STUDY OF THE ROLE OF THE AMYGDALA IN THE EFFECTS OF STRESS ON SENSORY PROCESSING RELATED TO THE URINARY BLADDER JENNIFER J.

Transcription

1 STUDY OF THE ROLE OF THE AMYGDALA IN THE EFFECTS OF STRESS ON SENSORY PROCESSING RELATED TO THE URINARY BLADDER by JENNIFER J. DEBERRY TIMOTHY J. NESS, COMMITTEE CHAIR JAMES E. COX CANDACE FLOYD ALAN RANDICH DIANE TUCKER A DISSERTATION Submitted to the graduate faculty of The University of Alabama at Birmingham, in partial fulfillment of the requirements for the degree of Doctor of Philosophy BIRMINGHAM, ALABAMA 2010

2 Copyright by Jennifer J. DeBerry 2010

3 STUDY OF THE ROLE OF THE AMYGDALA IN THE EFFECTS OF STRESS ON SENSORY PROCESSING RELATED TO THE URINARY BLADDER JENNIFER J. DEBERRY BEHAVIORAL NEUROSCIENCE ABSTRACT Interstitial cystitis (IC) is a chronic visceral condition of the urinary bladder characterized by pelvic/suprapubic pain, and urinary frequency and urgency. There is no documented cause for IC, but a prominent role for stress in its pathophysiology and presentation are well-documented. In the clinical setting, IC pain-related symptomatology is exacerbated during periods of stress. Numerous laboratory studies of humans and animals have similarly demonstrated stress-induced visceral hypersensitivity. The amygdala is highly connected with physiological stress response systems and pain modulatory pathways, and its connectivity with these systems places it in a unique anatomical position for mediating the reciprocal relationship between pain and affective processes. This set of studies examined the role of the amygdala central nucleus (CeA) in modulation of urinary bladder nociceptive responses and physiological/behavioral indices of stress. Urinary bladder distension (UBD)-evoked visceromotor responses (VMRs), plasma corticosterone concentration, and spatiotemporal (% open arm time) and ethological behaviors (stretch-attend postures [SAPs], freezing, rearing) on the elevated plus maze (EPM) were measured in rats with CeA lesions following acute exposure to a footshock stressor and following acute chemical stimulation of the CeA with corticosterone. CeA lesions abolished acute footshock-induced bladder hyperalgesia and significantly decreased footshock-induced corticosterone release. Lesions significantly increased and decreased the frequency of SAPs and freezing, respectively. Acute corticosterone iii

4 stimulation of the CeA significantly facilitated VMRs in a fashion similar to acute footshock exposure, but did not significantly affect corticosterone release before or after EPM testing. CeA stimulation with corticosterone significantly increased freezing behavior on the EPM, but did not significantly affect any other anxiety-like behaviors. Spinal c-fos expression in response to UBD following corticosterone stimulation of the CeA was quantified. UBD significantly increased spinal c-fos, and corticosterone stimulation of the CeA significantly reduced it. These findings indicate that the CeA plays a significant role in modulation of visceral nociceptive responses (via an as-of-yet undefined spinal mechanism) and HPA axis activity in response to acute experimental manipulations. iv

5 TABLE OF CONTENTS Page ABSTRACT... iii LIST OF TABLES... ix LIST OF FIGURES...x LIST OF ABBREVIATIONS... xii CHAPTER 1 INTRODUCTION Why Study Stress and Bladder Pain? Purpose Hypotheses and Specific Aims Summary Definition of Terms REVIEW OF LITERATURE Brief Historical Perspective Multidimensionality of Pain Physiological Stress Response Systems Overview Hypothalamic-Pituitary-Adrenal Axis Sympatho-Adrenal Axis Locus Coeruleus/Norepinephrine System Stress and Mechanisms of Hypersensitivity in Interstitial Cystitis Nociceptive Pathways Overview Ascending Pathways Descending Pathways The Amygdala Overview Role of the Amygdala in Stress Role of the Amygdala in Nociception Summary Preliminary Conceptual Model...27 v

6 3 EXPERIMENTAL METHODS Selection of Species Anesthesia Footshock Stressor Overview Acute Footshock Procedure Measurement of Fecal Pellet Output Assessment of Somatic Nociceptive Responses Mechanical Testing Thermal Testing Noxious Visceral Distension and Response Measures Overview Urinary Bladder Distension and Visceromotor Response Procedure Amygdala Lesion Procedure Flinch-Jump Threshold Procedure Amygdala Stimulation Procedure Elevated Plus Maze Overview Elevated Plus Maze Procedure Enzyme-Linked Immunosorbent Assay (ELISA) Procedure c-fos Western Blotting Overview Western Blot Procedure Statistical Analyses Statistical Significance Somatic Nociceptive Threshold Measures Visceromotor Response Measures Elevated Plus Maze Measures Flinch-Jump Thresholds Enzyme-Linked Immunosorbent Assay Analyses Western Blot Analyses Histological Analyses Central Nucleus Lesion Sites Central Nucleus Stimulation Sites METHODOLOGICAL DEVELOPMENT Purpose Selection of Stress Paradigm and Rat Strain Overview Methodological Study 1: Acute and Chronic Water Avoidance in High- and Low-Anxiety Rat Strains Methodological Study 2: Acute Footshock in Sprague- Dawley Rats Preliminary Studies Related to the Amygdala...62 vi

7 4.3.1 Overview Methodological Study 3: Chronic Amygdala Stimulation Enhances Visceromotor Responses to Urinary Bladder Distension Methodological Study 4: Dose-Response Study of Drugs onto the Amygdala Summary Restatement of Hypotheses and Specific Aims RESULTS SPECIFIC AIMS 1 AND Purpose Research Hypotheses and Findings Specific Aim Specific Aim Overview Specific Aim 2 Sub Aim Specific Aim 2 Sub Aim Summary Related to Specific Aims 1 and RESULTS SPECIFIC AIMS 3 AND Purpose Research Hypotheses and Findings Specific Aim Specific Aim Overview Specific Aim 4 Sub Aim Specific Aim 4 Sub Aim Summary Related to Specific Aims 3 and RESULTS SPECIFIC AIM Purpose Research Hypotheses and Findings Summary Related to Specific Aim DISCUSSION Summary of Results Relationship Between Current Findings and Existing Literature Research Findings Related to Visceral Nociceptive Responses Research Findings Related to a Spinal Mechanism of Amygdala Action Research Findings Related to Plasma Corticosterone Concentration Research findings Related to Anxiety-Like Behavior Discussion of Results Acute Footshock: A Stressor or a Noxious Stimulus? vii

8 8.3.2 What is the Function of a Spinal Mechanism? The Central Nucleus and Other Important Neuroanatomical Mediators of Physiological and Behavioral Nociceptive and Stress-Related Responses Strengths and Limitations Strengths Limitations A Revised Conceptual Model Future Directions Conclusions LIST OF REFERENCES APPENDIX A IACUC NOTICE OF APPROVAL B IACUC NOTICE OF APPROVAL MEMORANDUM C IACUC NOTICE OF APPROVAL FOR PROTOCOL MODIFICATION D IACUC PROTOCOL MODIFICATION WORKSHEET viii

9 LIST OF TABLES Table Page 1-1 Animal studies of stress-induced urinary bladder hypersensitivity Effect of acute footshock on visceral nociceptive responses Effect of chronic CeA stimulation on visceral nociceptive responses Group designations for Specific Aims 1 and Effect of CeA lesions on footshock-induced visceral nociceptive responses Effect of CeA lesions on acute footshock-induced plasma corticosterone concentration Effect of CeA lesions on acute footshock-induced spatiotemporal anxietylike behavior Effect of CeA lesions on acute footshock-induced ethological anxiety-like behavior Effect of acute CeA stimulation on visceral nociceptive responses Effect of acute CeA stimulation on plasma corticosterone concentration Effect of acute CeA stimulation on anxiety-like behavior ix

10 LIST OF FIGURES Figure Page 2.1 Stress-related mechanistic model of IC Original conceptual model of bidirectional spinal nociceptive modulatory mechanisms Footshock apparatus UBD-evoked visceromotor and pseudaffective responses Effect of acute WA stress on visceral nociceptive responses Effect of chronic WA stress on visceral nociceptive responses Effect of acute WA stress on somatic nociceptive responses Effect of chronic WA stress on somatic nociceptive responses Effects of acute and chronic WA stress on fecal pellet output Effect of chronic footshock on visceral nociceptive responses and plasma corticosterone concentration Effect of acute footshock on visceral and somatic nociceptive responses Effect of acute footshock on plasma corticosterone concentration and fecal pellet output Effect of chronic CeA stimulation on visceral nociceptive responses Effect of CeA lesions on visceral nociceptive responses Effect of CeA lesions on flinch and jump thresholds Effect of CeA lesions on acute footshock-induced plasma corticosterone Concentration Effect of CeA lesions on acute footshock-induced anxiety-like behavior...87 x

11 5.5 Histological representation of CeA lesions for Specific Aim Effect of CeA lesion misses on visceral nociceptive responses Histological representation of CeA lesions for Specific Aim Effect of acute CeA stimulation on visceral nociceptive responses Effect of acute CeA stimulation on plasma corticosterone concentration Effect of acute CeA stimulation on anxiety-like behavior Histological representation of CeA stimulation sites for Specific Aim Histological representation of CeA stimulation sites for Specific Aim Effects of CeA stimulation on UBD-evoked spinal c-fos expression Histological representation of CeA stimulation sites for Specific Aim A revised conceptual model: functional anatomical pathways supporting CeA modulation of nociceptive and stress-related responses xi

12 LIST OF ABBREVIATIONS ACTH ADR AMYG ANOVA AP CeA CeLC CRD CRF DH DNA ELISA EMG EPM HNCS HPA HR HYP IC l-stt adrenocorticotrophic hormone adrenal medulla amygdala analysis of variance arterial pressure amygdala central nucleus laterocapsular central nucleus colorectal distension corticotrophic-releasing factor dorsal horn deoxy-ribonucleic acid enzyme-linked immunosorbent assay electromyographic elevated plus maze heterotopic noxious conditioning stimulus hypothalamic-pituitary-adrenal heart rate hypothalamus interstitial cystitis lateral spinothalamic tract xii

13 LC M mmhg mrna m-stt NE OD PAG PB PBS PIT PKA PSDC PVN SD SIA SIH SPT SRT STT TBST UBD VH locus coeruleus medial spinal cord millimeters of mercury messenger ribonucleic acid medial spinothalamic tract norepinephrine optical densities periaqueductal gray parabrachial nucleus painful bladder syndrome pituitary gland protein kinase A postsynaptic dorsal column paraventricular nucleus of the hypothalamus Sprague-Dawley stress-induced analgesia stress-induced hyperalgesia spinoparabrachial tract spinoreticular tract spinothalamic tract Tris-buffered saline urinary bladder distension ventral horn xiii

14 VMR WA WKY visceromotor response water avoidance Wistar Kyoto xiv

15 CHAPTER 1 INTRODUCTION 1.1 Why Study Stress and Bladder Pain? Visceral pain comprises a significant portion of reported chest and abdominal pain, which is a primary stated reason that patients seek urgent physician care (Cervero and Laird, 2004; Ness, 2003). Interstitial cystitis (IC) is a chronic visceral condition of the urinary bladder that is characterized by pelvic and/or suprapubic pain and urinary frequency and urgency. Although symptoms vary across individuals, pain often becomes the most incapacitating symptom as the disease progresses (Rothrock et al., 2001). In 1988, the National Institute of Diabetes and Digestive and Kidney Diseases established a set of inclusion and exclusion criteria for IC to be used as a guideline for clinical research as a means of ensuring homogeneity among study participants (Group consensus, 1988). These strict guidelines were not established to define the disease itself, and the broader term painful bladder syndrome (PBS) has been used more recently to describe patients with painful urinary bladder symptoms that may not meet each of the criteria. Currently, epidemiologic studies estimate the incidence of IC/PBS to be 5-7 in 10,000, affecting an estimated 1.3 million Americans with a 9:1 female-to-male ratio (Curhan et al., 1999; Jones and Nyberg, 1997; Parsons, 2002). The IC Database Study has reported that of those diagnosed with classic IC, 93.6% patients present with varying degrees of pain (Simon et al., 1997). 1

16 There is currently no documented cause for the development of IC and, as such, effective treatment strategies remain undefined. A variety of etiologies have been proposed that involve peripheral and/or central mechanisms, including a disruption in urinary bladder structure (Slobodov et al., 2004), abnormal neuronal function (Nazif et al., 2007), infiltration of inflammatory cells into the bladder and enhanced mast cell activation (Letourneau et al., 1992; Sant et al., 2007), the presence of antiproliferative substances and/or infectious agents in the bladder (Keay et al., 2003; Haarala et al., 1999), and recurrent urinary tract infections (Peters et al., 2009) or the occurrence of similar developmental insults to the bladder that may alter sensory development (Randich et al., 2006; DeBerry et al., 2007). While the etiology is likely multi-faceted, a prominent role for stress in the pathophysiology and presentation of IC also has been welldocumented (Hayes et al., 1976; Mayer et al., 2001; Spanos et al., 1997; Van Dijken et al., 1992; Robbins et al., 2006). Patients with a diagnosis of IC are often co-morbidly diagnosed with anxiety-related disorders, and flares in patient symptomatology and increased daily life stress are related (Weissman et al., 2004; Rothrock et al., 2001). Stress is a common human experience and a notion that we transfer to animal experience, yet there remains considerable debate over a precise definition of stress. A stressor can be generally defined as a physically or psychologically demanding situation in which an organism is required to adapt, adjust, or cope. Exposure to a stressor results in a psychological state referred to as stress, and evokes an emotional state of anxiety that is often associated with changes in arterial blood pressure (AP), heart rate (HR), and respiratory rate (Ferrell-Torry and Glick, 1993). Exposure to stressors can trigger a myriad of biochemical, physiological, and behavioral changes, and what comprises an 2

17 adequate stress response is dependent upon factors that can be greater than simply the degree of demand imposed by the stressor itself. The global purpose of our physiological stress response systems is to restore and maintain homeostasis, and disease states related to stress, including those characterized by pain, may result, at least in part, from an impaired normal stress response or an overall heightened response to stress. While peripheral and spinal nociceptive mechanisms are important for enhanced pain following exposure to an aversive event or stressor, higher brain function is necessary to transform individual emotional state, cognitive factors, or memory of previous sensory events into modulation of peripheral afferent input. The amygdala is a forebrain limbic structure involved in attaching emotional valence to sensory stimuli, and its connectivity with physiological stress response systems and pain modulatory pathways places it in a notable position suitable for mediating the reciprocal relationship between pain and affective processes (Gauriau and Bernard, 2002; Price, 2002). There has been considerable focus in recent years on the role of the amygdala as a neural mediator of the relationship between stress and exacerbation of pain, particularly in visceral nociceptive systems (Stam et al., 2002; Greenwood-Van Meerveld et al., 2001; Traub et al., 1996). Most of this work has focused on irritable bowel syndrome (IBS), a disorder of the gastrointestinal system. To date, only a few animal studies have been published examining stress-induced exacerbation of urinary bladder pain (Table 1-1), and no reports regarding the role of the amygdala in stress-induced exacerbation of bladder pain per se have been published to our knowledge (although see Qin et al., 2003). 3

18 Table 1-1. Animal studies of stress-induced urinary bladder hypersensitivity.* Black LV, Ness Effects of oxytocin and prolactin on stressinduced bladder hypersensitivity in female TJ, Robbins MT. rats. Robbins MT and Ness TJ. Robbins MT, DeBerry J, Ness TJ. Footshock-induced urinary bladder hypersensitivity: role of spinal corticotropin-releasing factor receptors. Chronic psychological stress enhances nociceptive processing in the urinary bladder in high-anxiety rats. J Pain, 10: , J Pain, 9: , Physiol Behav, 91: , * studies were identified using the search terms stress, bladder, pain in PubMed. 1.2 Purpose Stress/anxiety is a significant factor impacting pain symptomatology and quality of life in people suffering from disorders of the urinary bladder characterized by pelvic pain, such as IC. Evidence within current literature suggests a potential role for the amygdala in stress-induced exacerbation of visceral pain. The current set of studies begins a novel line of basic science research examining the role of the amygdala in the relationship between stress and visceral pain with a focus on the urologic system. These studies use converging lines of evidence to address the following general questions: (1) is the amygdala a neuroanatomical component of the underlying pathway(s) mediating enhanced nociception following exposure to an experimental stressor, and (2) does visceral nociceptive modulation by the amygdala in response to stress occur, at least in part, via a spinal mechanism. 1.3 Hypotheses and Specific Aims The general hypothesis that guided the current set of studies was as follows: The amygdala plays a role in the activation of a spinal mechanism of acute footshock-induced urinary bladder hypersensitivity in rats. 4

