Animal Care Committee approved all experimental protocols.  2.2 General Treatment Schedule Three days after arrival, rats were weighed and handled for one day prior to surgery and hormone replacement. Animals were allowed 10 days for post-surgical recovery before behavioral testing. Rats were acclimatized to the defensive burying chamber containing a neutral mousetrap (Catchmaster Mouse Size Wood Snap Trap, AP&G Co., Brooklyn, NY) for 20 minutes on 3 consecutive days. On the fourth day, half the rats were then exposed to a loaded mousetrap that was remotely triggered when the rat was approximately 2 cm from the device. After the aversive test, rats were placed in an adjacent room for recovery and then deeply anesthetized for perfusion and tissue collection one hour after testing. 2.2.1 Surgery Testes were removed from all animals under isofluorane anesthesia. Testes were delivered separately through a small scrotal incision and removed by ligating and severing the vas deferens and spermatic artery. Closing the scrotal incisions with non-absorbable sutures completed the gonadectomies.  Testosterone replacement was performed at the same time, using Silastic capsules (0.062 mm inner diameter, 1.25 mm outer diameter; Dow Corning, Midland, MI) packed with either  11  10 or 30 mm of crystalline testosterone (Sigma Aldrich, Oakville, Canada). Two subcutaneous Silastic capsules were implanted bilaterally, adjacent to the dorsal midline in every rat; the high testosterone replaced group received two 30 mm capsules and the low replaced group received one 10 mm capsule and one 10mm empty capsule. These implants were to mimic plasma testosterone concentrations at the high and low levels of the physiological range observed in adult male rats (Williamson and Viau, 2008). 2.2.2 Behavioral Testing The burying test apparatus was composed of 2 separate acrylic chambers measuring 32 × 47 × 46 cm (W × L × H). Before and after each test, the chambers were filled with clean ß-wood chip bedding to a uniform depth of 5 cm. Each chamber contained a single mousetrap that was bolted to an acrylic mount standing 5 cm high to prevent movement. During 3 days of acclimatization, cage mates were placed in separate chambers that were adjacent to each other, and exposed to a neutral mousetrap for 20 min each day for 3 consecutive days. On the “snap” test day, the mousetrap was remotely triggered in only one of the two chambers. This created treatment groups consisting of animals that were castrated and replaced with either high or low testosterone, and placed in a chamber to be snap-exposed (snap) or not (yoked). The purpose of the yoked group during the test day was to differentiate between trap and non-trap specific behavior. This group is necessary because the threatening nature of the trap is considered quite mild, which makes it difficult to determine what behaviors are specific to the snap. Finally, there were high and low-T replaced transport groups, where animals were not exposed to the chamber at all. These animals were not exposed to the defensive burying paradigm on the test day, to account for cellular activation induced by physically transporting animals from one room to another. Behaviors were recorded using a JVC video camera. Video recording started when the rat was placed in the chamber  12  away from the mousetrap. Analysis of behavior was done using the event recorder Hindsight program (v1.5).  2.3 Analysis 2.3.1 Hormone Analysis Plasma testosterone was measured using an RIA kit from ICN Biomedicals, Inc. (Costa Mesa, CA), with 125I-testosterone as tracer and 25 µl of sample. The testosterone antibody cross-reacts 100% with testosterone and slightly with 5 -dihydrotestosterone (3.40%), 5 -androstane-3ß,17ß-diol (2.2%), and 11-oxotestosterone (2%) but does not cross-react with progesterone, estrogen, or the glucocorticoids (all <0.01%). The intra- and interassay coefficients of variation were approximately 4% and 10%, respectively. The standard curve 50% effective concentration was 1.75 ng/ml, and the detection limit of the assay was 0.25 ng/ml. The standard curve ED50 for the testosterone RIA was 1.2 ng/ml, with upper and lower detection limits of 7.5 ng/ml and 0.1 ng/ml, respectively. Plasma corticosterone was measured using an RIA kit from ICN Biomedicals, Inc., with [125I]corticosterone as tracer and 5 μl sample diluted 1:200. The corticosterone antibody cross-reacts 100% with corticosterone, slightly with deoxycorticosterone (0.34%), testosterone and cortisol (0.10%) but does not cross-react with the progestins or estrogens (<0.01%). The intra- and inter- assay coefficients of variation were approximately 4% and 14%, respectively. The standard curve 50% effective concentration was 16 μg/dl, and the detection limit of the assay was 0.63 μg/dl. 2.3.2 Behavioral Analysis Multiple behaviors were examined to detect any differences in high or low testosterone-replaced and snap or yoke-exposed animals. These behaviors include burying, freezing, grooming, stretch- attend postures (SAP), rearing, centerline crossings, and trap-reaction scores.  13  Burying behavior includes pushing, shoveling, or forward treading motion of the snout and forepaws. In this study, burying is a measure is of cumulative burying, which encompasses digging, prodding and swimming behavior in an attempt to displace bedding towards the trap. Freezing behavior is characterized by absolute immobility. Most freezing behavior is expressed within the first 5 minutes of the study in reaction to the stimulus. However, freezing behavior may be seen during the first habituation day due to novelty. Stretch-attend posture constitutes an elongation of the body towards an aversive source, followed by a retraction of the body to its normal length. Grooming behavior includes scratching, licking, or cleaning any part of the body (De Boer and Koolhaas, 2003) and rearing is displayed as a vertical stretch attend, with the animals standing on its two hind legs stretching its body towards the top of the chamber. Reactivity scores are a control measure to ensure the intensity and averseness of the stimuli was uniformly perceived between all animals (Treit et al, 1980). Based on a five-point scale (Gray et al, 1981) reactivity scores were: no discernible reaction (score = 0); startle, but no immediate withdrawal (score = 1); startle and withdrawal to the far end of the chamber (score = 2); jumping and/or squealing followed by rapid withdrawal (score = 3); and finally, a reflexive jump to the far end of the chamber (score = 4). 2.3.3 In Situ Hybridization Relative levels of AVP mRNA responses to testosterone replacement and mousetrap exposure were assessed using in situ hybridization.  One hour after the last exposure to the defensive burying chamber, animals were deeply anesthetized with a lethal dose of chlorohydrate (35% w/v, 350 mg/kg, intraperitoneal) for perfusion. Saline (125 ml) and 4% paraformaldehyde fixative (500 ml) were sequentially perfused via the ascending aorta at a flow rate of 20–25 ml/min. Brains were post- fixed for 4 h in fixative and then cryoprotected overnight in 15% sucrose in 0.1 M  14  potassium phosphate-buffered saline (KPBS). Five 1/5 series of frozen coronal sections (30 µm) were collected and stored in antifreeze (30% ethylene glycol and 20% glycerol in 0.05 M sterile KPBS) at –  20oC until processing.  The in situ hybridization approach measures the relative expression levels of the AVP transcript using a [33P] UTP-labeled (Amersham, Arlington Heights, IL) antisense cRNA probe encoding the AVP gene. The AVP probe contained a 230-bp cDNA fragment encoding the 3′ end of the gene (Dr. D. Richter, University of Hamburg, Germany). The autoradiographic signal was optimized by exposing the hybridized slides to liquid emulsion for a specific length of time (BST and MeA= 30 days) to ensure that the hybridized signal was within the linear range of the assay. This also allowed for relative comparisons in optical density (OD) measures to be made through t rostrocaudal extent of each cell group of interest. Bright and dark-level images were captured using a Retiga 1300 CCD digital camera (QImaging, Burnaby, BC) and cataloged using a Macintosh OS X- driven Open Lab Image Improvision software v 3.0.9 (Quorum Technologies, Guelph, ON). Captured darkfield images of AVP mRNA hybridized slices were exported to Image J (v. 1.3.5) for densitometric analysis. Lastly, selected images were exported to Adobe Photoshop (v. 7.0) for final assembly at a resolution of 300 dpi. 2.3.4 Immunohistochemistry Fos-immunoreactivity (-ir) was used as a marker of cellular activation using a primary antiserum (1:45,000) raised against amino acids 4-17 of the human c-Fos protein (Ab-5, lot number 4191-1-1; Oncogene Research Products). Immunohistochemistry was performed using a conventional nickel- intensified, avidin-biotin-immunoperoxidase procedure (Vectastain Elite ABC kit; Vector Laboratories). Free-floating sections were pretreated with hydrogen peroxide (0.3%) to quench endogenous peroxidase activity and then with sodium borohydride (1%) to reduce free aldehydes before the addition of the Fos antisera. Total cell number estimates of Fos-ir cells within the medial  15  amygdala, posterior bed of the stria terminalis, PVN and the lateral septum were determined by counting the number of Fos-positive cells by averaging each bilateral region of interest and then totaling the number of cell counts by slice number. Finally, the total cell number was multiplied by a factor of five to account for slice frequency (1-in-5 sections). 2.3.5 Statistics All behavioral, hormone, AVP, and c-Fos data were analyzed using two-way ANOVA. Statistical comparisons were made using the Tukey-Kramer post-hoc test because groups were not of equal sizes. Statistical comparisons and total analysis of recordings, tissue processing, and image capturing were made observer-blind.             16  3. Animals With High and Low Testosterone Replacement are Analyzed for Differences in Behavior During 3 Days Of Habituation  3.1 Introduction Rats exhibit defensive behaviors when threatened. Since rodents are neophobic, one such threat includes exposure to a complex novel object and/or environment. For example, a neutral mousetrap or un-lit flashbulb (which represent complex objects) have been shown to induce unconditioned defensive burying, whereas non-complex objects such as a pipette or non-electrified prod protruding from the wall of a bedding-filled chamber do not (Pinel, 1985). In the same experiment, it was shown that a habituation trial was able to significantly reduce unconditioned burying. Therefore, when employing novel object exposure it is imperative to habituate the animals in advance of testing behavioral responses to an aversive stimulus (Terlecki et al, 1979; Linfoot et al, 2009). This allows us to interpret changes in defensive behavior as a function of the aversive test condition rather than a product of novelty. We hypothesize that castrated animals replaced with high or low testosterone will show behavioral markers consistent with some form of habituation to 3 trials of neutral mousetrap exposure. Since burying is not the only behavior that can be measured, several other behaviors were assessed such as rearing, grooming, stretch-attending, freezing, line crossing and trap-zone duration. This allowed us to gain a full perspective of the animal’s shift from neophobia and for detecting differences in behavior as a function of testosterone replacement.    17  3.2 Results Before animals were placed in a chamber and exposed to a loaded mousetrap, they were placed in the defensive burying chambers containing a single neutral mousetrap, for 20 min each day for three consecutive days. This was to establish a baseline behavior that is independent of novelty- induced behavior. 3.2.1 Testosterone Replacement  Testosterone was successfully replaced within range of the normal physiological levels of testosterone in the intact male rat (0.035-2.5 ng/mL) (Williamson and Viau, 2008).  Two-way ANOVA confirmed differential testosterone replacement between animals designated as high- and low-T replacement groups (F (1, 28) = 205.5; P < 0.001; Figure 1). A significant main effect of trap condition was also revealed (F (1, 28) = 10.99; P = 0.003), in addition to a testosterone x trap condition interaction (F (1, 28) = 7.46; P = 0.011). Post hoc analysis suggested greater plasma testosterone levels in high-T replaced animals in the yolk compared to the snap condition (P < 0.001). 3.2.2 Habituation Multiple exposures to the neutral (unloaded) mousetrap exposure (habituation) could involve changes in multiple behaviors, in addition to those sensitive to testosterone replacement. Two-way ANOVAs were used to compare behaviors between the first and third day of neutral trap exposure, as well as between low- and high-T replaced animals.  There were no significant main effects of habituation (Hab) on burying (F (1, 60) = 0.02; P > 0.5), freezing (F (1, 60) = 1.72; P = 0.194), grooming (F (1, 60) = 1.39; P = 0.242), and latency to approach the trap (F (1, 60) = 0.71; P = 0.404). However, significant main effects of Hab occurred with respect to rearing (F (1, 60) = 20.4; P < 0.001), line crossings (F (1, 60) = 13.5; P < 0.001), and stretch-attend postures (F (1, 60) = 25.8; P < 0.001). As summarized in Table 1, Tukey-Kramer post hoc analysis attributed these significant differences as a product of comparisons between H1 and H3  18  trials in each case. Table 1 also summarizes main effects of testosterone replacement on behavior. A significant effect was found for rearing (F (1, 60) = 25.8; P < 0.001) and burying  (F (1, 60) = 25.8; P < 0.001), but not for line crosses, grooming, stretch-attend, freezing or latency to approach (P > 0.05 in all cases). Post hoc revealed significantly more rearing in high-T replaced animals for both H1 (P = 0.024) and H3 (P = 0.058) days of habituation. However, there was a higher incidence of burying in the Low-T group compared to High-T replaced animals on H1 (P = 0.004) and a trend on H3 (P = 0.083).  