19 Specific sub-hypotheses that refined and developed this general hypothesis were the following: 1. Lesions of the amygdala central nucleus (CeA) alter the expression of bladder hyperalgesia and physiological/behavioral indices of stress induced by acute exposure to footshock. 2. Acute corticosterone stimulation of the CeA induces visceral nociceptive phenomena and alterations in stress-related physiological and behavioral responses similar to that seen following acute exposure to footshock. 3. Corticosterone stimulation of the CeA enhances spinal c-fos expression produced by urinary bladder distension (UBD). The Specific Aims used to address these sub-hypotheses were the following: Specific Aim 1. The hypothesis that CeA lesions attenuate acute footshock-induced bladder hypersensitivity will be tested. Bilateral lesions will be performed, and UBD-evoked abdominal electromyographic (EMG) responses following acute exposure to footshock will be quantified. Flinch and jump thresholds in response to footshock will be assessed as a control measure. Specific Aim 2. The hypothesis that CeA lesions attenuate physiological and behavioral indices of stress following acute footshock exposure will be tested. Bilateral lesions will be performed. Acute footshock-induced plasma corticosterone and anxiety-like behavior on an elevated plus maze (EPM) will be measured. Specific Aim 3. The hypothesis that acute corticosterone stimulation of the CeA 5

20 enhances bladder nociceptive responses will be tested. Bilateral application of corticosterone to the CeA will be performed, and UBD-evoked abdominal EMG responses will be quantified. Specific Aim 4. The hypothesis that acute corticosterone stimulation of the CeA enhances the expression of physiological and behavioral indices of stress will be tested. Bilateral application of corticosterone to the CeA will be performed, and anxiety-like behavior will be measured on an EPM. Plasma corticosterone concentration will be measured at baseline and following EPM testing. Specific Aim 5. The hypothesis that acute corticosterone stimulation of the CeA alters c- Fos expression in the spinal cord in response to UBD will be tested. Bilateral application of corticosterone to the CeA will be performed, and Western blots assessing c-fos expression under conditions of UBD and no UBD will be quantified. 1.4 Summary Women who suffer from IC experience a pronounced impact of stress on their pain symptomatology. There is currently a limited understanding with regard to how stress- and anxiety-related affective and cognitive processes are translated into increased visceral pain. Although findings from previous studies have repeatedly demonstrated this phenomenon in the gastrointestinal system, there have been few studies examining stressinduced hyperalgesia of the urological system; furthermore, no specific underlying neuroanatomical substrates have yet been elucidated. This study was designed to examine the effects of stress on nociception of the urinary bladder, with the goal of 6

21 elucidating whether the amygdala is involved in a spinal mechanism related to stressinduced urinary bladder hyperalgesia. follows. 1.5 Definition of Terms The conceptual and operational definitions of the major study concepts were as Stressor: A physically or psychologically demanding situation where demand is characterized in terms of requiring an organism to adapt, cope, or adjust to that situation. Stress: The psychological state that occurs as a reaction to experiencing a stressor. Anxiety: A feeling of apprehension that can range from concern to dread; an indistinct emotion with a cause that is not always easily identifiable (Taylor-Loughran, 1989). Pain: An unpleasant sensory and emotional experience associated with actual or potential tissue damage, or described in terms of such damage (adapted from Merskey, 1964). Nociceptive stimulus: a tissue-damaging stimulus or stimulus that would produce tissue damage if maintained (Price, 2002). 7

22 CHAPTER 2 REVIEW OF LITERATURE 2.1 Brief Historical Perspective The recognition that endogenous substrates exist at which exogenous compounds, such as morphine, exert their analgesic action suggested long ago the existence of intrinsic mechanisms that function to modify nociception. In 1976, several independent laboratories reported within a short period of time that nociceptive thresholds can also be altered by environmental manipulations (Akil et al., 1976; Hayes et al., 1976). It is now accepted that exposure to stressors can produce bidirectional modulation, i.e., inhibition or facilitation, of pain in humans and nociceptive responses in animals, and these phenomena are respectively termed stress-induced analgesia (SIA) and stress-induced hyperalgesia (SIH). A review of the literature related to stress-induced changes in nociception reveals distinct characteristics of SIA and SIH, with one prominent distinction being that they typically, although not exclusively, occur in divergent tissues and sensory fibers. In animal studies, stress-induced nociceptive inhibition has been predominately observed in somatic or superficial sensory systems while stress-induced nociceptive facilitation typically occurs in deep tissue such as muscle or viscera. SIA and SIH also typically differ in their temporal characteristics, such that SIA tends to be more transient (seconds to minutes) than SIH, which has been reported to last as long as days (Bradesi et al., 2005). Although both phenomena have been reliably demonstrated in laboratory animals, stress-induced exacerbation of pain-related symptomatology in 8

23 human clinical studies is the standard rather than the exception, and has been associated with a variety of conditions (Zautra et al., 1997; Affleck et al., 1997; Schmidt-Ott et al., 1998; Nilsen et al., 2007; Thomason et al., 1992; Daney, 1998). Because of this, there is a significant focus in current research on stress-induced changes in human pain sensitivity, and a significant portion of this work is related to visceral systems. 2.2 Multidimensionality of Pain Current concepts of pain underscore the importance of recognizing pain as more than a purely sensory phenomenon. The experience of pain most commonly begins with transmission of information about a noxious stimulus via nociceptive pathways to the brain, where sensory, cognitive, and emotional information are integrated. As a result of this integration, pain is a multidimensional, subjective experience where an organism s unique history, biology, and surrounding environment can impact how a sensory stimulus is perceived. Psychophysical studies have demonstrated the existence of distinct dimensions of pain that are typically described along two axes: the sensory dimension comprises spatial localization, temporal discrimination, and intensity encoding properties, and the affective dimension represents the perceptual unpleasantness of a stimulus and the behavioral and autonomic reactions it evokes (Fernandez and Turk 1992; Melzack and Casey 1968; Price et al., 1987). In addition to reflecting the degree of unpleasantness of the sensory experience, the affective dimension accounts for an individual s emotions and cognitive processes about the short- and long-term implications of that experience, or secondary pain affect (Price 2000; 2002). Overall perception of pain and the intensity of noxious input are not necessarily linearly related, and psychophysical studies have 9

24 demonstrated that pain intensity, pain unpleasantness, and secondary pain affect can vary independently in relation to nociceptive stimulus intensity and can be individually modified by psychological factors. At the most basic level, simple temporal characteristics of a noxious stimulus can alter ratings of pain unpleasantness while pain intensity ratings remain stable, such that long and brief duration stimuli of the same intensity are rated as relatively more and less unpleasant, respectively (Price et al., 1987). A more complex relationship between the sensory and affective dimensions of pain emerges upon the introduction of various individual psychological factors. In a study examining the influence of individual personality traits on pain, Harkins et al. (1989) reported a selective effect of neuroticism, defined as the enduring tendency to experience negative emotional states (Eysenck and Prell, 1951), on secondary pain affect in response to clinical and experimental pain. Participants with a high (versus low) personality inventory score of neuroticism had higher ratings of secondary pain affect; however, neuroticism did not significantly influence pain unpleasantness or pain intensity. The nature of the relationship between the sensory and affective dimensions of pain is further underscored in two experiments using hypnotic suggestions to alternately increase and then decrease pain intensity or pain unpleasantness. These studies reveal that unpleasantness ratings change in both conditions, regardless of which dimension was actually targeted by the suggestions (Rainville et al., 1999), indicating that the affective dimension of pain can be altered both in series, i.e., in response to, and in parallel to alterations in the sensory dimension (Price, 2000). 10

25 Notably, there is divergence in the affective quality of visceral versus somatic sensation, where visceral stimuli are commonly described as more unpleasant and have the tendency to evoke stronger emotional responses than somatic stimuli. A study of healthy individuals comparing visceral stimulation of the esophagus to cutaneous thermal stimulation of the anterior chest wall supports this notion. Visceral stimulation evoked significantly higher ratings of pain unpleasantness than pain intensity at a variety of distension pressures, but intensity and unpleasantness ratings for thermal stimulation were statistically equivalent, and trended in the opposite direction (Strigo et al., 2002). A second set of data from the same studies shows that when participants described their experience using the McGill Pain Questionnaire, the total number of words chosen to describe the visceral stimulus was greater, and this difference was driven by the use of affective words. Although visceral stimuli generally produce a more diffuse sensation that is difficult to localize and can be referred to somatic structures, this perceptual difference in the affective quality of visceral versus somatic pain appears, at least in part, to be centrally driven (Strigo et al., 2003; Dunckley et al., 2005). In summary, the perceptual experience of pain can be stratified into two primary dimensions related to sensory (intensity) and affective (unpleasantness) modalities. Psychological variables can influence one s experience of pain, and the perception of pain is largely related to individual differences in affective states. In this way, psychological variables such as stress/anxiety may trigger or exacerbate an episode of pain, help maintain a pain disorder, and contribute to the distress or disability experienced as a result of a painful disorder. 11

26 2.3 Physiological Stress Response Systems Overview An organism s response to stress is generated by a network of integrated brain structures, in particular the hypothalamic paraventricular nucleus (PVN), amygdala, and periaqueductal gray (PAG) (Mayer, 2000). These structures receive input from somatic and visceral afferents as well as from cortical areas and project to pituitary and medullary nuclei that mediate neuroendocrine and autonomic output. Central stress systems are under feedback control through ascending serotonergic and noradrenergic projections from the raphe nuclei and locus coeruleus (LC), respectively. Circulating glucocorticoids of adrenal origin also exert an inhibitory influence on central stress systems via central receptors. Inadequate control of glucocorticoid release can promote the development of physiological and psychological disruptions, including anxiety disorders (e.g., Berger et al., 2006). These integrated networks are activated in response to various stressors, and their output initiates responses within the hypothalamic-pituitary-adrenal (HPA) axis, sympatho-adrenal axis, LC/norepinephrine (NE) system, and endogenous pain modulatory systems. Although there have been multiple classifications of distinguishable types of stressors, such as positive (eustress) and negative (distress), physical (interoceptive) and psychological (exteroceptive), Herman and Cullinan (1997) have postulated that there are two generalized stress pathways. Limbic-sensitive stressors require higher order processing prior to initiation of a stress response; limbic-insensitive stressors pose an immediate physiological threat and thus do not require interpretation by higher order 12

27 brain structures, which may explain why particularly severe stressors can elicit analgesia in decerebrate animals (Millan, 2002). The contribution of specific regulatory networks within the central nervous system to the regulation of corticosterone under conditions of stress is also influenced by similar factors (Herman et al., 2003). While reactive stressors (those that increase demand on the system via a sensory stimulus, such as pain, injury, or immune challenge) tend to involve the brainstem, the bed nucleus of the stria terminalis, and specific hypothalamic nuclei, anticipatory stressors (those that tap into innate memory programs such as social challenges or unfamiliar situations) tend to engage limbic system regions, particularly the hippocampus, amygdala, and medial prefrontal cortex (Herman et al., 2003) Hypothalamic-Pituitary-Adrenal Axis Acute stress activates cells within the PVN to release corticotrophin-releasing factor (CRF), which travels through the infundibulum to the pituitary gland where it stimulates secretion of adrenocorticotropic hormone (ACTH) into the bloodstream. ACTH binds to receptors in the adrenal cortex and stimulates secretion of cortisol in humans and corticosterone in rodents into the bloodstream. This cascade of events is under negative feedback regulation by corticosterone itself, such that circulating corticosterone decreases synthesis and release of CRF from PVN neurons thereby inhibiting the release of ACTH from the anterior pituitary (Dallman et al., 1987). In the basal, non-stressed state, secretion of corticosterone follows a circadian rhythm with a distinct rise at the time of awakening and a steady decline over the course of the day 13

28 (Horrocks et al., 1990). The amplitude of CRF pulsatile release is increased during periods of acute stress and results in increased secretion of ACTH and corticosterone. Classically, corticosterone exerts its effects to maintain homeostasis via ubiquitous cytoplasmic glucocorticoid receptors, although more recent evidence supports the involvement of mineralocorticoid receptors in regulation of stress responses as well (Berger et al., 2009). Upon ligand binding, these receptors can translocate into the nucleus and interact with specific response elements within DNA to activate hormoneresponsive genes. Once receptors are activated, they can inhibit other transcription factors, such as c-jun/c-fos and nuclear factor kappa B (NF-kB), which are components of the apoliprotein-1 (AP-1) transcription complex that has been implicated as a key regulator of biological processes (Piechaczyk and Blanchard, 1994). In some cases, corticosterone can also exert rapid, non-genomic effects via cell-surface glucocorticoid and mineralocorticoid receptors (Norman et al., 2004) Sympatho-Adrenal Axis The sympathetic nervous system (SNS) is an integral component of an organism s physiological stress response, and much of what is known about the sympatho-adrenal axis response to stress originates from seminal work by Cannon (1929) and Selye (1936). The classic sympatho-adrenal response has been characterized as fight or flight, and is considered a rapid onset/offset mechanism that affects multiple organ systems. However, it is now understood that sympatho-adrenal axis activity can be sustained and can produce long-term changes at the cellular level that may contribute to hyperalgesia (Khasar et al., 2009; Choudhry et al., 2009). 14

29 Sympathetic innervation of peripheral organs is from efferent preganglionic (cholinergic) fibers with cell bodies in the intermediate zone of the spinal cord, where they form two intermediolateral columns. These neurons receive direct input from the hypothalamus, pons, and medulla, and from other spinal segments and dorsal horn interneurons. The preganglionic fibers synapse either with other cholinergic neurons in the adrenal medulla or postganglionic (primarily adrenergic) neurons in the sympathetic chain ganglia that innervate smooth muscle. Neuronal transmission in sympathetic ganglia results in the release of epinephrine from the adrenal medulla and NE from sympathetic nerve terminals, and the amount of circulating NE is thought to reflect the degree of SNS activity (Axelrod and Reisine, 1984). NE and epinephrine release can also be modulated by neuropeptide release from preganglionic fibers, interneurons (enkephalins), and primary afferent collaterals (substance P) (Elfvin et al., 1993) Locus Coeruleus/Norepinephrine System The LC and other noradrenergic cell groups in the medulla and pons release NE when activated, acting as a global alarm system that enhances neuroendocrine (HPA axis) and autonomic (sympatho-adrenal axis) responses to stress (Dronjak et al., 2007; Banihashemi and Rinaman, 2006) and activates neurons within the amygdala (Kaneko et al., 2008). CRF and NE stimulate release of each other via reciprocal connections, and there is an autoregulatory negative feedback loop in the LC/NE system similar to that of the HPA axis, where NE acts at α 2 -adrenergic receptors to limit its own release (Bandoh et al., 2004). 15