Finally, the only behavior to show a significant testosterone x hab interaction was for line crossings (F (1, 60) = 4.14; P = 0.046). Post hoc test revealed significantly fewer line crosses in Low T animals during H3 (P= 0.020).  3.3 Discussion Typically, rats show unconditioned burying behavior towards complex novel objects (Terleki et al, 1980; Linfoot, 2009), making habituation a requirement when exposing animals to a mousetrap (Pinel, 1980). As seen in previous experiments, if a habituation trial is not included in the design then changes in burying behavior could represent an artifact of novelty rather than a specific reaction to the test phase of the paradigm (Terlecki et al, 1980). Although we saw no change in burying behavior for either the high- or low-T replaced groups across habituation (Table 1), we did see changes in markers for risk-assessment (stretch attend posture), locomotion (centerline crossings) and exploratory behavior (rearing). The lack of change in burying across days may suggest that the novelty of the trap was perceived equally as aversive as the novelty of the chamber. Therefore the burying results alone do not reflect the animal’s ability to habituate to their novel environment. This result may have been the product of introducing the neutral mousetrap at the same time as the open box chamber, perhaps diluting the   19       L o w  T H ig h T 0 .0 0 .5 1 .0 1 .5 2 .0 2 .5 Y o k e S n a p * * T e s to s te r o n e  ( n g /m L ) #  Figure 1. Mean ± SEM plasma testosterone concentrations showing successful high- and low-T replacement using Silastic capsules. In addition, high-T yoked animals show higher level of testosterone than high-T, snap animals. *P < 0.05 vs. Low-T; # P < 0.05 vs. high-T, yoke counterpart  (n = 8 per group).           20       Table 1. Post hoc analyses summarizing main effects of habituation and testosterone replacement.  Effects of habituation between day 1 (H1) and day 3 (H3) were detected for rearing and SAP in both low and high T replacement groups. Effects of testosterone were detected for burying and rearing behaviors on H1. Behaviors are measured as duration in seconds (s) or frequency (#). “*”; P < 0.05 compared to H1 within testosterone replaced group.  “†”; P < 0.05 compared to Low-T on the same day of habituation.    Low T High T  H1 H3 H1 H3 Behavior Rearing (s) 187.5 ± 16.2 241.5 ± 12.9* 210.45 ± 13.9† 260.37 ± 15.9*† Line Crosses (#) 37.5 ± 3.1 29.4 ± 2.1* 36.1 ± 1.6 32.8 ± 1.2† SAP (s) 5.4 ± 0.9 1.1 ± 0.5* 5.5 ± 1.0 2.1 ± 0.9* Grooming (s) 11.7 ± 2.4 13.4 ± 2.6 6.6 ± 2.2 12.1 ± 4.5 Freezing (s) 0.3 ± 0.3 3.3 ± 3.0 0.3 ± 0.6 1.1 ± 1.4 Bury (s) 85.0 ±12.9 75.1 ± 13.8 40.9 ±9.2† 49.9 ± 6.1 Latency to Approach (s) 7.9 ± 0.7 8.9 ± 2.8 11.8 ± 2.9 7.51 ± 0.9        21  behavior targeted towards the trap. Future experiments may require a two-step habituation procedure, one for the chamber and one for the complex object. Rearing is a behavior that is rarely measured in the literature. Most studies employ it as a measure of explorative escape, indicative of an active avoidance response and seen predominantly in active coping animals (Koolhaas, 1999; Linfoot et al, 2009). In this study, rearing was shown to increase across the duration of habituation for both high and low-T replaced animals. However, high-T replaced animals showed significantly more rearing than low-T animals. Since an increase in rearing represents proactive escape, these results may suggest a shift to a more active form of behavior. The number of centerline crosses has been used as a measure of general activity (Linfoot et al, 2009). It is important to measure general locomotion to control for activity that may influence other behavioral measures, including defensive burying (Koolhaas, 1999). Despite the lower number of line crosses in low-T animals, they show a significantly higher duration of burying. This reassures that the increase in burying activity seen in low-T animals was not a result of increased general activity.  However, even though the line cross results are significant, the magnitude of this result is very subtle (29.4 line crosses in low-T and 32.8 in high-T, Table 1). It is noteworthy here, that none of these behaviors are exclusive to one another due to the fixed time in which the behaviors are being recorded, and therefore the significance between high and low-T line-crosses on H3 may be an artifact of low-T animals spending a lot more time rearing. As mentioned, stretch-attend posture is an important risk-assessment behavior used to locate or explore an unfamiliar environment or stimulus source. Stretch-attend results show a dramatic decrease during habituation and are perhaps the key indication that the animals habituated. Previous experiments use stretch-attend postures in combination with other behaviors that may indicate anxiety (Pinel and Mumby, 1994). Since anxiety-like behavior is quite ambiguous,  22  coupling stretch-attends with other behaviors can confirm a shift towards or away from anxiety (Pinel and Mumby 1994; De Boer and Koolhaas, 2003). Thus, the decrease in stretch attends in both high and low-T replaced animals, with the increase in rearing and decrease in centerline crosses represents a unique profile describing how animals have adapted to their environment. More specifically, as animals become more familiar with the defensive burying chambers over the 3 days of exposure, we see an expected decrease in locomotion as risk assessment declines. Concurrently, there is an increase in explorative escape. The fact that these responses were similar for both high and low-T replaced groups suggests that the ability to become familiar with the environment is not influenced by testosterone. Therefore, despite showing no differences in burying behavior across the three days of habituation, animals did show behavioral changes indicating they habituated to the environment. Testosterone’s effects seem to be limited to the relative magnitude of the behaviors. In particular the greater amount of rearing and decrease in burying that was reflected in high-T compared to low-T replaced animals. Since low-T replaced animals bury more and rear less than high-T animals, high-T may play a role in reducing neophobia as seen with decrease burying and promoting explorative escape when placed in a novel environment (Table 1). Therefore under habituation conditions, testosterone seems to have the opposite effect that what was predicted. Although testosterone was replaced to create high and low groups, there is an unexpected decrease in testosterone within the high-T snap compared to high-T yoke animals. Currently in the literature, there is no evidence to suggest that, when compared to a yoked group, stress will cause significantly less testosterone in animals with clamped T levels one hour after testing. Acute effects of stress to increase the metabolism of testosterone may explain this phenomenon.  