30 2.4 Stress and Mechanisms of Hypersensitivity in Interstitial Cystitis IC is a clinical example of a functional visceral condition characterized by hypersensitivity. Mechanisms that can result in visceral hypersensitivity in multiple systems, and that often overlap, have been elucidated at multiple levels of nociceptive processing using animal models. Heightened perception of pain could result from (i) peripheral sensitization, i.e., an increased signal arising from peripheral primary afferents, (ii) central sensitization, i.e., amplification of a normal peripheral signal at the spinal level or spinal-bulbo-spinal amplification, or (iii) changes in descending inhibitory and facilitatory pathways driven by supraspinal sites, and stress-related effects on visceral hypersensitivity have been identified at each of these levels (Gue et al., 1997; Chung et al., 2007; de Lange et al., 2005; Stam et al., 2002). While peripheral mechanisms may be an important factor in the onset and/or maintenance of stress-induced visceral pain, chronic, sustained hypersensitivity is likely to involve the contribution of central mechanisms. The brain has multiple avenues through which to modulate perception of afferent information that can be influenced by cognitive factors, individual emotional state, or memories of previous sensory events. It has been hypothesized that stress/anxiety-related affective responses, including symptomrelated anxiety, can significantly alter perceptual responses to painful stimuli via multiple brain mechanisms including attentional, opioidergic, and descending modulatory networks (Dunckley et al., 2007; Phillips et al., 2003; Schweinhardt et al., 2008). Enhanced activity in emotional arousal pathways is believed to play a role in stress-induced visceral hyperalgesia, and this concept is not novel (Mayer et al., 2002, 2006; Azpiroz et al., 2007; Twiss et al., 2009). Seminal experiments by Pavlov (1910) 16

31 and Cannon (1929) confirmed the existence of a brain-gut interaction, and more recent studies examining the interactions between the central nervous system and the gastrointestinal system have highlighted the role of stress in the modulation of many gastrointestinal disorders. It has also more recently been observed that anxiety and stress may initiate and worsen urinary symptoms and functional urinary disorders associated with pain such as IC (Rothrock et al., 2001; Lutgendorf et al., 2000). In 2001, Mayer et al. proposed a stress-related mechanistic model of IBS that may be applicable to IC. In this model, stress factors may contribute at three different levels to the development of the disorder (Figure 2.1). At Level One, stress-related risk factors include genetic predisposition, early life experiences, and pathological stress or permanent enhancement of stress responsive systems. Indeed, a genetic linkage study has provided evidence that both IC patients and their first-degree relatives have approximately a three-fold increase in panic disorder as compared to individuals without IC and their first degree relatives (Weissmann et al., 2004). Furthermore, there is evidence that IC patients have altered autonomic function, diurnal cortisol variations, and increased CRF, factors indicative of pathological stress and altered stress responsiveness (Lutgendorf et al., 2002, 2004; Dimitrakov et al., 2001; Klausner and Steers, 2004). An increased presence of and activity within sympathetic nerves supplying the bladder and elevated levels of urinary NE have been identified in individuals with IC, and greater severity of bladder-related symptoms is associated with more numerous sympathetic fibers (Hohenfellner et al., 1992; Lundeberg et al., 1993). Sympathetic activation may also contribute to IC symptoms by exacerbating local inflammation through acutely increased mast cell degranulation (Sant et al., 2007). Exposure to experimental stressors 17

32 in rats increases both the number of mast cells and the degree of mast cell degranulation within the bladder (Spanos et al., 1997; Ercan et al., 1999; Alexacos et al., 1999). At Level Two of Mayer s stress-related mechanistic model, the presence of psychosocial stressors is considered a trigger factor. Clinical laboratory studies have also demonstrated a positive relationship between stress and IC pain (Lutgendorf et al., 2000). Although IC patients have higher bladder pain ratings at baseline than control participants, a significant increase in IC-related pain occurred following a 25 minute mental stress task compared to baseline, and this change did not occur in the control group. A positive relationship between stress and IC symptoms, including pain, has also been reported in a self-report, life stress model, where IC patients experienced more stress and pain symptoms than controls and pain increased as a function of disease severity (Rothrock et al., 2001). The latter finding fits into Level Three of the stressrelated mechanistic model, where symptom-generated stress may act as a perpetuating factor of the symptoms. 2.5 Nociceptive Pathways Overview The net experience of pain results from a balance between two opposing, inhibitory and facilitatory, influences. Any factor that results in a shift between the balance of inhibition and facilitation can, then, influence the degree of nociception or experience of pain. Modulation of nociceptive transmission can occur at multiple levels within the central nervous system. Inhibitory and facilitatory modulation can occur locally within the spinal cord via interneuronal or propriospinal connections, or through 18

33 ascending and descending nociceptive pathways. The ascending pathways included in the scope of this discussion are the spinothalamic tract (STT), spinoreticular tract (SRT), spinoparabrachial tract (SPT), and postsynaptic dorsal column (PSDC) pathway. The primary network for descending modulation includes an excitatory projection from the midbrain PAG to brainstem structures, including the nucleus raphe magnus (NRM), nucleus reticularis gigantocellularis (NGC), and LC. Projections from several populations of neurons within these regions make connections within the spinal cord that can either directly or indirectly excite or inhibit dorsal horn neurons Ascending Pathways There are multiple ascending pathways by which nociceptive information is transmitted to supraspinal sites. The traditional ascending nociceptive pathway is the ventrolateral STT. STT neurons respond to a variety of nociceptive stimuli, including noxious mechanical and thermal cutaneous stimulation, noxious chemical stimulation of muscle, and noxious mechanical, thermal, and chemical stimulation of the viscera (Horn et al. 1999; Milne et al. 1981). In humans and monkeys, the STT comprises axons originating from neurons scattered throughout the spinal cord and that cross segmentally prior to ascending via the anterolateral quadrant (Willis 1985 for review). Studies in rat have demonstrated a similar organization (Giesler et al., 1979). Based on anatomical experiments using horseradish peroxidase labeling techniques, the STT has been functionally subdivided based on whether ascending fibers relay primarily through medial versus lateral thalamic nuclei (e.g., Berkley 1980; Kobayashi 1998). These functional subdivisions are referred to as the medial STT (m-stt) and lateral STT (l- 19

34 STT), respectively. The m-stt is thought of as the more primitive ascending pathway, and is present in lower order animals as well as mammals; in contrast, the l-stt is believed to be phylogenetically newer and is significantly more developed in mammals, particularly primates (Kevetter and Willis, 1984). Specifically, the m-stt is a polysynaptic pathway composed of short fibers with heavy collateral branching that targets medullary, pontine, and midbrain sites prior to entering medial thalamic nuclei (Liu, 1986). Neuronal transmission along this pathway activates numerous subcortical limbic structures, and the m-stt is viewed as important for affective and motivational aspects of pain. Conversely, fibers within the l-stt are fewer in number and longer, and project directly to ventrobasal thalamic nuclei prior to activating primary somatosensory cortex. This apparently dedicated pathway is of value for sensory-discriminative aspects of pain such as stimulus localization and intensity encoding. As mentioned above, many STT neurons have bifurcating axonal projections, and some SRT axons are collateral branches of STT neurons (Kevetter and Willis, 1983). Ascending fibers within the SRT are those that reach medullary and pontine sites without a relay through thalamic sites and include many neuronal groups. Some SRT fibers overlap with SPT fibers and project to the parabrachial nucleus, through which input arising from visceral primary afferents is transmitted and which has extensive projections to subcortical limbic structures including the amygdala (Han and Neugebauer, 2004). SRT fibers are involved in affective aspects of pain and control of descending modulation from the NRM and reticular formation (Pezet et al., 1999; Menetrey et al., 1980), and the SPT has been implicated in affective nociceptive transmission as well as autonomic and endocrine responses to pain (Bernard et al., 1996; see Benarroch, 2001). 20

35 The PSDC is now accepted as the major efferent pathway for visceral nociception of the colon, bladder, and uterus (Berkley and Hubscher, 1995; Al-Chaer et al., 1996, 1999; Willis et al., 1999; Ness, 2000; Robbins et al., 2006). Dorsal column lesions can prevent potentiation of the visceral nociceptive responses and inhibit exploratory activity induced by noxious visceral stimulation and reduce thalamic neuronal responses to visceral distension (Palecek and Willis, 2003; Ness, 2000; Al-Chaer et al., 1996). The predominance of visceral nociceptive neurons that transmit sensory information via the PSDC is contained in lamina X (Al-Chaer et al., 1996) Descending Pathways The primary network for descending nociceptive modulation includes the PAG, NRM, NGC, NGC pars alpha, and LC. While spinal nociceptive systems are under tonic descending inhibition, descending neuronal projections from multiple populations of neurons can either directly or indirectly excite or inhibit dorsal horn neurons. The primary descending spinal pathways for inhibition and facilitation of spinal neuronal responses are the dorsolateral and ventrolateral funiculi, respectively (Basbaum et al., 1978). Numerous neurotransmitters have been implicated in descending inhibition and facilitation (Millan, 2002). The principal system for descending inhibition involves activation of supraspinal sites and subsequent transport and spinal release of serotonin and/or NE and involvement of spinal opioids (Millan, 2002). There is substantial overlap in the neurochemical substrates of descending inhibition and facilitation (e.g., Zhuo and Gebhart, 1991), and divergent effects on spinal nociceptive transmission can be produced 21

36 through activation of unique receptor subtypes. Additional neurotransmitters may be unique to facilitatory influences (Buhler et al., 2008; Guo et al., 2006). 2.6 The Amygdala Overview It is understood that the amygdala acts to link sensory stimuli with affective processes. Due to its connectivity with brain structures known to be integral for pain processing and stress-related responses, the amygdala is uniquely positioned to participate in the relationship between stress and pain by linking physical sensation and cognitive-emotional constructs Role of the Amygdala in Stress The amygdala is a limbic forebrain structure linked to anxiety, fear, and stress responses (Gallagher and Chiba, 1996). It comprises several distinct nuclei, including the lateral nucleus, basolateral nucleus, medial nucleus, and CeA. The CeA is the area from which the principle projection neurons to the basal forebrain, hypothalamus, and brainstem structures arise, and has been shown to exert regulatory influences over the HPA axis stress response system (Gauriau and Bernard, 2002; Feldman and Weidenfeld, 1998; Shepard et al., 2003). Large amygdaloid lesions or CeA lesions markedly reduce activation of the HPA axis following stress, while electrical stimulation of the CeA enhances it (Allen and Allen, 1974; Beaulieu et al., 1986, 1987, 1989; Feldman et al., 1994; Lilly et al., 2000; Van de Kar et al., 1991; Feldman et al., 1982). Thus, unlike other limbic system mechanisms of HPA axis regulation (i.e., hippocampus, prefrontal 22

37 cortex), the CeA appears to play a facilitatory, rather than inhibitory, role in the HPA axis response to stress (Bhatnagar and Dallman, 1998). Similar to the PVN, the amygdala is rich with both glucocorticoid and mineralocorticoid receptors, with the highest density found in the CeA (Reul and de Kloet, 1985; Sapolsky et al., 1983). Stereotaxic application of corticosterone onto the CeA elevates CRF mrna in the CeA and basal CRF mrna in the PVN (Shepard et al., 2000,2003). It also increases indices of anxiety on the EPM, an effect that is significantly inhibited by antagonism of either glucocorticoid or mineralocorticoid receptors within the CeA (Shepard et al., 2000; Myers and Greenwood-Van Meerveld, 2007). Paradoxically, while electrical stimulation of the CeA produces increased plasma levels of corticosterone and inhibits HPA axis activity via negative feedback to the HPA system itself, chemical stimulation of the CeA via application of corticosterone does not elevate peripheral corticosterone (Dunn and Whitener, 1986; Feldman et al., 1982; Shepard et al., 2003). Thus, direct chemical stimulation of the amygdala facilitates HPA axis activation in the absence of elevated circulating corticosterone as a negative feedback mechanism. Chronic stimulation of the CeA with corticosterone or aldosterone, an endogenous mineralocorticoid, produces enhanced dorsal horn neuronal responses to colorectal distension (CRD) and UBD, and enhanced nociceptive responses to CRD (Qin et al., 2003a,b,c; Greenwood-Van Meerveld et al., 2001; Myers and Greenwood-Van Meerveld, 2007; Myers et al., 2007). However, there are no published studies examining the effects of this type of CeA stimulation either in an acute setting or on visceral nociceptive responses to UBD. 23

38 The influence of the amygdala on stress responsiveness appears to be stressorspecific and region-specific, although this idea is not universally accepted (Sawchenko et al., 2000; Herman and Cullinan, 1997; see Pacak et al., 1988). Studies have demonstrated distinct and contrasting patterns of neural activation within the amygdala in response to stress that seem to be dependent upon the nature of the stressor (Dayas et al., 2001). This would appear to be a consistent extension of the dual stressor pathway hypothesis proposed by Herman and Cullinan (1997) Role of the Amygdala in Nociception Anatomical and electrophysiological studies implicate multiple neural pathways that provide the amygdala with sensory information. Somatic information from the spinal dorsal horn is transmitted via projections through the lateral thalamus to cortical areas (primary/secondary somatosensory cortex, insular cortex) then primarily to the basolateral amygdala (Shi and Cassell, 1998; Shi and Davis, 1999; Smith et al., 2000). The CeA, particularly the lateral capsular division of the CeA, contains a high number of neurons that respond predominately to noxious stimuli (Heinricher and McGaraughty, 1999; Gauriau and Bernard, 2002). The CeA receives nociceptive information via direct spino-amygdalar projections and via the SPT, which is reported to be involved in visceral pain (Burstein and Potrebic, 1993; Gauriau and Bernard, 2002; Bernard et al., 1994). Basic science studies have demonstrated a role for the amygdala in nociceptive facilitation using several animal models (Han and Neugebauer, 2004; Ikeda et al., 2007; Greenwood-Van Meerveld 2001; Qin et al., 2003a,b,c; Myers and Greenwood-Van Meerveld, 2007; Myers et al., 2007; Suarez-Roca et al., 2008). Pain-related plasticity 24

39 within the amygdala has been demonstrated in rats following acute-onset models of arthritis and colitis, in a model of neuropathic pain, and in a cyclophosphamide-induced cystitis pain model in mice (Neugebauer et al., 2003; Han and Neugebauer, 2004; Ikeda et al., 2007; Nishii et al., 2007). Neurons within the lateral nucleus of the CeA became sensitized in vivo to afferent inputs arising from the knee joint following kaolin/carrageenan-induced arthritis in rats, and similar neurons display synaptic plasticity and hyperexcitability in vitro using the same model (Ji and Neugebauer, 2007; Bird et al., 2005). In a neuropathic model, CeA neurons demonstrated a significant increase in postsynaptic current amplitude following spinal nerve ligature (Ikeda et al., 2007). Studies in the gastrointestinal system have demonstrated that chemical stimulation of the CeA produces behavioral and spinal neuronal hypersensitivity in response to CRD (Qin et al., 2003a,b; Myers and Greenwood-Van Meerveld, 2007), but to date, only two published studies have addressed the involvement of the amygdala in nociceptive modulation related to the urinary bladder. Neuroendocrine peptide expression is upregulated in the CeA following cyclophosphamide-induced cystitis of the urinary bladder, and chemical stimulation of the CeA via stereotaxic application of corticosteroids results in facilitation of spinal dorsal horn neuronal responses to bladder distension (Nishii et al., 2007; Qin et al., 2003c). The latter electrophysiological study employed the UBD paradigm developed by Ness et al. (2001) that was used in the current set of studies to measure nociceptive behavior related to the bladder. Differential contributions of the amygdala to cutaneous versus deep tissue nociception are not entirely clear. Specific amygdaloid subnuclei receive relatively domain-specific projections, and basic science studies indicate that the CeA is involved 25

40 in various types of deep tissue sensation including joint pain, neuropathic pain, and visceral pain. A study of c-fos mrna expression within the amygdala found that different patterns of expression emerge following somatic (intraplantar (i.pl.) injection of formalin) and visceral stimulation (intraperitoneal (i.p.) injection of acetic acid) (Nakagawa et al., 2003). I.pl. formalin markedly increased c-fos mrna in the contralateral basolateral nucleus, but not in the CeA, while i.p. acetic acid induced c-fos mrna bilaterally in the CeA, and to a much lesser degree in the basolateral nucleus. Further studies by the same group using a conditioned place aversion paradigm to assess negative affect associated with these stimuli indicated that discrete lesions of either the basolateral nucleus or CeA abolished conditioned place aversion following somatic stimulation while only CeA lesions reduced this measure following visceral stimulation (Tanimoto et al., 2003). It has been suggested that the basolateral nucleus integrates a variety of sensory inputs and then transmits this processed information to other limbic areas and amygdaloid nuclei, including the CeA (Price, 2002; LeDoux, 2000; Pare et al., 2004). As the primary output nucleus for the amygdala and with its diffuse efferents to forebrain and brainstem structures, activity within the CeA may reflect the sum of neuronal activity produced by different amygdaloid nuclei (Tanimoto et al., 2003). 2.7 Summary Various types of stressors can lead to bidirectional (inhibitory and excitatory) modulation of nociception at numerous anatomical levels and through diverse mechanisms, but clinically, exacerbation of pain is the standard rather than the exception. The overall experience of pain is multidimensional and reflects the sum of sensory, 26