Nonetheless, when considering physiologic effects of testosterone, it is unlikely that these hormone level differences in the high-T animals will have a significant effect on behavior.  23  4. Differential Effects of High and Low Testosterone Replacement on Coping and Behavior under Aversive Conditions 4.1 Introduction One contributing factor associated with an individual’s susceptibility to psychopathology is maladaptive coping behavior (Charney, 2004). Coping behavior is driven or modified by variations in central neurotransmitters in addition to steroid hormones. The most studied and well-known steroid hormone, which has pronounced effects on male and female behavior including coping, is testosterone (Frye and Seliga, 2001).  In this chapter, high and low testosterone-replaced animals were exposed to a mild stimulus source in a modified version of the defensive burying paradigm. As previously discussed, this test can be used to unmask individual differences in coping behavior. However, rather than using a shock prod to induce defensive burying, a remotely triggered mousetrap was presented to the animal. We predict testosterone replacement levels will increase burying behavior under aversive conditions, since testosterone has been shown to promote active coping behaviors. In addition to burying behavior, several other behaviors were assessed to examine additional aspects of the rat’s defensive repertoire as a function of testosterone replacement and trap conditions. The following experiment is an extension of the habituation stage of our paradigm. The same animals were placed in a chamber and exposed to a loaded mousetrap and recorded for 20 minutes on day 4 of the experiment. The behavioral effects of trap conditions and testosterone in comparison to the last day of habituation will serve as a measure of baseline behavior.    24  4.2 Results Aversive trap exposure is likely to have an effect on multiple behaviors, and those behaviors may differ with testosterone replacement levels. The behaviors analyzed were: burying, freezing, grooming, rearing, centerline crosses, stretch attend, and time spent in the trap zone. A two-way analysis of variance was used to compare behaviors between trap conditions (H3, yoke, or snap) and testosterone replacement. 4.2.1 Burying There was no effect of trap (F (2, 58) = 0.91; P = 0.410), a significant main effect of testosterone (F (1, 58) = 9.1; P = 0.004), and no significant testosterone x trap interaction (F (2, 58) = 0.13; P > 0.5). Finally, post hoc analysis confirms that low-T animals buried significantly more than high-T animals (P = 0.02) on the last day (H3) of habituation (Figure 2). 4.2.2 Freezing No effect of testosterone (F (1, 58) = 0.25; P > 0.5) or trap x testosterone interaction (F (2, 58) = 1.59; P = 0.212) was detected. Trap condition yielded significant effects (F (2, 58) = 5.36; P = 0.007) on freezing behavior (Figure 3). In Low-T animals, post hoc analysis confirmed significantly more freezing in the yoke (P = 0.036) and snap condition (P = 0.015) compared to H3.  In the high-T group, freezing duration was likewise significantly higher during yoke (P = 0.008) and snap (P = 0.005) exposure compared to H3. 4.2.3 Grooming Main effects of trap (F (2, 58) = 0.58; P > 0.5), testosterone (F (1, 58) = 0.01; P > 0.5) and testosterone x trap interactions (F (2, 58) = 0.35; P > 0.5) failed to reach significance (Figure 4).    25      L o w  T H ig h T 0 2 0 4 0 6 0 8 0 1 0 0 H a b itu a tio n Y o k e S n a p B u r y  D u r a ti o n  ( s ) *  Figure 2. Mean ± SEM burying duration (seconds) in high- and low-T rats under habituation, snap or yoke conditions. Low-T animals bury more during habituation than high-T animals (P = 0.02). Habituation (n = 16), Snap (n = 8) and Yoke (n = 8) groups.        26      L o w  T H ig h T 0 5 1 0 1 5 2 0 2 5 H a b itu a tio n Y o k e S n a p * F r e e z e  D u r a ti o n  ( s ) * * *  Figure 3. Mean ± SEM duration of freezing behavior (seconds) in high- and low-T rats under habituation, snap or yoke conditions. Similar freezing patterns were shown in both testosterone-replaced groups in response to the snap. *P < 0.05 vs. habituation. Habituation (n = 16), Snap (n = 8) and Yoke (n = 8) groups.         27  4.2.4 Rearing There were significant main effects of trap (F (2, 58) = 6.65; P = 0.003) and testosterone (F (1, 58) = 6.65; P = 0.017) on rearing behavior (Figure 5). However, there was no significant trap x testosterone interaction (F (2, 58) = 0.79; P = 0.457). In the low-T replacement group, post hoc analysis confirmed a significant increase in rearing behavior under the snap condition compared to H3 (P = 0.036). Within high-T replaced animals, there was significantly more rearing in the yoke condition compared to H3 (P = 0.033). As a function of testosterone replacement, high-T animals showed more rearing than low-T animals on H3 (P = 0.041), as well as under the yoked testing condition (P = 0.05). 4.2.5 Trap Zone With respect to time spent in the trap zone (Figure 6), two-way ANOVA revealed no significant effects of trap (F (2, 58) = 1.62; P = 0.2) and testosterone (F (2, 58) = 0.79; P = 0.457), and no significant trap x testosterone interaction (F (2, 58) = 0.79; P = 0.457). 4.2.6 Stretch-Attend Posture There was no significant main effect of testosterone (F (1, 58) = 1.84; P = 0.184), and no significant trap x testosterone interaction (F (2, 58) = 0.23; P = 0.23) for stretch attend posture (Figure 7). However, there was a significant main effect of trap condition (F (2, 58) = 3.88; P = 0.026). Post hoc analysis confirmed significant increases in stretch attend postures under the snap condition compared to habituation in low- (P = 0.036) and high-T replaced (P = 0.036) animals. 4.2.7 Centerline Crosses There was no significant effect of trap (F (2, 34) = 0.60; P > 0.5), and no significant trap x testosterone interaction (F (2, 34) = 1.17; P = 0.316) on the frequency of centerline crosses. However there was a main effect of testosterone to increase centerline crosses (F (2, 34) = 4.25; P = 0.044), regardless of trap condition (Figure 8).  28      L o w  T H ig h T 0 5 1 0 1 5 2 0 2 5 H a b itu a tio n Y o k e S n a p G r o o m in g  D u r a ti o n  ( s )  Figure 4. Mean ± SEM duration of grooming behavior (seconds) in high- and low-T rats under habituation, snap or yoke conditions. No significant differences in grooming patterns were seen between groups. Habituation (n = 16), Snap (n = 8) and Yoke (n = 8) groups.        29      L o w  T H ig h T 0 1 0 0 2 0 0 3 0 0 4 0 0 H a b itu a tio n Y o k e S n a p * * R e a r in g  D u r a ti o n  ( s ) * * #  Figure 5. Mean ± SEM duration of rearing behavior (seconds) in high- and low-T rats under habituation, snap or yoke conditions. In general, rats show more rearing behaviors with high-T replacement.  *P < 0.05 vs. low-T. Habituation (n = 16), Snap (n = 8) and Yoke (n = 8) groups.       30      L o w  T H ig h T 0 2 0 0 4 0 0 6 0 0 H a b itu a tio n Y o k e S n a p T r a p  Z o n e  ( s )  Figure 6. Mean ± SEM time spent in the trap zone (seconds) in high- and low-T rats under habituation, snap or yoke conditions. All groups spent comparable time on the side of the testing chamber containing the trap. Habituation (n = 16), Snap (n = 8) and Yoke (n = 8) groups.       31      L o w  T H ig h T 0 5 1 0 1 5 H a b itu a tio n Y o k e S n a p S tr e tc h  A tt e n d  ( s ) * *  Figure 7. Mean ± SEM duration of stretch-attend behavior (seconds) in high- and low-T rats under habituation, snap or yoke conditions. Rats in the snap condition show a significantly higher duration of stretch attends compared to those under yoke or habituation conditions (*P < 0.05). Habituation (n = 16), Snap (n = 8) and Yoke (n = 8) groups.        32      L o w  T H ig h T 0 1 0 2 0 3 0 4 0 H a b itu a tio n Y o k e S n a p L in e  C r o s s e s  ( # ) *  Figure 8. Mean ± SEM number (#) of line crosses in high- and low-T rats under habituation, snap or yoke conditions. Results indicate a main effect of testosterone increase centerline crosses regardless of trap conditions (P = 0.044). Habituation (n = 16), Snap (n = 8) and Yoke (n = 8) groups.         33  4.2.8 Reactivity There were no significant effects of trap (F (2, 34) = 3.18; P = 0.183) and testosterone (F (1, 34) = 1.3; P = 0.28), and no significant trap x testosterone interaction (F (2, 34) = 1.3; P = 0.287) on reactivity scores (Figure 9).  4.3 Discussion This experiment examined the effects of testosterone replacement in animals exposed to snap and yoked trap conditions. As with habituation, high-T animals continued to show smaller amounts of burying in general, but to comparable levels between snap and yoked conditions. This suggests a general effect of testosterone to reduce stimulus-induced burying behavior under aversive conditions. In addition, these results do not support the hypothesis that testosterone increases defensive burying in response to an aversive snap.  Direct (snap) or indirect (yoke) exposure to the mousetrap evoked a freezing response that was significantly greater than observed during habituation, but without any effect of testosterone. The data confirm that both groups identified and responded to the noise of the mousetrap when it was remotely triggered. However, since most of the freezing behavior was expressed within five minutes of the trap being triggered, it would be incorrect to interpret this freezing behavior as a higher level or cognitive choice of passive avoidance behavior. Rather, these results seem to reflect more of an instinctive survival reflex to a sudden sensory input, a similar behavioral pattern observed by Menard et al, 2004. Thus, no effect between high and low-T replaced groups was expected for freezing behavior.  Only those animals placed in the snap condition showed a significant increase in stretch attend postures. This result suggests that snap-exposed animals identified the source of the noise,   34      L o w  T H ig h T 0 1 2 3 4 Y o k e S n a p R e a c ti v it y  S c o r e  Figure 9. Mean ± SEM reactivity scores in high- and low-T rats under habituation, snap or yoke conditions. Scores are based on a 5-point scale, previously used by Gray et al, 1981. Comparable reactivity scores give insight to the uniformity of the perception of the stressor, as well as the overall magnitude of the response. Habituation (n = 16), Snap (n = 8) and Yoke (n = 8) groups.      35  as well as its aversive nature. Since stretch-attend postures were significantly lower in the yoke condition, stretch-attend posture in this study represents an extremely powerful and reliable behavioural measure for risk-assessment, as it was only seen under aversive (snap) conditions.  The results also show that rearing increases with testosterone replacement and exposure to the snap. This is consistent with animals wanting to explore and escape their environment. However, these results may also be influenced by not having a lid on the chamber (Lapiz-Bloom et al, 2009). Under aversive test conditions, assessment of line-crosses yielded no significant differences between groups. Since this behavior is a control measure for locomotor activity, this outcome was desired. Locomotion and sympathetic activities are positively correlated with defensive burying (Sjoifo et al, 1996). Therefore, a group difference in locomotion could represent a possible confound for detecting differences in defensive burying activity. Interestingly, during habituation low T animals buried more than high-T animals despite shwoing lower line-crosse frequencies. The lack of change in reactivity scores confirms that all animals experienced the same level of aversiveness to the snap. These results are expected since the snap noise is a very mild stimulus even for those indirectly exposed. This is indicated by the low magnitude of reactivity scores; all less than 2 (see Figure 10). Trap zone duration represents the amount of time the rats spend on the side of the chamber containing the trap.  Expectations were to see a decreased amount of time in the trap- zone for animals only in the snap condition. However, there was no change in the trap zone durations between snap and yolk animals. This may be explained by the mild nature of the stimulus, which may not be intense enough to deter the animals from moving towards the trap. It may also be explained by the duration of the experiment, in which significant differences in trap zone duration may have occurred within the first 5 minutes of trap exposure.  36  Grooming has been documented to increase after exposure to low to mild stressors, but is suppressed after severe stressors. Therefore, self-grooming is suppressed by aversive stimuli and its reemergence is thought to signal a shift to stress recovery (Menard et al, 2004). Our study shows no significant difference in grooming behavior between groups. Since our study did not involve a severe stressor, the results most likely reflect that the snap and yoke groups were of similar low severity and/or that testosterone does not drive grooming behavior. These data reflect effects of high or low testosterone replacement on rats exposed to a mild stressor in the defensive burying paradigm. Several behavioral effects of trap exposure arose, including a freezing response for both snap and yoked animals; increased stretch attends under the snap condition, as well as a generalized increase in rearing behavior. In addition, similar to habituation, rearing behavior was increased and burying was decreased in high-T compared to low- T. Finally, locomotor activity, reactivity and trap zone preference were not influenced by high or low testosterone replacement.          