41 cognitive, and emotional input. As a result, psychological variables such as stress and anxiety may trigger or exacerbate an episode of pain, help maintain a pain disorder, and contribute to the distress experienced as a result of a painful disorder. Stress activates a network of physiological systems aimed at restoring and maintaining homeostasis, and these systems are integrated with endogenous pain modulatory systems. People with chronic, functional visceral disorders that are characterized by pain report exacerbation of their pain-related symptoms by stress in experimental and real-life settings, and a stressrelated mechanistic model related to such disorders theorizes that stress-related riskfactors, trigger factors, and perpetuating factors may contribute. Ascending nociceptive pathways, particularly those transmitting sensory information related to the viscera, overlap with emotional arousal pathways and with supraspinal regions known to be involved in the affective experience of pain. The amygdala is one such structure, and its anatomical connectivity and involvement in both stress- and pain-related physiological responses positions it as a likely interface for modulation of stress-induced visceral hypersensitivity. 2.8 Preliminary Conceptual Model Overall, whether pain or analgesia is experienced is a result of the net output derived from two opposing forces: inhibition versus facilitation. The critical balance between the mechanisms of descending inhibition and descending facilitation can be modified as a function of internal and external environmental factors. This modification can be rapid and reversible, or it can be stable and persistent. With regard to SIA and SIH, several broad patterns emerge from a review of literature and are supported by 27

42 empirical evidence. SIA is exhibited primarily in superficial sensory systems in which stimuli can be readily localized and the intensity of which can be accurately encoded. The behaviors associated with SIA are decreased attention to focal stimuli and inhibition of stereotypical reflexive behaviors. In contrast, SIH predominates in deep tissue systems that lack localization and sensory-discriminative characteristics. SIH is associated with facilitation of behaviors; importantly, those requiring integration of multimodal information which rely on polysynaptic transmission through supraspinal sites. Based on the behavioral manifestations of SIA and SIH, a more apropos terminology for these phenomena may be stress-induced inhibition and facilitation, respectively. An intriguing phenomenon relative to stress-induced inhibition and stress-induced facilitation is that they can co-exist. This inherently points to the existence of separate mechanisms to subserve these phenomena. As mentioned in Section 2.5.2, there are multiple pathways through which nociceptive information is transmitted to brainstem and supraspinal central nervous system sites. The classic ascending pathway is the STT, which has two components. The m-stt is a diffuse, polysynaptic pathway that projects to numerous areas within the brainstem, midbrain, thalamus, and subcortical/cortical areas, including emotional-affective brain sites, prior to terminating in somatosensory cortex. In contrast, the l-stt is a monosynaptic pathway limited to one primary relay through the ventroposterolateral thalamus prior to terminating in somatosensory cortex. Stress-induced inhibition of behaviors in response to focused stimuli can be elicited within seconds following stress exposure (Ross and Randich, 1984), suggesting that sensory transmission may be occurring along a direct and hard-wired pathway. In contrast, manifestation of stress-induced facilitation is linked with polysynaptic pathways 28

43 related to affective processes. Stress-induced inhibition of reflex responses can co-occur with stress-induced facilitation of more complex behavior, resulting in a co-phenomenon with adaptive potential. However, stress-induced inhibition is transient and habituates over time, while stress-induced facilitation does not habituate and may, in fact, sensitize over time. While co-occurrence of these phenomena may serve an adaptive purpose, stress-induced facilitation in the absence of inhibition shifts the balance between these opposing phenomena in a direction that may be maladaptive. There are multiple potential points at which divergence of stress-induced phenomena could occur, and the first potential point is in ascending pathways originating in the dorsal horn of the spinal cord (Section 2.5.2). The existence of two subpathways in the classic STT is not a novel idea, and attribution of facilitatory and inhibitory effects of stress to these pathways offers one hypothesis for how these divergent phenomena may co-occur based on existing ideas about the neuroanatomy underlying nociceptive transmission. A schematic to illustrate a proposed mechanism of spinal divergence for inhibitory and facilitatory effects of stress is presented in Figure 2.2. Stress activates numerous neural structures that produce either inhibition or facilitation of ascending nociceptive transmission by acting on different systems of spinal origin. Modulation of transmission from spinal systems may occur through descending inhibitory or facilitatory pathways arising in the brainstem, or via descending modulatory connections acting on different receptor subtypes or inhibitory interneurons. 29

44 Figure 2.1. Stress-related mechanistic model of IC (adapted from Mayer et al., 2001). Stress-related factors may contribute at multiple levels to the development and/or maintenance of IC. Risk factors such as genetic predisposition, pathological stress, and early-in-life experiences may enhance an individual s sensitivity to trigger factors, i.e., psychosocial or physiological (infection, inflammation) stressors, while symptom-related anxiety may act as a perpetuating factor. 30

45 Figure 2.2. Original conceptual model reflecting a mechanism for spinal divergence of inhibition and/or facilitation of nociceptive responses during or following stress. The lateral and medial aspects of the ascending spinothalamic tract (STT) may correspond to ontologically distinct components of the STT referred to as NEO and PALEO, respectively. DH, dorsal horn; M, medial spinal cord; VH, ventral horn. 31

46 CHAPTER 3 EXPERIMENTAL METHODS 3.1 Selection of Species All studies were performed in virgin, female rats. Rats were chosen due to the extensive behavioral, neurochemical, neurophysiological and pharmacological literature associated with them and the close correlation that has been noted between findings in rats and primates (Willis, 1985). Female rats were specifically chosen due to the 9:1 female to male bias in the prevalence of chronic urological conditions that are associated with pain. All rats were obtained from Harlan (Prattville, AL, USA). In some methodological development studies (Section 4.2) Sprague-Dawley (SD) and Wistar Kyoto (WKY) rats weeks of age and weighing g were used. In all other studies, SD rats weighing g were used. All rats were housed in standard cages with food and water available on an ad libitum basis. A 12:12-h light:dark cycle was maintained, where lights were off between 6:00 pm and 6:00 am. There was one week between the time of any animal s arrival and the start of any surgical or experimental procedures. 3.2 Anesthesia For ethical and methodological reasons, rats were anesthetized during all surgical procedures and also during experiments where UBD was administered. Animals were anesthetized using inhalation induction of 5% isoflurane in oxygen delivered via a tight- 32

47 fitting mask, and surgical procedures were performed under deep anesthesia (2.5-3% isoflurane). Following completion of surgery, the anesthetic was reduced to %, where animals maintain vigorous flexion-withdrawal and cardiovascular reflexes in response to noxious stimuli, but do not demonstrate spontaneous movement of evidence of tonic sympathetic nervous system activation (e.g., piloerection) as demonstrated by multiple experiments in our laboratory (Randich et al., 2006a,b; DeBerry et al., 2007a,b; Robbins et al., 2007; Ness et al., 2001). For experiments involving expression of c-fos protein (Section 3.12 and Chapter 7), halothane was used in place of isoflurane to minimize anesthetic-related depression of c-fos expression in the spinal cord (Jinks et al., 2002). Surgical procedures were performed under deep anesthesia (2.5-3% halothane), and anesthesia was reduced to 2% and maintained during the 120 min UBD protocol since animals remained in the stereotaxic apparatus during this time. Clearly, no anesthetics, analgesics, or tranquilizers were used during any stressor paradigm, since the goal was to create a stressed condition and ultimately elucidate the mechanisms underlying stress-induced exacerbation of pain. The use of these agents during stressor exposure would have minimized this effect and negated the experiments. 3.3 Footshock Stressor Overview Electrical current applied to the footpads (footshock) is an established and readily controlled stressor, and factors such as predictability, frequency, intensity, and duration of exposure determine the characteristics of the resultant responses. The degree of 33

48 behavioral control an animal has over a stressor may be one of the most potent variables in determining the impact of an experimental stressor, because those that are perceived as uncontrollable produce a wide range of functional consequences that are not produced by equal intensities of those that are controllable (Brown et al., 2001; Maier and Watkins, 2005). Attempts to categorize animal models of stress have often distinguished between physical (reactive) and psychological (anticipatory) stressors (Zhou et al., 1993; Palermo- Neto et al., 2003; Herman et al., 2003). However, it is clear that physical stressors have psychological consequences and that psychological stressors can have physical consequences (Section 2.3.1). The use of the terminology systemic versus neurogenic presumes that any given stressor may exert both physical and psychological demands, and that the magnitude and relative proportion of these two elements distinguish one stressor from another (Emmert and Herman, 1999). This notion is consistent with the original concept of stress as a nonspecific phenomenon (Selye, 1976), and under this assumption any given stressor has the potential to activate multiple biological systems. Exposure to footshock results in a combination of sensory primary afferent activation and psychological stress produced by the unpredictable nature of the stimulus, and as such, would be considered a mixed or neurogenic model of stress, i.e., one that relies upon somatosensory and/or nociceptive pathways for its initial transduction and that involves a distinct cognitive and/or affective component (Li and Sawchenko, 1998). Many studies in the field of stress biology employ measurement of plasma ACTH and cortisol/corticosterone as indices of stressor-induced physiological activation (Section 2.3.2). Evidence shows that plasma levels of these hormones and CRF mrna 34

49 in the hypothalamus are enhanced following exposure to footshock (Kant et al., 1988; Zelena et al., 2009; Iwasaki-Sekino et al., 2009; Imaki et al., 1991). Acute exposure to footshock also activates cell groups in extra-hypothalamic areas, including the amygdala (Li and Sawchenko, 1998). A classic nociceptive phenomenon related to the exposure of animals to environmental stressors is SIA (Section 2.1). Exposure to footshock produces SIA in somatic/superficial sensory systems, and whether this analgesia is attributable to opioid and/or non-opioid mechanisms is dependent upon the intensity and duration of the footshock stimulus (Menendez et al., 1993; Zou et al., 2001; Jodar et al., 1995; Akil et al., 1976; Hayes et al., 1976). Laboratory studies in rats have shown that a single session of footshock can produce stable and long-lasting sensitization of behavioral, hormonal, and visceral (gastrointestinal) responses to a novel stressor (Stam et al., 2002; Bruijnzeel et al., 1999; van Dijken et al., 1993). Chronic exposure to footshock results in enhanced nociceptive processing related to the urinary bladder (Robbins et al., 2007), but the effects of acute footshock have not previously been examined in the urological system Acute Footshock Procedure Rats exposed to acute intermittent footshock were placed in an operant conditioning chamber (Figure 3.1) enclosed in a sound-attenuating cubicle and received intermittent footshock (15 min, 1.0 ma, 1 sec duration, total of 30 shocks) administered via a parallel rod floor on a variable-interval schedule. Prior to the day of testing, each rat was placed in an operant conditioning chamber for a 15 min accommodation session 35

50 twice daily for three days. The purpose for the accommodation sessions as a control procedure was to reduce stress-related variability due to animal handling and novel environment exposure (Section 4.2.3). Rats in the control condition were treated identically except did not receive any footshock while in the operant conditioning chambers. 3.4 Measurement of Fecal Pellet Output Fecal pellet output is a reliable measure of autonomic system activation and modulation of colonic motility (Tache et al., 2004). Fecal pellets were counted at the termination of each stress or control session. 3.5 Assessment of Somatic Nociceptive Responses Mechanical Testing A set of calibrated von Frey monofilaments was used in tests of mechanical sensitivity. Each rat was allowed to crawl into a glove. Once the rat was still, the tip of a nylon monofilament was applied to the lateral edge of the hindpaw. Mechanical stimuli were increased in a graded manner until the paw was withdrawn. A withdrawal threshold was defined as the mechanical stimulus that elicited a withdrawal reflex in two out of three applications. Paw withdrawal threshold was determined for both hindpaws, and these values were averaged to obtain a single measure of mechanical sensitivity. Baseline measures were taken prior to exposure to the experimental stressor, and poststressor measures were taken immediately following the final stressor exposure. 36

51 3.5.2 Thermal Testing The method described by Hargreaves et al. (1988) was used in tests of thermal sensitivity. Rats were confined within a clear plexiglass cage placed on an elevated piece of glass 3 mm thick. A radiant heat source consisting of a high-intensity projector lamp (50W) was positioned 6 mm under the glass floor. The beam was projected through an 11 x 11 mm 2 aperture and positioned so that it struck the glaborous skin and toe pads of the hindpaw. The latency to withdraw the hindpaw was measured. Three trials were conducted on each hindpaw (3 min inter-trial interval) prior to any stressor exposure and averaged to obtain a mean response latency for each hindpaw. These values were averaged to obtain a single measure of baseline sensitivity. Three trials were similarly conducted on each hindpaw immediately following the final stressor exposure and averaged to obtain a single post-stressor measure of thermal sensitivity. However, in studies involving acute footshock, only the first post-stressor measure is reported due to the rapid attenuation of the acute footshock-induced analgesic effect. 3.6 Noxious Visceral Distension and Response Measures Overview In basic science studies of visceral pain, pressure-controlled distension of the hollow organs is widely accepted as a noxious stimulus and produces pseudoaffective autonomic and motor reflexes (Woodsworth and Sherrington, 1904). One such reflex, the visceromotor response (VMR), is a common endpoint measure of nociception in response to visceral distension in rodents. In a series of studies, Ness and Gebhart (1987a,b; 1988a,b; 1989a,b) have extensively characterized this model using behavioral, 37

52 physiological and pharmacological techniques, and their findings have been supported by various other laboratory studies (c.f., Ide et al., 1997; Gschossmann et al., 2001; Traub et al., 2002; Gaudreau and Plourde, 2003). Specifically, the VMR is spino-bulbo-spinal reflex contraction of abdominal musculature in response to visceral distension, and is operationally defined as a sustained increase in myoelectrical activity quantified as the amplitude of abdominal EMG responses produced by contractions of the external oblique musculature (Ness et al., 2001). This model was initially characterized in studies of gastrointestinal nociception in response to graded pressure distension of the distal colon. CRD initiates passive avoidance learning in the unanesthetized rat, and the rate of learning acquisition increases as a function of distension pressure, presumably due to the increasingly noxious nature of higher distension pressures (Ness et al., 1991). CRD evokes reliable and reproducible VMRs in a stimulus-response dependent fashion, i.e., the threshold to evoke a nociceptive response is significantly greater than that necessary to evoke a nonnociceptive response (Ness and Gebhart, 1988). Passive avoidance behavior and VMRs are enhanced following colonic inflammation and attenuated by pharmacological treatments known to produce analgesia in humans (i.e., morphine) (Ness et al., 1991; (Ness and Gebhart, 1988, 2001). Inhibition of the VMR by analgesics occurs in a dosedependent fashion consistent with their clinical effects on pathological pain. This well-characterized model of gastrointestinal nociception has been adapted into a model for animal studies of urological nociception, although fewer studies overall have utilized UBD as a stimulus in visceral nociceptive studies. Similar to CRD, UBD produces vigorous and reliable increases in abdominal EMG responses in a stimulus- 38

53 response dependent manner (Castroman and Ness, 2001; Ness et al., 2001) (Figure 3.2), and these responses are likewise inhibited by analgesic treatments (intravenous [i.v.] morphine; i.v. and intravesical lidocaine) (Ness et al., 2001). Although there is significant between-animal variability in the VMR, within-animal responses are reliable and reproducible following an initial period of sensitization (Ness et al., 2001) Urinary Bladder Distension and Visceromotor Response Procedure Animals were anesthetized with isoflurane (5% induction, 2-3% maintenance) in oxygen via a tight-fitting mask and a 22-gauge polytetrafluoroethylene angiocatheter (Johnson and Johnson, Arlington TX) was placed into the bladder via the urethra and held in place by a tight suture around the distal urethral orifice. Platinum or silver wire electrodes were inserted into the external oblique musculature immediately superior to the inguinal ligament for recording of abdominal EMG activity. Following surgery, anesthesia was reduced until flexion reflexes were present in the hind limbs but spontaneous escape behaviors were absent (1-1.25% isoflurane). UBD consisted of pressure-controlled air distension of the urinary bladder via the transurethral catheter using a previously described distension control device (Anderson et al., 1987). An inline, pneumatically-linked, low volume pressure transducer was used to monitor intravesical pressure. Contraction of the abdominal and hind limb musculature, recorded as EMG activity, was measured via the electrodes using standard differential amplification and rectification and saved on a computer (Spike 2 software, Cambridge Electronic Design, UK). Approximately 15 min after initial anesthesia induction, three 60 mmhg distensions (3 min inter-trial interval) were administered to establish stable 39

54 responses (Castroman and Ness, 2001) and were followed by graded, constant-pressure air distensions of the urinary bladder (20 sec duration; 1 min inter-trial interval) at ascending pressures at intervals of 10 mmhg. Grass P511 amplifier settings were the following: EMG amplification factor=200; low frequency filter=10hz; high frequency filter=3khz; sample rate=10khz. EMG responses were quantified as: (rectified EMG activity during UBD - rectified baseline EMG prior to UBD) / (rectified baseline EMG). 3.7 Amygdala Lesion Procedure Rats were anesthetized with inhaled isoflurane (5%) in oxygen and then secured in a stereotaxic apparatus using non-puncture ear bars and a bite bar. Anesthesia was reduced to %. Hair along the dorsal surface of the head was clipped and the skin surrounding the surgical area was swabbed with a povidone-iodine solution. A midline skin incision was made along the top of the skull, beginning just behind the eyes and extending approximately 2 cm caudally. The epidermis was retracted and the fascia cleared away, and a flat-skull orientation was set using bregma and lambda as landmarks. A round, 2.0 mm burr attached to a Dremel device was used to drill bilateral holes in the skull at the coordinates of 2.4 mm posterior to bregma and approximately 4.0 mm lateral to the midline, as described by Paxinos and Watson (1986). An electrode (00 insect pin insulated to within 1.0 mm of the tip) was lowered 7.2 mm ventral to the brain surface, so that the tip was located within the central nucleus. Lesions were produced by passage of anodal 0.8 μa direct current for 9 sec. The control surgery procedure was identical except no electric current was passed. Gel foam was placed in the trephine holes and the 40

55 skin incision was closed. Animals were returned to their home cage to recover from the procedure and experiments were performed seven days post-surgery. Rats were administered acetaminophen via drinking water (80 mg/500 ml) daily for three days prior to and two days following surgery. They were also administered carprofen (5 mg/kg, subcutaneous [s.c.]) and buprenorphine (0.1 mg/kg, s.c.) upon completion of surgery, and again in the morning and afternoon on the day following surgery. Upon completion of the experiments, brain tissue was processed with cresyl violet staining, and light microscopy was used to verify lesion sites (Section 3.14). 3.8 Flinch-Jump Threshold Procedure The purpose of the flinch-jump procedure was to determine whether lesions of the amygdala impaired sensory transduction during exposure to acute intermittent footshock. Rats underwent bilateral lesions of the amygdala or control surgery (Section 3.7). Following recovery from surgery, each rat was placed in an operant conditioning chamber enclosed in a sound-attenuating cubicle for a 15 min session of accommodation to the apparatus twice daily over three days. On the fourth day and during the seventh and final session, each rat was presented with a single ascending series of electric shocks (100 ms) delivered via the grid floor. The initial shock intensity was 0.00 ma and subsequent shocks increased in increments of 0.05 ma. The inter-shock interval was 30 sec, but presentation of a shock was dependent upon the rat maintaining all four paws in contact with the grid floor. A flinch response was defined as the lowest shock intensity that elicited a withdrawal of a single forepaw from the grid floor. A jump response was defined as the lower of two successive shock intensities that elicited simultaneous 41

56 withdrawal of both hindpaws from the grid floor. Three trials were conducted for each rat and the thresholds were averaged for statistical analyses. 3.9 Amygdala Stimulation Procedure Rats were anesthetized with inhaled isoflurane (5%) in oxygen and then secured in a stereotaxic apparatus using non-puncture ear bars and a bite bar. Anesthesia was reduced to 2.5-3%. Hair along the dorsal surface of the head was clipped and the skin surrounding the surgical area was swabbed with a povidone-iodine solution. A midline skin incision was made along the top of the skull, beginning just behind the eyes and extending approximately 2 cm caudally. The epidermis was retracted and the fascia cleared away, and a flat-skull orientation was set using bregma and lambda as landmarks. A round, 2.0 mm burr attached to a Dremel device was used to drill bilateral holes in the skull at the coordinates of 2.4 mm posterior to bregma and approximately 4.0 mm lateral to the midline, as described by Paxinos and Watson (1986). For acute amygdala stimulation in UBD studies, a 25-gauge stainless steel microinjector loaded with corticosterone (15 μg/0.5 μl) or cholesterol (15 μg/0.5 μl) was lowered 7.0 mm ventral to the brain surface to the dorsal margin of the CeA. The solution was injected in one hemisphere and the microinjector was removed and re-positioned for injection in the opposite hemisphere. The microinjector was left in place in each hemisphere for one min following drug injection, and approximately two min passed between injections. Rats remained anesthetized and in the stereotaxic apparatus during UBD testing, which began 30 min following the second injection. 42

57 For acute amygdala stimulation in EPM studies, bilateral indwelling guide cannulae were positioned 2.4 mm posterior to bregma and 4.0 mm lateral to the midline and held in place with screws and dental cement. Rats were administered acetaminophen via drinking water (80 mg/500 ml) daily for three days prior to and two days following surgery. They were also administered carprofen (5 mg/kg, s.c.) and buprenorphine (0.1 mg/kg, s.c.) upon completion of surgery, and again in the morning and afternoon on the day following surgery. Rats recovered from the procedure for four days. On the day of testing, corticosterone (15 μg/0.5 μl) or cholesterol (15 μg/0.5 μl) was injected through the guide cannulae 30 min prior to behavioral assessment. The dose of corticosterone that was administered acutely was determined in a preliminary dose-response experiment (Section 4.3.3). Direction of drug placement to the dorsal margin of the CeA in all stimulation studies allowed this region to be bathed in drug without being damaged by the microinjector or guide cannula. Upon completion of all experiments, brain tissue was processed and light microscopy was used to verify the site of drug placement. Injection of dye was used in rats with indwelling cannulae to verify that the cannulae remained patent throughout the recovery period Elevated Plus Maze Overview The EPM is an elevated maze with open and enclosed arms that is used both as a reliable measurement instrument of anxiety-like behavior in animals and for its utility in the detection of anxiolytic effects and screening of benzodiazepine-related compounds. In the formerly stated use, the EPM provides a behavioral assay for the study of 43

58 anatomical sites and mechanisms underlying anxiety behavior, and it has been used as a model of state (unconditioned) anxiety (Korte and DeBoer, 2003). The use of the EPM as an anxiogenic paradigm is based on a procedure by Montgomery (1958) using a Y- shaped maze, where rats consistently explored an enclosed alley more than an open alley when allowed to access the alleys from their home cage. This task was modified into the plus-shaped form of the maze by Handley and Mithani (1984), who described the assessment of anxiety behavior in rats by using the ratio of time spent on the open arms to time spent on the closed arms, relying on the proclivity of rodents toward dark, enclosed spaces (approach) and an unconditioned fear of open spaces (avoidance) (Barnett, 1975). Behavioral responses on the EPM are readily assessable and quantifiable by an independent observer. Conventional measures are spatiotemporal in nature, for example the ratio or percent of time spent in open:closed arms. Assessment of ethological behaviors, such as freezing, rearing, and stretch-attend postures (SAPs), are often determined simultaneously and can reveal differences that are not evident using spatiotemporal scoring alone. A measure of spontaneous motor activity (total number of arm entries or frequency of rearing) is also generally used. The EPM has been evaluated for construct validity and predictive validity. Construct validity represents whether an observable dependent variable such as time spent in an open arm measures an unobservable construct, such as anxiety. Behavioral measures have shown that the EPM is bi-directionally sensitive to anxiolytic drugs (particularly benzodiazepines) (Handley and Mithani, 1984; Lister, 1987; Pellow et al., 1985) as well as compounds that are anxiogenic in man (Lister, 1987; Pellow et al., 1985; 44

59 Pellow and File, 1986). With regard to predictive validity, or the extent to which a dependent measure predicts behavior on a related measure, increased open arm activity on the EPM occurs in rodents that also demonstrate increased central square entries in an open field test for anxiety-related behavior (Frye et al., 2000), and plasma corticosterone is typically increased with open arm exposure (e.g., File et al., 1994) and is positively correlated with risk assessment behavior, i.e., (SAPs), in the EPM (Mikics et al., 2005; Albrechet-Souza et al., 2007) Elevated Plus Maze Procedure The elevated maze was constructed of wood and consisted of one open arm (50 x 10 cm) and one enclosed arm (50 x 10 x 27 cm) with a junction area (central area) of 10 x 10 cm, elevated to a height of 52 cm above the floor. During assessment of anxiety-like behaviors, rats were placed facing the closed arm in the central area and their behavior was video recorded for 5 min. Spatiotemporal and ethological behaviors were quantified. The spatiotemporal measure consisted of % of time spent in the open arm, and ethological measures included frequency of and/or time spent rearing and freezing. Quantification of spatiotemporal and ethological behaviors was performed by the experimentor at the time of testing and again in a blinded fashion using the recorded video with an inter-rating reliability of >90%. The EPM was also employed as a behavioral stressor in studies involving acute delivery of corticosterone to the CeA. In these studies, rats remained on the EPM for a total of 15 min, i.e., for an additional 10 min following cessation of the period when spatiotemporal and ethological behaviors were assessed. 45

60 3.11 Enzyme-Linked Immunosorbent Assay (ELISA) Procedure Fifteen min following acute application of either corticosterone or cholesterol to the CeA (Section 3.9), or 15 min following EPM testing (Section 3.10), blood was collected from anesthetized rats via cardiac puncture into heparinized vials and centrifuged at 14,000 g for 15 min. Plasma was extracted and plasma samples (50 μl) were aliquotted onto a 96-well plate in duplicate. Corticosterone acetylcholinesterase tracer (50 μl) and corticosterone antiserum (50 μl) were added to each well. The plate was covered and incubated for two hrs at room temperature on an orbital shaker, then emptied and rinsed five times with wash buffer. Ellman s reagent (200 μl) was added to each well and corticosterone acetylcholinesterase tracer (5 μl) was added to the Total Activity wells. The plate was covered and developed in the dark on an orbital shaker for min. Optical densities (OD) at 412 nm were obtained using a plate reader (BioRad Laboratories, Hercules, CA). A standard curve was plotted and the concentration of corticosterone in each sample was calculated from the curve based on its OD value c-fos Western Blotting Overview The proto-oncogene c-fos is a member of a family of immediate early genes and is frequently used to monitor cellular activation within the nervous system. Neuronal excitation leads to a rapid and transient induction of c-fos following voltage-gated calcium entry into the cell (Morgan and Curran, 1986). c-fos codes for the transcription factor c-fos, a component of the AP-1 transcription complex that has been implicated as a key regulator of biological processes such as cell proliferation, differentiation, 46

61 apoptosis and response to stress (Piechaczyk and Blanchard, 1994). Various types of peripheral stimulation of primary sensory neurons in the rat induce c-fos expression in postsynaptic neurons of the spinal dorsal horn (Hunt et al., 1987). Upon activation, c-fos dimerizes with a member of the jun family of immediate early genes, and mitogenactivated protein kinase pathways regulate both the amounts and trans-activating capacities of the Fos and Jun component proteins of the AP-1 complex in a stimulusspecific manner Western Blot Procedure Four groups of rats were used in these experiments to examine the effects of CeA stimulation and bladder distension on spinal c-fos expression. Rats were administered either corticosterone or cholesterol and either UBD or no UBD. Group designations represent drug treatment with CORT and CHOL for corticosterone and cholesterol, respectively. Following bilateral drug injection (Section 3.9), anesthesia was reduced to 2% and half of the rats in each group were administered a series of UBD (Section 3.6.2) of 60 mmhg (20 sec duration, 3 min inter-trial interval) over the course of 120 min (total of 60 distensions). The remaining rats were left anesthetized for 120 min but were not administered UBD. At the termination of the distension/control procedures, rats were deeply anesthetized and spinal cords were hydraulically extruded. Lumbar segments were isolated and immediately frozen in liquid nitrogen and stored at -20 C until spinal cord tissue was homogenized in 50 mm Tris buffer. The homogenate was centrifuged at 14,000 g for 15 min at 4 C and the supernatant was decanted and stored at -20 C until 47

62 the time of use. Brains were removed and processed for histological verification of injection site (Section 3.14). The concentration of protein in the homogenate was measured using a bicinchoninic acid protein assay (Pierce Protein Research Products, Rockford, IL, USA). An equal amount of protein from each sample (70 µg) was fractionated by 10% (w/v) sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred onto a nitrocellulose membrane (Bio-Rad Laboratories, Hercules, CA, USA). Membranes were washed (three five min in 1% Tween-20 in Tris-Buffered Saline; TBST) and nonspecific membrane binding was blocked using 5% (w/v) non-fat dry milk in TBST for 60 min at room temperature. Membranes were incubated overnight at 4 C with a primary polycolonal affinity purified antibody (1:200 in 5% non-fat milk/tbst) raised against a peptide mapping within an internal region of c-fos (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA). Membranes were washed (three five min in TBST) to remove unbound primary antibody and incubated for 60 min at room temperature with a goat anti-rabbit IgG secondary antibody conjugated with horseradish peroxidase (1:6000 in 5% non-fat milk/tbst; Santa Cruz, San Francisco, CA, USA) and washed again (four five min in TBST). A chemiluminescent detection method (Amersham ECL Western Blotting Detection Reagents, GE Healthcare, Buckinghamshire, UK or SuperSignal West Pico Chemiluminescent Substrate, Thermo Scientific Life Science Research Products, Rockford, IL, USA) was used and membranes were exposed to autoradiography film (HyBlot CL; Denville Scientific, Inc., Metuchen, NJ, USA). The membranes were washed (three five min in TBST), stripped (Restore Western Blot Stripping Buffer, Thermo Scientific Life Science Research Products, Rockford, IL, USA), and washed 48

63 again (three five min in TBST). Each membrane was re-probed using the protocol described above with a primary antibody for β-actin (1:75000 in 5% non-fat milk/tbst) and a donkey anti-mouse IgG secondary antibody conjugated with horseradish peroxidase (1:5000 in 5% non-fat milk/tbst). A peptide neutralization protocol was performed on one membrane as a control procedure. A c-fos blocking peptide (sc-253 P, Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA) was incubated overnight at 4 C with the primary antibody. Then this solution was applied to the membrane and the above protocol was followed Statistical Analyses Statistical Significance Statistical significance for all analyses was set at p 0.05, and Holm s correction (Holm, 1979) was used to maintain family-wise α for each set of individual group comparisons Somatic Nociceptive Threshold Measures Quantitative comparisons of group mean mechanical data were performed using non-parametric Mann-Whitney U tests, and comparisons of group mean thermal data were performed using t-tests Visceromotor Response Measures For Chapters 5-7, quantitative comparisons of group mean abdominal EMG responses were performed using repeated-measures ANOVA on distension pressures of 49