37  5. Differential Effects of High and Low Testosterone Replacement on Vasopressin mRNA Expression and Cellular Activation of limbic Regions 5.1 Introduction Differences in Fos protein and vasopressin (AVP) mRNA in limbic-related structures are useful markers to help explain the behavioral results seen under aversive conditions. Fos is a neuronal marker that can be used as an indicator of cellular activation.  Thus, stimulus induced staining of this protein provides a functional anatomical map of activated circuits (Hoffman et al, 1993).  Stimulus- or testosterone-dependent changes in the pattern of Fos induction could be used to explain, at least anatomically, the nature by which high and low burying animals respond or cope to stressful challenges. AVP is highly dependent on testosterone levels within limbic regions of the brain, explained by the high concentrations of androgen receptors within AVP-synthesizing cells of the MeA and pBST. Therefore, we also examined whether differences in defensive behaviors could be reflected by differences in AVP expression based on levels of testosterone replacement.  We also looked at downstream changes in Fos protein expression, since the septum seems to be crucial in regulating defensive behavior, as well as in receipt of AVP containing projections from the pBST and MeA. Fos expression in the septum and hippocampus in mice would appear to be dependent on AVP (Giri et al, 1990). Whether testosterone-dependent differences in AVP account for differences in Fos induction in the septum during defensive burying testing has not been considered.    38  5.2 Results Aversive trap exposure is likely to have an effect on Fos protein induction and perhaps AVP expression in multiple brain regions, and the magnitude of these responses may differ with testosterone replacement.  However, the physical act of transporting the animals from room to room may also have an effect. The following analysis compares changes in Fos protein and AVP mRNA levels as a function of testosterone replacement in transport control animals and those exposed to different trap conditions. 5.2.1 Fos-ir in the MeA and pBST Analysis of Fos protein levels revealed a general effect of trap exposure (snap or yolk) to increase the number of Fos positive cells in the MeA relative to the transport condition (F (2, 34) = 4.12; P = 0.04, Figure 10).  However, there was no significant effect of testosterone replacement (F (1, 34) = 0.38; P > 0.5) or a significant testosterone x trap interaction (F (2, 34) = 0.06; P > 0.5).  For the pBST (Figure 11), there were no significant effects of trap (F (2, 34) = 1.74; P = 0.191) and testosterone replacement (F (1, 34) = 1.22; P = 0.275), and no significant trap x testosterone interaction (F (2, 34) = 0.58; P > 0.5). 5.2.2 Fos-ir in the ventral LS (LSv) Two-way ANOVA revealed a general effect of trap exposure (snap or yolk) to increase the number of Fos positive cells in the LSV (Figure 12) relative to the transport condition (F (2, 34) = 7.11; P= 0.003). However, there was no significant effect of testosterone replacement (F (1, 34) = 0.03; P > 0.5) or a significant testosterone x trap interaction (F (2, 34) = 0.29; P > 0.5). 5.2.3 AVP mRNA expression in the MeA and pBST There were significant main effects of trap exposure (F (2, 34) = 3.12; P = 0.050) and high testosterone replacement (F (2, 34) = 6.56; P = 0.015) to increase AVP mRNA expression in the MeA (Figure 13), but no significant testosterone x trap interaction (F (2, 34) = 0.04; P > 0.05).  For the  39  pBST, there were significant effects of trap exposure (F (2, 34) = 4.12; P = 0.025) and high testosterone replacement (F (1, 34) = 5.66; P  = 0.023) to increase AVP mRNA expression relative to transport controls (Figure 14), but no interaction (F (2, 34) = 0.06; P > 0.05). Post-hoc analyses for the effect of trap exposure on AVP mRNA in the pBST, confirmed higher levels of AVP mRNA in animals under the snap condition compared to those under yolk exposure (Figure 14). 5.3 Discussion The first part of the results provides insight into the relative expression levels of Fos protein in limbic regions of rats exposed to a snap, yoke or transport condition. As a marker of cellular activation, Fos expression in this context provides a useful measure for functional anatomical mapping of circuits related to stress-induced coping behavior (Hoffman et al, 1993). Fos induction within the MeA and pBST showed no difference between snap and yoke conditions or between high or low testosterone replaced animals. However, only when the data is compared against transport control animals, does a significant effect of chamber exposure appear. This suggests a general stimulatory effect of chamber placement to increase the cellular activation of the pBST and MeA. The lateral septum plays an important role in defensive behavior. Several studies show that lesions of the septum abolish defensive behavior (Gray et al, 1981; Caffe et al, 1987; Pesold and Treit, 1992; Canteras et al, 1995; Menard and Treit, 1996; Dong and Swanson, 2004; Singewald et al, 2011). Similar to the pBST and MeA, the present results indicate a general effect of yoke and snap conditions to increase Fos expression in the lateral septum, regardless of testosterone replacement (Figure 12).  Thus, the findings reflect a general involvement of several interconnected limbic- related structures in response to our version of the defensive burying paradigm. On the basis of previous anatomical studies, most of the inputs to the LS originating from the pBST and MeA contain AVP. Considering the dependency of AVP on testosterone and  40      L o w  T H ig h T 0 1 0 0 0 2 0 0 0 3 0 0 0 4 0 0 0 T ra n s p o rt Y o k e F o s  M e A  ( c e ll  c o u n t) S n a p * *  Figure 10. Mean ± SEM Fos-ir cell counts in the MeA of high- and low-T rats under transport, snap or yoke conditions. A two-way ANOVA reveals a main effect of trap exposure (P = 0.04) to increase Fos responses compared to transport controls. Habituation (n = 16), Snap (n = 8) and Yoke (n = 8) groups.        41       L o w  T H ig h T 0 1 0 0 0 2 0 0 0 3 0 0 0 T ra n s p o rt Y o k e F o s  p B S T  ( c e ll  c o u n t) S n a p  Figure 11. Mean ± SEM Fos-ir cell counts in the pBST of high- and low-T rats under transport, snap or yoke conditions. Results fail to show any significant effects of testosterone replacement or trap conditions on Fos responses. Transport (n = 4), Snap (n = 8) and Yoke (n = 8) groups.         42      L o w  T H ig h T 0 2 0 0 4 0 0 6 0 0 8 0 0 1 0 0 0 T ra n s p o rt Y o k e F o s  L S .v  (c e ll  c o u n t) S n a p * * * *  Figure 12. Mean ± SEM Fos-ir cell counts in the LS.v of high- and low-T rats under transport, snap or yoke conditions. A two-way ANOVA reveals a main effect of trap exposure (P = 0.05) to increase Fos responses compared to transport controls. Transport (n = 4), Snap (n = 8) and Yoke (n = 8) groups.          43       L o w  T H ig h T 0 2 4 6 T ra n s p o rt Y o k e A V P  M e A  ( O D ) S n a p *  Figure 13. Mean ± SEM relative AVP mRNA levels in the MeA of high- and low-T rats under transport, snap or yoke conditions. Results reveal a main effect of high-T replacement to increase AVP expression (P = 0.015). Transport (n=4), Snap (n =8) and Yoke (n = 8) groups.        