64 40-80 mmhg, which are within the noxious range (Ness et al., 1991). Previously published studies related to urological hypersensitivity conditions have similarly analyzed pressures within a noxious range (i.e., Randich et al., 2006). Post-hoc effects were assessed by simple contrast analyses of group data at each distension pressure within the same range (40-80 mmhg) Elevated Plus Maze Measures Quantitative comparisons of spatiotemporal and ethological measures between two groups were made using independent samples t-tests. Comparisons for > two groups were made using ANOVA followed by simple contrast analyses of group data Flinch-Jump Thresholds Flinch and jump thresholds were compared using ANOVA Enzyme-Linked Immunosorbent Assay Analyses For comparisons of plasma corticosterone levels, a standard curve was plotted and the concentration of corticosterone in each sample was calculated from the curve based on its OD value. Group mean comparisons of sample concentrations between two groups were performed using independent samples t-tests. For similar comparisons of > two groups, ANOVA was used followed by simple contrast analyses. 50

65 Western Blot Analyses All autoradiography films were converted to digital images for analyses and optical densitometry was performed using NIH ImageJ. The immunoreactive bands of interest were determined based on molecular weight (Kaleidescope Precision Plus Protein Standards, Bio-Rad Laboratories, Hercules, CA, USA) and included a band corresponding to c-fos (62 kd) and a band corresponding to β-actin (43 kd), a proteinloading control that is ubiquitously expressed in all eukaryotic cells. First, the OD of each c-fos band was calculated. Then, a corrected OD value was calculated by subtracting an average of the OD values of the background regions above and below each c-fos band. The same procedure was performed to obtain corrected OD values for bands corresponding to ß-actin. A normalized value was obtained to account for any error in protein loading by calculating a ratio of the corrected c-fos value to the corrected ß-actin value. Finally, each normalized value was divided by a control value. The control value was an average of the normalized values from two common samples that were run on every gel. This final calculation was performed to allow for between-groups comparisons of values that were obtained from samples run on separate gels Histological Analyses Central Nucleus Lesion Sites Brain tissue from rats with CeA lesions was harvested and processed for cresyl violet staining. The locations of lesions were determined using light microscopy in a blinded fashion by two independent observers. Micrographs of brain tissue were 51

66 matched to templates (Paxinos and Watson, 1986) and the obliterated areas were assessed as a function of total CeA area. Lesions ranged from % of total CeA area (Mean = 81.41%, SEM = 1.66%. Data from cases without bilateral CeA lesions were not included in statistical analyses Central Nucleus Stimulation Sites In studies involving microinjection of drug to the CeA, the placement of microinjection cannulae tracks was determined histologically using light microscopy. Cases were included in data analyses if both cannulae tracks were visible within the appropriate plane (2.40 mm posterior to bregma; Paxinos and Watson, 1986) with cannulae tips located dorsal to the CeA and no obvious damage to the CeA due to cannulae placement. Data from cases that did not meet this criterion were not included in statistical analyses. 52

67 Figure 3.1. Footshock was administered on a variable-interval schedule (1 ma, 1 sec, 30 shocks in 15 min) through the wire-grid floor of an operant conditioning chamber (below) enclosed in a sound-attenuating cubicle. 53

68 Figure 3.2. Urinary bladder nociception was indexed by the amplitude of EMG responses resulting from contractions of abdominal oblique musculature during 20 sec periods of graded UBD. UBD produces vigorous increases in EMG responses, arterial blood pressure (AP), and heart rate (HR). Adapted from Ness et al.,

69 CHAPTER 4 METHODOLOGICAL DEVELOPMENT 4.1 Purpose Prior to undertaking the current set of studies, several questions that could not be answered through a literature review required investigation. Specifically, it was unclear precisely what type, intensity, and duration of stressor were optimal, and it was unknown what rat strain would work best for the stressor characteristics chosen. This chapter describes the preliminary experiments and data that helped answer these questions. 4.2 Selection of Stress Paradigm and Rat Strain Overview The WKY strain of rats has been proposed as a putative animal model of stress vulnerability (Maillot et al., 2000; Tache et al., 1999). Compared to other rat strains, WKY rats exhibit enhanced neurochemical, behavioral, and physiological sensitivity to stress, such as an exaggerated HPA axis response, attenuated brain noradrenergic response, reduced locomotor activity in behavioral assessments of anxiety-like behavior, and a heightened response to adverse environments (Gentsch et al., 1987; Pardon et al., 2003). WKY rats also exhibit greater susceptibility to gastric ulceration following stress (Pare, 1992; Pare and Redei, 1993), suggesting potential relevance to gastrointestinal pain disorders characterized by pain and heightened stress responses, such as IBS. Indeed, a study in the year 2000 of visceral nociceptive responses demonstrated that WKY rats 55

70 displayed more robust VMRs to CRD compared to two rat strains (SD and Fisher-344) considered low to moderate anxiety strains, as determined by acoustic startle reflex and open-arm exploration on the EPM (Gunter et al., 2000). Following this study in 2005, Mayer and colleagues further demonstrated that repeated exposure to water avoidance (WA) stress, a pure psychological stressor, produced visceral hyperalgesia of the gastrointestinal system in high-anxiety WKY rats (Bradesi et al., 2005). Based on these collective findings, our original studies of stress-induced visceral hyperalgesia sought to determine whether WA stress produced similar changes in the urologic system as in the gastrointestinal system, and whether development of stress-induced visceral hypersensitivity was, in fact, dependent upon rat strain. In three preliminary studies, behavioral and physiological responses to two different stress paradigms were characterized Methodological Study 1: Acute and Chronic Water Avoidance in High- and Low- Anxiety Rat Strains Study 1 was conducted to determine the effects of acute (1 day) versus chronic (10 day) exposure to WA stress on bladder hypersensitivity, and whether there are strain differences (WKY versus SD) in WA stress-induced bladder hypersensitivity. WA exposure per se is considered a purely psychological stressor where no sensory stimulus is presented. Chronic WA exposure has been shown to increase anxiety-like behavior and colonic visceral nociception in the stress-sensitive WKY strain of rats (Bradesi et al., 2005), but prior to our studies there were no reports on the effect of acute WA exposure 56

71 or on WA stress-related effects in SD rats. The data and figures presented in this section have been previously published (Robbins et al., 2007). WA exposure took place between 9:00 am and 12:00 pm. Rats were placed on a pedestal (10 x 8 x 8 cm 3 ) affixed to the center of the floor of a plexiglass tank (45 x 25 x 25 cm 3 ). In the stress condition, the tank was filled with water to within 1 cm of the top of the pedestal. In the control condition, the tank was left waterless. Rats were placed on the pedestal for one hr/daily for ten days in the chronic paradigm, and for a single one hour session in the acute paradigm. Somatic nociceptive threshold testing (Section 3.5) was performed in each rat prior to the first (or only) WA session, and again after the final (or only) session. Abdominal EMG responses to UBD (Section 3.6.2) were measured approximately 15 min following the second assessment of somatic thresholds. Abdominal EMG responses to UBD following acute WA stress or WA control exposure were not significantly different from each other in either strain of rats (Figure 4.1) (WKY: F(1,18)=0.301, p=0.590; SD: F(1,25)=1.643, p=0.212). Chronic WA stress produced urinary bladder hypersensitivity in anxiety-prone WKY rats, manifested as significantly more vigorous abdominal EMG responses to UBD relative to the responses obtained from rats in the control condition (Figure 4.2) [F(1,16)=5.351, p<.05]. Post-hoc tests revealed significant group differences at UBD pressures of mmhg (p values<.05). Visceral hypersensitivity was not evident in the low/moderate-anxiety SD rats following 10 days of WA stress (Figure 4.2) [F(1,9)=0.001, p=0.971]. Stress-induced analgesia to somatic stimuli was observed after acute WA stress exposure, as shown in Figure 4.3. The mean change in mechanical paw withdrawal thresholds of WKY and SD rats in the WA stress condition was significantly greater than 57

72 that from rats in the corresponding WA control conditions (WKY: Mann-Whitney U=85.000, p<.01; SD: Mann-Whitney U= , p<.01). Baseline mechanical withdrawal thresholds between rats in the stress and control groups did not differ (WKY: Mann-Whitney U=73.00, p=0.953; SD: Mann-Whitney U= , p=0.415). The mean change in thermal paw withdrawal thresholds reflected stress-induced thermal analgesia in WKY rats, but not in SD rats (WKY: t(18)=3.090, p<.01; SD: t(30)=1.301, p=0.203), and baseline thermal withdrawal thresholds between stress and control rats did not significantly differ (WKY: t(18)=-1.266, p=0.219; SD: t(30)=1.891, p=0.069). Following chronic WA stress, no significant changes in group mean withdrawal thresholds for WKY or SD rats were observed in mechanical (WKY: Mann-Whitney U=44.000, p=0.709; SD: Mann-Whitney U=12.000, p=0.580) or thermal (WKY: t(16)=0.647, p=0.527; SD: t(9)=0.409, p=0.692) somatic responses. These results are depicted in Figure 4.4. Baseline thresholds between stress and control groups did not differ. There was a significant effect of acute WA stress on fecal pellet output, where stressed rats of both strains had greater fecal pellet output than their control counterparts, which had no fecal pellet output at all (WKY: Mann-Whitney U= , p<.05; SD: Mann-Whitney U=37.500, p<.01). These findings are presented in Figure 4.5. In the chronic paradigm, both strains of WA stress rats had enhanced fecal pellet output compared to their WA control counterparts on Day 1 and, as in the acute paradigm, control rats had no fecal pellet output (WKY: Mann-Whitney U=77.500, p<.05; SD: Mann-Whitney U=72.000, p<.01). On Day 10, neither strain of stressed rats had enhanced fecal pellet output compared to their control counterparts (WKY: Mann-Whitney U=68.500, p=0.084; SD: Mann-Whitney U=45.000, p=0.317). Furthermore, both groups of WKY rats (stress and 58

73 control) had significantly reduced fecal pellet output on Day 10 as compared to Day 1 (WA stress: Wilcoxon Z=-2.557, p<.05; WA control: Wilcoxon Z=-2.023, p<.05). In SD rats, the WA stress group, but not the WA control group, had significantly reduced fecal pellet output on Day 10 as compared to Day 1 (WA stress: Wilcoxon Z=-2.043, p<.05; WA control: Wilcoxon Z=0.000, p=1.000). These findings are presented in Figure 4.5. These results demonstrate that chronic, but not acute, exposure to a stressor that is purely psychological in nature augments urinary bladder nociceptive responses in WKY but not SD rats. The findings in WKY rats are consistent with those reported in the aforementioned study of the gastrointestinal system. However, that study did not examine WA-induced visceral hypersensitivity in SD rats. It was concluded from the results of this study that the divergent effect of WA stress in WKY and SD rats may be attributable to the stress-prone nature of each strain. It was evident that while WKY rats may be appropriate for studies utilizing a purely psychological stressor in a chronic setting, the augmented bladder nociceptive sensitivity observed in the control group as well as the stress group negated its usefulness for studies of acute stress-induced visceral hypersensitivity. To the contrary, acute stress-induced effects in response to a mild, psychological stressor were absent in SD rats, also negating its usefulness. The next study presents a series of follow-up experiments utilizing a mixed psychological/physiological (neurogenic) stress paradigm under the assumption that a more intense stressor would be necessary to evoke robust stress-induced nociceptive effects in SD rats. 59

74 4.2.3 Methodological Study 2: Acute Footshock in Sprague-Dawley Rats Imaki et al. (1991) described a chronic intermittent footshock paradigm that produced robust activation of the HPA axis as evidenced by upregulation of CRF mrna in the brain. Using this paradigm, our laboratory has demonstrated that chronic intermittent footshock can magnify physiological responses in a model of urinary bladder pain (Robbins and Ness, 2008) and produces an increase in the stress hormone corticosterone in blood plasma (Figure 4.6). An adaptation of this chronic intermittent footshock paradigm was used in the current studies of acute stress. In our initial studies of acute footshock stress, rats were simply exposed to a single session of footshock or no footshock. A subsequent study was performed in which all rats underwent multiple accommodation sessions to become habituated to the footshock apparatus. In study 2, SD rats were exposed to a single 15 min session of intermittent footshock. In the stress condition, rats were placed in an operant conditioning chamber enclosed in a sound-attenuating cubicle, and intermittent footshock (30 shocks/day over a 15 min period, 1.0 ma, 1 sec duration) was administered via a parallel rod floor under a variable-interval schedule. Rats in the control condition were treated in an identical manner but received no footshock while in the operant conditioning chamber. Approximately 15 min following the administration of footshock (or no footshock), rats were instrumented for assessment of bladder sensitivity and abdominal EMG responses to graded UBD were measured. A measure of fecal pellet output was taken at the end of each footshock or no footshock exposure as an estimation of autonomic system modulation of colonic motility. The original groups of stress and control rats did not undergo any habituation to the operant conditioning chamber prior to the day of testing, 60

75 and underwent thermal somatic testing before and after footshock/control exposure. A second set of stress and control rats were also tested but were accommodated to the operant conditioning chambers for six 15 min accommodation sessions prior to the day of testing. Additional groups were treated identically and used to assess somatic thermal response thresholds and plasma corticosterone concentrations. In rats without any prior accommodation to the operant conditioning chambers, there was no significant effect of acute footshock on urinary bladder nociceptive responses [F(1,13)=1.936, p=0.187]. When rats were accommodated to the apparatus prior to the day of testing to control for any confounding influence of a novel environment, repeated-measures two-way ANOVA revealed a significant main effect of footshock (footshock > control), and post-hoc tests revealed significant effects at pressures of mmhg. There was also a significant main effect of bladder distension (higher pressures > lower pressures), but no significant interaction between footshock and bladder distension. These results are presented in Table 4-1 and Figure 4.7. As was seen in bladder nociceptive testing, acute footshock had no effect on thermal nociceptive thresholds in rats without any accommodation to the stress apparatus [t(12)=-0.521, p=0.612]. However, in rats that underwent previous accommodation to the operant conditioning chambers, there was a significant stress-induced analgesic effect on thermal nociceptive responses (Table 4-1, Figure 4.7). Fecal pellet output data were collected from rats that underwent accommodation sessions prior to the day of testing. There was a significant effect of footshock on fecal pellet output (footshock > control: Mann-Whitney U=24.000, p<0.01). There was no fecal pellet output from any rats in the control condition. These results are presented in Figure 4.8. Following six accommodation 61

76 sessions, rats exposed to acute footshock had significantly elevated plasma corticosterone concentrations compared to their control counterparts [t(9)=4.033, p<.01] as seen in Figure 4.8. The results of this study indicated that acute exposure to footshock stress, a neurogenic stressor, augments urinary bladder nociceptive responses in low/moderate anxiety SD rats when rats are first accommodated to the conditioning apparatus. Exposure to a novel environment is considered a behavioral stressor and can induce stress-related nociceptive changes (Hayes et al., 1976). Exposure to the operant conditioning chamber on the day of testing without prior accommodation produced responses in the control group, presumably due to exposure to the novel environment. Following accommodation to the apparatus, the observed effects are more likely attributable to the introduction of the footshock stimulus and the unpredictable nature in which it is delivered. The presence of SIA along with elevated plasma corticosterone concentrations following acute footshock exposure validates this model as a physiological stress paradigm. Studies by others have reported similar findings (Kant et al., 1988; Zelena et al., 2009; Iwasaki-Sekino et al., 2009). 4.3 Preliminary Studies Related to Amygdala Overview The CeA is rich with glucocorticoid and mineralocorticoid receptors (Raul and de Kloet, 1985; Sapolsky et al., 1983), which bind the endogenous ligands corticosterone and aldosterone. In 2000, Shepard et al. demonstrated that direct administration of corticosterone to the amygdala via a sustained-release micropellet resulted in 62

77 upregulation of CRF mrna in the CeA. This provided evidence that CRF-containing cells within the CeA were activated. The same study by Shepard further demonstrated that stereotaxic delivery of exogenous glucocorticoids to the amygdala increased indices of anxiety, and this was not a non-specific effect, because the same dose of corticosterone applied to the CA3 hippocampal region did not produce an effect. Subsequent studies by others have shown that chronic stimulation of the CeA with either corticosterone or aldosterone produces enhanced visceral nociceptive responses to CRD and enhanced spinal neuronal responses to CRD and UBD (Qin et al., 2003a,b,c; Myers et al. 2005,2007). Based on these findings, two preliminary studies were conducted to help develop an acute amygdala stimulation model. It is important to note that direct acute administration of corticosterone to the CeA in the present studies was intended as a method for chemically stimulating this area and not necessarily as a model of acute stress Methodological Study 3: Chronic Amygdala Stimulation Enhances Visceromotor Responses to Urinary Bladder Distension With the ultimate goal of modifying the aforementioned chronic stimulation paradigm into an acute stimulation paradigm, a study was first performed to determine whether chronic corticosteroid application onto the amygdala also augmented visceral nociceptive responses to UBD. Briefly, rats were prepared for stereotaxic surgery and a 25-gauge stainless steel microinjector loaded with a 30 μg micropellet of corticosterone, aldosterone or cholesterol was lowered 7.0 mm ventral to the brain surface toward the dorsal margin of 63

78 the CeA. The micropellet was expelled and the microinjector removed. Surgery was completed and rats were returned to their home cage to recover from the procedure. UBD testing was performed seven days post-surgery. Compared to delivery of cholesterol, chronic delivery of both corticosterone and aldosterone resulted in enhanced abdominal EMG responses to UBD (Figure 4.9). A repeated-measures ANOVA at noxious distension pressures (40-80 mmhg) including data collected from rats administered either cholesterol, corticosterone, or aldosterone revealed a significant main effect of drug (corticosterone and aldosterone > cholesterol) and a significant main effect of distension pressure (higher pressures > lower pressures), with no significant interaction. Post-hoc comparisons revealed a significant difference between aldosterone and cholesterol at pressures of 40, 70, and 80 mmhg (Table 4-2). A previously published study utilizing this technique for drug administration to the CeA demonstrated an area of drug spread of 750 μm from the micropellet implantation site at five days post-implantation (Shepard et al., 2000). The current study did not assess drug spread, but it is presumed either that the drug is still present at seven days postimplantation or that the effects of the drug are sustained at least until this timepoint, since visceral nociceptive responses were significantly enhanced at seven days postimplantation in other studies (Qin et al., 2003b; Myers et al., 2005; Myers and Greenwood-Van Meerveld, 2007; Myers et al., 2007; Myers and Greenwood-Van Meerveld, 2009) and in the present study. This study confirms previous findings in the gastrointestinal system that chronic application of corticosteroids onto the CeA enhances visceral nociceptive processing. It also provides a behavioral nociceptive correlate to the enhanced lumbosacral dorsal horn 64

79 neuronal responses that were observed by Qin et al. (2003c) in response to UBD in the presence of corticosteroids on the CeA Methodological Study 4: Dose-Response Study of Drugs onto the Amygdala After confirming that urological nociceptive responses were indeed influenced by chronic application of exogenous corticosteroids onto the amygdala, a small doseresponse study was performed to determine an appropriate drug dose for acute application of corticosterone. Briefly, rats were instrumented with a transurethral catheter and abdominal EMG electrodes for UBD testing and then prepared for stereotaxic surgery. A stainless steel microinjector loaded with a soluble corticosterone complex (5 μg or 15 μg /0.5 μl) or cholesterol (15 μg /0.5 μl) was lowered 7.0 mm ventral to the brain surface toward the dorsal margin of the CeA. The solution was injected in one hemisphere, and after one min the microinjector was removed and resituated onto the opposite hemisphere for drug injection, left in place for one min following injection and then removed. UBD testing began 30 min following drug injection. As depicted in Figure 4.9, a repeated-measures ANOVA revealed a significant effect of corticosterone on VMRs to distension pressures within the noxious range (40-80 mmhg) at the 15 μg dose [F(1,6)=3.825, p<.05]. Post-hoc tests using Holm s correction (1979) to maintain family-wise α were significant at pressures of 40, 50, 70, and 80 mmhg. Significant results were not observed with the 5 μg dose [F(1,6)=0.315, p=0.595]. Although this study was performed in a preliminary fashion, the results obtained with n=4/group yielded results sufficient to determine an effective acute dose. 65

80 The low dose (5 μg) in the current study was approximated by dividing the chronic dose (30 μg) by the number of days it remained in place (seven). The high dose (15 μg) was chosen simply because it is intermediate to the low dose that did not produce significant results and the chronic dose. 4.4 Summary Several questions related to experimental methodology required investigation prior to undertaking the Specific Aims for testing the subhypotheses related to the overall general hypothesis proposed in Chapter 1. First, the studies presented in this chapter provided the preliminary evidence necessary for deciding which rat strain, experimental stressor, and duration of exposure to that stressor would be used in Specific Aims 1 and 2. Confirmatory physiological and behavioral evidence (plasma corticosterone concentration, stress-induced somatic analgesia, fecal pellet output) that acute footshock (30 shocks delivered over a period of 15 min, 1 ma, 1 sec duration on a variable-interval schedule) can be considered a valid experimental stressor was demonstrated. It was also found that exposing rats to acute footshock without first accommodating them to the footshock apparatus presents the confounding variable of exposure to a novel environment, which itself is considered an experimental stressor. Second, the studies confirmed that chronic application of glucocorticoids to the CeA modulates visceral nociception and provided novel evidence that this model of CeA stimulation is effective for modulation of nociceptive responses in the urological system. It was also determined that corticosterone produced a stronger effect than aldosterone, and that acute application 66

81 at the dose of 15 μg is more effective than a dose of 5 μg. These findings were necessary to perform the studies addressed in Specific Aims 3, 4, and Restatement of Hypotheses and Specific Aims The general hypothesis that guided the current set of studies was as follows: The amygdala plays a role in the activation of a spinal mechanism of acute footshock-induced urinary bladder hypersensitivity in rats. Specific sub-hypotheses that refined and developed this general hypothesis were the following: 1. Lesions of the CeA alter the expression of bladder hyperalgesia and physiological/behavioral indices of stress induced by acute exposure to footshock. 2. Acute corticosterone stimulation of the CeA induces visceral nociceptive phenomena and alterations in stress-related physiological and behavioral responses similar to that seen following acute exposure to footshock. 3. Corticosterone stimulation of the CeA enhances spinal c-fos expression produced by UBD. The Specific Aims used to address these sub-hypotheses were the following: Specific Aim 1. The hypothesis that CeA lesions attenuate acute footshock-induced bladder hypersensitivity will be tested. Bilateral lesions will be performed, and UBD-evoked abdominal EMG responses following acute exposure to footshock will be quantified. Flinch and jump thresholds in response to footshock will be assessed as a control measure. 67

82 Specific Aim 2. The hypothesis that CeA lesions attenuate physiological and behavioral indices of stress following acute footshock exposure will be tested. Bilateral lesions will be performed. Acute footshock-induced plasma corticosterone and anxiety-like behavior on an EPM will be measured. Specific Aim 3. The hypothesis that acute corticosterone stimulation of the CeA enhances bladder nociceptive responses will be tested. Bilateral application of corticosterone to the CeA will be performed, and UBD-evoked abdominal EMG responses will be quantified. Specific Aim 4. The hypothesis that acute corticosterone stimulation of the CeA enhances the expression of physiological and behavioral indices of stress will be tested. Bilateral application of corticosterone to the CeA will be performed, and anxiety-like behavior will be measured on an EPM. Plasma corticosterone concentration will be measured at baseline and following EPM testing. Specific Aim 5. The hypothesis that corticosterone stimulation of the CeA alters c-fos expression in the spinal cord in response to UBD will be tested. Bilateral application of corticosterone to the CeA will be performed, and Western blots assessing c-fos expression under conditions of UBD and no UBD will be quantified. Chapter 5 will address the experiments of Specific Aims 1 and 2; Chapter 6 will address the experiments of Specific Aims 3 and 4; Chapter 7 will address the experiments of Specific Aim 5. 68

83 Figure 4.1. Group mean EMG responses to UBD (10-50 mmhg) in SD and WKY rats. Rats were exposed to WA stress (open circles) or the WA control condition (solid circles) for 1 hr prior to UBD testing. Sample sizes ranged from 12-16/group. There was no significant difference between the acute WA stress and control conditions in either SD (upper panel) or WKY (lower panel) rats. 69

84 Figure 4.2. Group mean EMG responses to UBD (10-50 mmhg) in SD and WKY rats. Rats were exposed to WA stress (open circles) or the WA control condition (solid circles) for 1 hr/daily for 10 days prior to UBD testing. Sample sizes ranged from 5-10/group. * indicates p <.05 that the chronic WA stress condition > the chronic WA control condition in WKY rats at UBD pressures of mmhg (lower panel). There was no significant difference between the chronic WA stress and control conditions in SD rats (upper panel). 70

85 Figure 4.3. Group mean change scores for somatic mechanical and thermal withdrawal thresholds in SD and WKY rats. Baseline withdrawal thresholds were measured, then rats were exposed to WA stress (open bars) or the WA control condition (solid bars) and withdrawal thresholds were measured again. Sample sizes ranged from 5-16/group. * indicates p <.05 that the acute WA stress condition > the acute WA control condition for mechanical withdrawal change scores in SD rats (upper left panel) and for mechanical and thermal withdrawal change scores in WKY rats (lower panels). There was no significant difference in thermal withdrawal change scores between acute WA stress and control conditions in SD rats (upper right panel). Mechanical or thermal baseline measures between acute WA stress groups and their respective WA control groups were not statistically different. 71

86 Figure 4.4. Group mean change scores for somatic mechanical and thermal paw withdrawal thresholds in SD and WKY rats. Baseline withdrawal thresholds were measured on Day 0, then rats were exposed to chronic WA stress (open bars) or the WA control condition (solid bars) 1 hr/day for 10 days and withdrawal thresholds were measured again. Sample sizes ranged from 5-10/group. There was no significant difference in mechanical (upper panel) or thermal (lower panel) baseline measures or post-stress measures on Day 10 between chronic WA stress groups and their WA control counterparts. 72

87 Figure 4.5. Group mean number of fecal pellets expelled during acute and chronic WA stress (open circles) or the WA control condition (solid circles) by WKY and SD rats. Rats were exposed to WA stress or the WA control condition for 1 hr (acute) or for 1 hr/daily for 10 days (chronic) and fecal output was measured. Sample sizes ranged from 12-16/group for acute studies and 5-10/group for chronic studies. * indicates p <.05 within respective strains that the acute WA stress condition > the acute WA control condition, and that the chronic WA stress condition > the chronic WA control condition. 73

88 Figure 4.6. Group mean EMG responses to UBD (10-60 mmhg) and plasma corticosterone concentration following chronic footshock (1 ma, 1 sec, 30 shocks in 15 min) (open circles) or no footshock (solid circles) once daily for 7 days prior to UBD testing. Blood was drawn for measurement of plasma corticosterone concentration from separate groups of rats treated identically immediately following the final footshock/no footshock exposure. Sample sizes ranged from 10-11/group for UBD testing and 6-7/group for measurement of plasma corticosterone concentration. ** indicates p <.01 that the chronic footshock group had greater UBD-evoked EMG responses than the chronic no footshock group at pressures of mmhg (upper right panel). * indicates p <.05 that the chronic footshock group had significantly enhanced plasma corticosterone compared to the chronic no footshock group (lower panel). Typical examples of EMG responses to a 50 mmhg UBD stimulus are presented from a rat exposed to chronic footshock and chronic no footshock (upper left panel). Adapted from Robbins et al.,

89 Figure 4.7. Group mean EMG responses to UBD (10-80 mmhg) (upper panel) and somatic thermal withdrawal thresholds (lower panel). Rats were exposed to footshock (1 ma, 1 sec, 30 shocks in 15 min) (open circles) or no footshock (solid circles) once following six accommodation sessions and prior to UBD testing. Thermal paw withdrawal thresholds were measured in separate groups of rats at baseline (pre) and again following the same acute footshock (open bars) or no footshock (solid bars) procedures (post). All sample sizes equaled 7/group. * indicates p <.05 that acute footshock > acute no footshock at UBD pressures of mmhg (pressures < 40 were not analyzed). There was no significant difference between somatic thermal pre and post measures in the acute no footshock group. # indicates p <.05 that post > pre in the acute footshock group. 75

90 Figure 4.8. Group mean plasma corticosterone concentration (upper panel) and fecal pellet output (lower panel). Rats were exposed to footshock (1 ma, 1 sec, 30 shocks in 15 min) (open circles) or no footshock (solid circles) once following six accommodation sessions. The number of fecal pellets expelled during the acute footshock/no footshock session was measured in the same rats used for UBD testing (Figure 4.7). Blood was drawn for measurement of plasma corticosterone concentration from separate groups of rats that were treated identically immediately following acute footshock/no footshock. Sample sizes equaled 5-7/group. * indicates p <.05 that acute footshock > acute no footshock. 76

91 Figure 4.9. Group mean EMG responses to UBD (10-80 mmhg) from rats with chronic, bilateral drug micropellet implants targeted to the CeA and containing 30 μg of corticosterone (CORT, open circles), aldosterone (ALD, open squares), or cholesterol (CHOL,solid trianges) (upper panel). Rats underwent seterotaxic surgery for bilateral micropellet implantation and UBD-evoked EMG responses were measured one week later. Sample sizes equaled 7/group. Group mean EMG responses to UBD (10-80 mmhg) from rats following acute application of CORT (15 µg, open circles; 5 µg, open triangles) or CHOL (15 µg, solid circles) (lower panel). Rats underwent bilateral stereotaxic application of drug and UBD-evoked EMG responses were measured 30 min later. Sample sizes equaled 4/group. * indicates p <.05 that chronic CORT and chronic ALD > chronic CHOL at UBD pressures of mmhg (pressures < 40 were not analyzed). # indicates p <.05 a main effect of drug where acute CORT (15 µg, but not 5 µg) > acute CHOL at pressures of mmhg (pressures < 40 mmhg were not analyzed). 77

92 Table 4-1. Statistical analyses of UBD-evoked EMG responses (pressures of mmhg) and somatic thermal paw withdrawal thresholds (pre and post measures) in rats exposed to acute footshock or no footshock. * indicates p <.05 significant main effects of footshock, UBD, time, and a significant footshock time interaction. Holm s procedure was used to maintain family-wise α in simple contrast analyses of distension pressure. Repeated-measures ANOVA Simple Contrast Analyses Footshock vs. No Footshock Visceromotor Response Df F p mmhg FOOTSHOCK (1,12) * Footshock > No Footshock p <.05 UBD (4,48) * FOOTSHOCK UBD (4,48) Repeated-measures ANOVA Simple Contrast Analyses Somatic Thermal Threshold df F p Pre NS Footshock vs. No Footshock p <.05 FOOTSHOCK (1,24) * TIME (1,24) * Post FOOTSHOCK TIME (1,24) * Footshock > No Footshock p <.05 78

93 Table 4-2. Statistical analyses of UBD-evoked EMG responses (pressures of mmhg) in rats following chronic stimulation of the CeA with corticosterone, aldosterone, or cholesterol. * indicates p <.05 significant main effects of drug treatment (DRUG) and UBD. Holm s procedure was used to maintain family-wise α in simple contrast analyses of distension pressure. Repeated-measures ANOVA Simple Contrast Analyses Corticosterone vs. Aldosterone vs. Cholesterol Visceromotor Response df F p mmhg DRUG (2,19) * Aldosterone > Cholesterol (40, 70, 80 mmhg) p <.05 UBD (4,76) * Corticosterone vs. Cholesterol NS DRUG UBD (8,76)

94 CHAPTER 5 RESULTS SPECIFIC AIMS 1 AND Purpose The purpose of Specific Aims 1 and 2 was to determine whether lesions of the CeA alter acute footshock-induced urinary bladder hypersensitivity or the expression of physiological and behavioral indices of stress, respectively. All rats underwent bilateral electrolytic lesioning of the CeA or control surgery (Section 3.7) followed by a single 15 min session of acute intermittent footshock or no footshock one week later after six accommodation sessions (Section 3.3.2). For Specific Aim 1, rats were instrumented for measurement of UBD-evoked abdominal EMG responses (Section 3.6.2). Specific Aim 2 was stratified into two Sub Aims. For Specific Aim 2 Sub Aim 1, rats were deeply anesthetized, and blood was drawn for measurement of plasma corticosterone concentration (Section 3.11). For Specific Aim 2 Sub Aim 2, rats were placed on the EPM for quantification of anxiety-like behavior (Section ). Flinch and jump thresholds were assessed (Section 3.8) as a control measure to ensure that lesions or control surgery did not impair sensory transduction of the footshock stimulus. Group designations were as follows: Table 5-1. Group designations for Specific Aims 1 and 2. Lesion Treatment Footshock Treatment Group Designation CeA Lesion Footshock Lesion-FS CeA Lesion No Footshock Lesion-NFS Control Surgery Footshock Control-FS Control Surgery No Footshock Control-NFS None None Naive* *flinch-jump experiments only 80