44       L o w  T H ig h T 0 5 1 0 1 5 2 0 T ra n s p o rt Y o k e A V P  p B S T  ( O D ) * S n a p * *  Figure 14. Mean ± SEM relative AVP mRNA levels in the pBST of high- and low-T rats under transport, snap or yoke conditions. Results reveal a main effect of high-T replacement to increase AVP expression (P = 0.023), as well as relatively higher levels of AVP mRNA in snap compared to yolk animals (P = 0.021), regardless of testosterone replacement. Transport (n = 4), Snap (n = 8) and Yoke (n = 8) groups.        45  concentration of androgen receptors in the pBST and MeA, alterations in testosterone secretion could still play an important role in modulating information flow to the lateral septum. As indicated in Figures 13 and 14, there was an increase in AVP expression in the snap condition in both high and low testosterone replaced animals. This suggests that either testosterone is not directly involved in the AVP response to snap exposure, or that testosterone may simply have a permissive effect on the capacity of these cells to respond in a context dependent manner. Since we know that testosterone is an absolute requirement for AVP expression within the pBST and MeA, the latter remains entirely possible. This idea is consistent with previous findings to show that testosterone can regulate aggression in a dose dependent manner, whereas only minimal amounts of the steroid are required for males to express reproductive behavior (Albert et al, 1990).             46  6. Differential Effects of High and Low Testosterone Replacement on Hypothalamic-Pituitary-Adrenal (HPA) Axis Activation 6.1 Introduction Excitatory information sent to the paraventricular nucleus (PVN) of the hypothalamus, in particular the mediodorsal parvocellular part of the nucleus, facilitates secretagogue release to the anterior pituitary to mount a neuroendocrine stress response. More specifically, corticotrophin releasing hormone (CRH) and AVP in the PVN synergize at the level of the anterior pituitary to stimulate the release of adrenocorticotropin (ACTH), which then stimulates the synthesis and release of glucocorticoid hormones from the adrenal glands (Williamson et al, 2005). Limbic circuits, including those involving the MeA, pBST and septum, project to the PVN and are capable of influencing the activity of the HPA axis.  Thus, we extended our study to include assessments of Fos protein and AVP mRNA expression within the PVN, as well as plasma levels of corticosterone, the principle glucocorticoid hormone in the rat.  Changes in the activity of the HPA axis activity have not been attributed to individual differences in defensive burying, since the stimulus is very mild and burying behavior actually dampens the stress response (Koolhaas et al, 2010).  Therefore, we anticipate no differences in HPA axis responses solely on the basis of trap exposure. However, different levels of testosterone replacement have been shown to exert differential effects on the stress-induced expression of AVP and Fos in the PVN (Williamson and Viau, 2008). Thus, these markers could provide some indication of an interaction between burying behavior and high and low testosterone replacement. The following compares changes in Fos protein and AVP mRNA expression levels in the PVN as a function of testosterone replacement in transport control animals and those exposed to yolk and snap mousetrap conditions.   47  6.2 Results 6.2.1 AVP PVN For AVP mRNA within the dorsomedial part of the PVN regulating the HPA axis (Figure 15), there were no main effects of trap (F (2, 34) = 0.49; P > 0.5) and testosterone (F (1, 34) = 0.02; P > 0.5), and no significant interaction between testosterone x trap (F (2, 34) = 0.23; P > 0.5). 6.2.2 Fos PVN Analysis of Fos-ir induction in the PVN (Figure 16) indicated no significant effect of testosterone replacement (F (1, 34) = 2.60; P = 0.12), and no significant testosterone x trap interaction (F (2, 34) = 0.51; P > 0.5). However, there was a significant effect of trap condition (F (2, 34) = 3.52; P = 0.04). Post hoc analysis confirmed a significant increase from control (transport) levels in the yoke condition (P = 0.042), regardless of testosterone replacement. 6.2.3 Corticosterone For plasma corticosterone concentrations at 1 hour after trap testing and transport (Figure 17), there were no main effects of trap (F (2, 34) = 0.97; P = 0.37) and testosterone (F (1, 34) = 0.05; P > 0.5), and no significant interaction between testosterone x trap (F (2, 34) = 0.16; P > 0.5).  6.3 Discussion  Although it was expected that corticosterone would show no differences, what was not anticipated were differences in PVN activation. Animals exposed to the yoked condition showed significantly more Fos activation in the PVN compared to transport controls. Frank et al, 2006 previously demonstrated high anxiety rats to show higher PVN Fos responses, more freezing behaviors, but lower corticosterone responses to novel cage exposure compared to animals selected for low anxiety traits. The loud audible snap in the yolk condition of our paradigm is ambiguous and  48       L o w  T H ig h T 0 5 1 0 1 5 T ra n s p o rt Y o k e A V P  P V N  ( O D ) S n a p  Figure 15. Mean ± SEM relative AVP mRNA levels in the PVN of high- and low-T rats under transport, snap or yoke conditions. No significant differences in AVP mRNA levels were found between groups. Transport (n = 4), Snap (n = 8) and Yoke (n = 8) groups.         49      L o w  T H ig h T 0 1 0 2 0 3 0 4 0 T ra n s p o rt Y o k e F o s  P V N .m p d  ( c e ll  c o u n t) S n a p * *  Figure 16. Mean ± SEM Fos-ir cell counts in the PVN.mpd of high- and low-T rats under transport, snap or yoke conditions. Results indicate a main effect of trap exposure to increase Fos responses (P = 0.05), regardless of testosterone replacement. Transport (n=4), Snap (n = 8) and Yoke (n = 8) groups.         50        L o w  T H ig h T 0 1 0 0 2 0 0 3 0 0 4 0 0 T ra n s p o rt Y o k e C o r ti c o s te r o n e  ( n g /m L ) S n a p  Figure 17. Mean ± SEM plasma corticosterone concentrations (ng/mL) in high- and low-T rats under transport, snap or yoke conditions. No significant differences in corticosterone responses were revealed. Transport (n=4), Snap (n = 8) and Yoke (n = 8) groups.        51  lacks controllability.  This could represent more of a stressor in yolk verses snap animals, resulting in greater activation of the PVN.  Because we sampled plasma corticosterone levels 1 hour following testing, perhaps greater differences in HPA output may have occurred closer to mousetrap exposure. It was interesting that there was no difference in the relative expression of AVP mRNA within the PVN despite differences in testosterone replacement and trap conditions. There are several possibilities of this such as the context and severity of the paradigm.  However, one likely explanation is that the two replacement doses were both within the low end of the normal physiological range for testosterone. Therefore, testosterone levels in the high-T group may not have been sufficiently high enough to alter AVP, as well as Fos responses to the challenges imposed by the trap conditions.           52  7. General Discussion To date there have been no previous studies encompassing relations between testosterone replacement, limbic AVP and Fos responses in the context of defensive behavior. Several studies have previously described relationships between these parameters, including those examining testosterone and defensive burying (Frye et al 2002; Fernandez-Guasti and Martinez-Mota, 2005); testosterone and stress-induced Fos (Williamson and Viau, 2008); defensive burying and Fos (Menard et al, 2004); testosterone and AVP (Van Leeuwen et al, 1984; Zhou et al, 1994; Watters et al, 1998; de Vries and Miller, 1998; Kalsbeek et al, 2002; Veenema et al, 2010); and AVP and defensive behavior (Ebner et al, 1999; Ebner et al, 2005; Egashira, 2007; and Bleickardt et al, 2008). Thus, several uncertainties remain as to the extent to which each of these factors may be linked to account for individual differences in defensive behaviors.  In the current study it was shown that all testosterone-replaced animals were capable of habituating to a novel environment and presented with a complex object over 3 consecutive 3 days. Although burying behavior was not a reliable indicator for habituation, we observed a unique behavioral adaptation from increased risk assessment and locomotion, as well as an increase in explorative escape. It is believed that this behavior shift, as shown in Illustration 1, may be the result of tempered stress responses to initial open chamber box exposure, isolation and transport of animals. Under these conditions, the results suggest that testosterone’s actions may be limited to altering the magnitude of behaviors associated with habituation.  Moreover, high testosterone replacement decreased burying and increased the amount of rearing (Table 1). This suggests an anxiolytic effect of testosterone to promote proactive exploration in our paradigm.  Unlike previous defensive burying studies, a unique aspect of our study was the use of a remotely snapped mousetrap as an aversive stimulus source. The trap provided a non-painful, acute and mild stimulus, which allowed us to explore several different aspects of rat behavior. The limited  53       Illustration 18. Schematic showing a common behavioral shift seen from the first day of habituation (H1) to the last day of habituation (H3) for both testosterone replaced groups. Locomotion is represented by the number of centerline crosses; explorative escape as rearing; and risk assessment as stretch attend postures.        54  ability of the trap to evoke a response has shown to be a reliable way to induce individual differences in animal behavior (Pinel et al, 1995; Linfoot et al, 2009). In addition, given the audible nature (snap) of the mousetrap exposure, it was possible to test the behavior of the yoked group of animals in the neighboring chamber. It is known from stress and coping research that animals placed in an uncontrollable and stressful environment, such as in a shock-prod chamber without bedding material, will mount a greater endocrine response than animals in the same conditions with bedding (De Boer et al, 1989; Bondi et al, 2007). Conversely, rats given a wooden dowel to chew on in a restraint paradigm show significantly lower corticosterone compared to rats without a dowel (Ono et al, 2008). In both of the experiments, manipulating the ability for animals to cope had profound effects on the endocrine system. In our paradigm, the yoked group also allowed us to test if the animals in the neighboring chamber would experience a similar endocrine response based on being unable to control the stimulus. Despite showing increased freezing in response to the snap, indicating that the animals identified the noise, there were no major differences in stress hormone levels or meaningful HPA responses in any of test groups. Interestingly, while animals exposed directly to the snap showed stimulus-specific increases in stretch-attend behavior, indicative of identifying the mousetrap as a stimulus source, the high-T yoked animals reared significantly more than low-T yoked animals. This result parallels the habituation data, supporting the role of elevations in testosterone to increase proactive exploration.  Limbic AVP is highly dependent on circulating testosterone levels. Differences in AVP levels between high- and low-T animals were expected, since animals in this study were separated based on testosterone replacement. Our analysis showed a general effect of testosterone to increase AVP mRNA in both the MeA and pBST; however post hoc analysis failed to find a significant comparison within or between groups. The lack of significance between groups may be explained by how robust  55  individual variations in AVP circuitry may be, even with static testosterone replacement. Surprisingly, there were significantly higher AVP mRNA levels in the pBST within both high- and low- T snapped animals compared to the yoked groups. This result is very interesting and may reflect a role for testosterone, even at very low concentrations, as a general requirement for the limbic system to express AVP in a context dependent manner. Further studies are required to determine the nature by which testosterone and AVP may come to interact on defensive behavior. A downstream target of the limbic AVP system is the septum. Septal Fos expression was significantly higher in animals that were exposed to the test compared to transport groups. Although a powerful neuroendocrine tool when used in combination with lesions, drug treatments, and double labeling techniques (Hoffman et al, 1993), Fos is limited to the detection of circuits without directionality, or quality. However, lesion studies of the lateral septum provide evidence that septal activation is indeed required for the expression of defensive behaviors (Blanchard and Blanchard, 1979; Pesold and Treit, 1992; Everts et al, 1997; Singewald et al, 2011). Therefore, one possibility is that the Fos activation is representing an anatomical profile that drives defensive behaviors. However, Fos activation can only confirm an involvement of these regions as a general response to exposure to the paradigm, but nonetheless provides impetus for further investigation.  The results of this study have shown that testosterone may have a role in reducing signs of neophobia and altering the magnitude of adaptive behaviors under habituation. After exposure to the snap, animals with high testosterone show less burying behavior and more rearing than low-T animals, and even more so with direct exposure to the snap. 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