95 5.2 Research Hypotheses and Findings Specific Aim 1 Specific Aim 1 tested the hypothesis that CeA lesions will attenuate acute footshock-induced bladder hypersensitivity. As indicated in Figure 5.1 and Table 5-2, a repeated-measures ANOVA of noxious distension pressures revealed significant main effects of lesion and footshock, and a significant lesion footshock interaction. No other interactions were significant. Post-hoc analyses of pressures mmhg indicated that rats in the Control-FS condition had significantly greater responses than rats in the Control-NFS, Lesion-FS, and Lesion-NFS conditions. Group Lesion-FS did not significantly differ from group Lesion-NFS. An additional analysis of EMG responses of naive rats that were exposed to footshock and the Control-FS group indicated no difference at similar pressures. There were no significant differences among rats with lesions, rats that underwent control surgery, or naive rats in either flinch thresholds [F(2,13)=0.951, p=.412] or jump thresholds [F(2,13)=0.318, p=.733] (Figure 5.2). Histological representation of lesion sites for this Specific Aim is presented in Figure Specific Aim Overview. Specific Aim 2 Sub Aim 1 tested the hypothesis that CeA lesions will attenuate plasma corticosterone concentration following acute footshock exposure, and Specific Aim 2 Sub Aim 2 tested the hypothesis that CeA lesions will 81

96 attenuate anxiety-like behavior on the EPM following acute footshock exposure. Histological representation of lesion sites for this Specific Aim is presented in Figure Specific Aim 2 Sub Aim 1. The findings presented in Figure 5.3 and Table 5-3 indicate significant main effects of footshock and lesion, and a significant footshock lesion interaction, indicating that the magnitude of footshock-induced enhancement in plasma corticosterone was significantly reduced by CeA lesions. Posthoc contrasts demonstrated that Control-FS had significantly greater circulating corticosterone compared to all other groups, Lesion-FS had significantly greater circulating corticosterone than Control-NFS, and Lesion-NFS did not differ from Lesion- FS or Control-NFS Specific Aim 2 Sub Aim 2. The spatiotemporal measure analyzed was % of time spent on the open arm. There were significant main effects of footshock and lesion, but no significant interaction between these two variables. After correcting for multiple comparisons, the only post-hoc contrast that met statistical significance indicated that Control-FS exhibited a significantly reduced % of time in the open arm. Lesion-FS and Control-NFS tended to spend less time on the open arm compared to Lesion-NFS, but these comparisons did not meet statistical significance. The results from Specific Aim 2 Sub Aim 1 are presented in Figure 5.4 and Table 5-5. Ethological measures (SAPs, freezing, and rearing) were also quantified. There were significant main effects of footshock and lesion on SAPs, where footshock exposure decreased and lesions increased the frequency of SAPs. The interaction was not significant. Post-hoc analyses of SAPs showed that Control-FS exhibited significantly fewer stretch-attend postures than Control-NFS and Lesion-FS, with no other significant 82

97 group differences. Post-hoc analyses of freezing indicated that Control-FS demonstrated significantly more freezing behavior than Control-NFS of Lesion-FS, and Lesion-FS exhibited significantly more freezing than Lesion-NFS. There were no statistically significant effects on rearing. The results from Specific Aim 2 Sub Aim 2 are presented in Figure 5.5 and Table Summary Related to Specific Aims 1 and 2 The goals of Specific Aims 1 and 2 were met. It was definitively demonstrated that lesions of the CeA abolished acute footshock-induced bladder hypersensitivity without affecting baseline sensory processing. CeA lesions also significantly attenuated footshock-induced corticosterone release, the primary physiological index of HPA axis activation, and anxiety-like behaviors. 83

98 Figure 5.1. Group mean EMG responses to UBD (10-80 mmhg) from rats with CeA lesions (Lesion, square symbols), rats that underwent control surgery (Control, circle symbols), or naive rats (Naive, open triangles) (upper panel). Rats underwent stereotaxic surgery (with or without bilateral electrolytic lesions), and were exposed to acute footshock (FS; 1 ma, 1 sec, 30 shocks in 15 min; open symbols) or no footshock (NFS, solid symbols) one week later following six accommodation sessions. UBDevoked EMG responses were measured approximately 15 min following footshock or no footshock exposure. Sample sizes ranged from 6-8/group. * indicates p <.05 that Control-FS differed from all other groups at pressures of mmhg (pressures < 40 were not analyzed). Data from the Naive-FS group are the same as presented in Figure 4.7. Control-FS and Naive-FS did not significantly differ. Representative CeA lesions (approximately 2.40 mm posterior to bregma) from a rat exposed to acute footshock are indicated by white arrows (lower panel). 2 mm 84

99 Figure 5.2. Group mean flinch and jump thresholds from rats with CeA lesions (Lesion, open circles), rats that underwent control surgery (Control, solid circles), or naive rats (Naive, solid triangles). Rats underwent stereotaxic surgery (with or without bilateral electrolytic lesions), and flinch and jump thresholds were measured one week later following six accommodation sessions. Sample sizes ranged from 5-6/group. There were no significant differences in flinch or jump thresholds between any groups. 85

100 Figure 5.3. Group mean plasma corticosterone concentration from rats with CeA lesions (solid circles) or that underwent control surgery (open circles). Rats underwent stereotaxic surgery (with or without bilateral electrolytic lesions), and were exposed to acute footshock (1 ma, 1 sec, 30 shocks in 15 min) or no footshock one week later following six accommodation sessions. Blood was drawn for measurement of plasma corticosterone 15 min following footshock or no footshock exposure. Sample sizes ranged from 6-8/group. * indicates p <.05 that control footshock differed from all other groups. 86

101 Figure 5.4. Group mean anxiety-like behavior from rats with CeA lesions (Lesion) or that underwent control surgery (Control). Rats underwent stereotaxic surgery (with or without bilateral electrolytic lesions) and were exposed to acute footshock (FS; 1 ma, 1 sec, 30 shocks in 15 min) or no footshock (NFS) one week later following six accommodation sessions. 30 min after FS or NFS, rats were placed on the EPM and % of open arm time (upper panel) and ethological behaviors (lower panel) were recorded for 5 min. Sample sizes ranged from 5-6/group. * indicates significantly different from all other groups (all p s <.05). # indicates significantly different from Lesion-NFS (p <.05). 87

102 Figure 5.5. Histological representation of CeA lesions for Specific Aim 1. Bilateral electrolytic lesions were performed, and rats were exposed to acute footshock or no footshock one week later following six accommodation sessions. UBD-evoked EMG responses were measured approximately 15 min following footshock or no footshock exposure. The black dotted lines represent lesion hits. The blue dotted lines represent lesion misses in rats exposed to footshock. UBD-evoked EMG responses in these rats are presented in Figure

103 Figure 5.6. Individually plotted EMG responses to UBD (10-80 mmhg) in rats with lesions that missed the CeA. The data presented are from 3 rats that were exposed to acute footshock (FS, solid symbols) and from 2 rats in the control footshock condition (NFS, open symbols). 89

104 Figure 5.7. Histological representation of CeA lesions for Specific Aim 2. Bilateral electrolytic lesions were performed and rats were exposed to acute footshock or no footshock one week later following six accommodation sessions. 15 min following footshock or no footshock exposure, blood was drawn for measurement of plasma corticosterone concentration or anxiety-like behavior was assessed. 90

105 Table 5-2. Statistical analyses of UBD-evoked EMG responses (pressures of mmhg) in rats with CeA lesions and control rats following acute exposure to footshock (FS) or no footshock (NFS). * indicates significant main effects of footshock and lesion and a significant footshock lesion interaction (p s <.05). Holm s procedure was used to maintain family-wise α in simple contrast analyses. Repeated-measures ANOVA Simple Contrast Analyses Lesion vs. Control Surgery Footshock vs. No Footshock Visceromotor Response df F p mmhg FOOTSHOCK (1,21) * Control-FS > Control-NFS p <.05 LESION (1,21) * Lesion-FS vs. Lesion-NFS NS FOOTSHOCK LESION (1,21) * Control-FS > Lesion-FS p <.05 FOOTSHOCK UBD (4,84) Control-NFS vs. Lesion-NFS NS LESION UBD (4,84) Control-FS > Lesion-NFS p <.05 LESION FOOTSHOCK UBD (4,84) Lesion-FS vs. Control-NFS NS 91

106 Table 5-3. Statistical analyses of plasma corticosterone concentration (pg/ml) in rats with CeA lesions and control rats following acute exposure to footshock (FS) or no footshock (NFS). * indicates significant main effects of footshock and lesion, and a significant footshock lesion interaction (all p s <.05). Holm s procedure was used to maintain family-wise α in simple contrast analyses. ANOVA Simple Contrast Analyses Lesion vs. Control Surgery Footshock vs. No Footshock Plasma Corticosterone Concentration df F p Control-FS > Control-NFS p <.05 FOOTSHOCK (1,25) * Lesion-FS vs. Lesion-NFS Control-FS > Lesion-FS NS p <.05 LESION (1,25) * Control-NFS vs. Lesion-NFS NS FOOTSHOCK LESION (1,25) * 92

107 Table 5-4. Statistical analyses of % time spent in the open arm of the EPM in rats with CeA lesions and control rats following acute exposure to footshock (FS) or no footshock (NFS). * indicates p <.05 main effects of footshock and lesion. Holm s procedure was used to maintain family-wise α in simple contrast analyses. ANOVA Simple Contrast Analyses Lesion vs. Control Footshock vs. No Footshock % Open Arm Time Control-FS vs. Control-NFS NS Lesion-FS vs. Lesion-NFS NS FOOTSHOCK (1,22) * Control-FS vs. Lesion-FS NS LESION (1,22) * Lesion-NFS > Control-NFS p <.05 FOOTSHOCK LESION (1,22)

108 Table 5-5. Statistical analyses of the frequency of stretch-attend posture (SAP), freezing, and rearing behavior during EPM testing in rats with CeA lesions and control rats following acute exposure to footshock (FS) or no footshock (NFS). * indicates significant main effects of footshock and lesion on SAP and freezing, and a significant footshock lesion interaction on freezing (all p s <.05). Holm s procedure was used to maintain family-wise α in simple contrast analyses. ANOVA Simple Contrast Analyses Lesion vs. Control Footshock vs. No Footshock Stretch-Attend Posture df F p Stretch-Attend Posture FOOTSHOCK (1,22) * Control-FS < Control-NFS p <.05 LESION (1,22) * Lesion-FS vs. Lesion-NFS NS FOOTSHOCK LESION (1,22) Control-FS vs.< Lesion-FS p <.05 Control-NFS < Lesion-NFS p <.05 Freezing FOOTSHOCK (1,22) * LESION (1,22) * Freezing FOOTSHOCK LESION (1,22) * Control-FS > Control-NFS p <.05 Rearing Lesion-FS > Lesion-NFS p <.05 Control-FS > Lesion-FS p <.05 FOOTSHOCK (1,22) Control-NFS vs. Lesion-NFS NS LESION (1,22) FOOTSHOCK LESION (1,22)

109 CHAPTER 6 RESULTS SPECIFIC AIMS 3 AND Purpose The purpose of Specific Aims 3 and 4 was to determine whether acute chemical stimulation of the CeA alters urological nociceptive responses or the expression of physiological and behavioral indices of stress, respectively. All rats underwent bilateral stereotaxic administration to the CeA of either corticosterone or cholesterol (Section 3.9). For Specific Aim 3, rats were instrumented for measurement of UBD-evoked abdominal EMG responses (Section 3.6.2). Specific Aim 4 was stratified into two Sub Aims. For Specific Aim 4 Sub Aim 1, plasma corticosterone was measured at baseline and 15 min following exposure to the EPM (Section 3.11). For Specific Aim 4 Sub Aim 2, anxiety-like behavior was assessed on the EPM 15 min following drug injection (Section ). 6.2 Research Hypotheses and Findings Specific Aim 3 Specific Aim 3 tested the hypothesis that acute chemical stimulation of the CeA with corticosterone will enhance bladder nociceptive responses relative to treatment with cholesterol. The findings in Figure 6.1 and Table 6-1 indicate significant main effects of drug treatment and distension pressure (40-80 mmhg). The drug distension pressure interaction was not significant. No post-hoc analyses met statistical significance after 95

110 correction for multiple comparisons. Histological representation of microinjection sites for this Specific Aim is presented in Figure Specific Aim Overview. Specific Aim 4 Sub Aim 1 tested the hypotheses that acute chemical stimulation of the CeA with corticosterone will enhance plasma corticosterone concentration following EPM exposure relative to treatment with cholesterol, but will not change baseline corticosterone release. Specific Aim 4 Sub Aim 2 tested the hypothesis that acute chemical stimulation of the CeA will enhance anxiety-like behavior on the EPM. Histological representation of microinjection sites for this Specific Aim is presented in Figure Specific Aim 4 Sub Aim 1. The findings presented in Figure 6.2 and Table 6-2 indicate no significant main effects of or interaction between drug treatment and time (pre- versus post-epm) on plasma corticosterone concentration Specific Aim 4 Sub Aim 2. Figure 6.3 and Table 6-3 present data related to anxiety-like behavior. There was no significant effect of corticosterone treatment on the % of time spent in the open arm of the EPM. Ethological behaviors (SAPs, freezing, and rearing) were also quantified. Corticosterone treatment significantly increased the frequency of freezing episodes and the amount of time spent freezing, but did not significantly alter SAPs. Rearing also was not affected, indicating that enhanced freezing behavior can not be attributed to locomotor disruption. 96

111 6.3 Summary Related to Specific Aims 3 and 4 The goal of Specific Aim 3 was met in that acute chemical stimulation of the CeA with corticosterone produced a statistically significant increase in bladder sensitivity at UBD pressures of mmhg. In general, the hypotheses addressed by Specific Aim 4 were not supported. Freezing behavior was significantly augmented by acute CeA stimulation, but % of open arm time and SAPs were not. Although plasma corticosterone concentration was unaffected at baseline as was hypothesized, EPM exposure failed to induce a significant increase in corticosterone release. 97

112 Figure 6.1. Group mean EMG responses to UBD (10-80 mmhg) in rats following acute application of corticosterone (CORT, open circles) or cholesterol (CHOL, solid circles) to the CeA. Rats underwent bilateral stereotaxic delivery of drug (15 μg/0.5 μl) and UBD-evoked EMG responses were measured 30 min later. Sample sizes equaled 6/group. * indicates p <.05 a main effect of drug treatment at distension pressures of mmhg. 98

113 Figure 6.2. Group mean plasma corticosterone concentration at baseline (upper panel) and following EPM testing (lower panel) in rats rats following acute application of corticosterone or cholesterol to the CeA. Blood was drawn for baseline measurement of corticosterone concentration, and rats underwent stereotaxic surgery for bilateral cannulae implantation. Four days later, drug (15 μg/0.5 μl) was applied to the CeA via the implanted cannulae, and rats underwent EPM testing 30 min later followed by a second blood draw 15 min later. Sample sizes ranged from 6-7/group. There was no significant difference between groups in plasma corticosterone concentration at either timepoint. Plasma Corticosterone Concentration (pg/ml) Corticosterone Baseline Cholesterol Plasma Corticosterone Concentration (pg/ml) Corticosterone Post EPM Cholesterol 99

114 Figure Group mean anxiety-like behavior from rats following acute application of corticosterone (CORT) or cholesterol (CHOL) to the CeA. Rats underwent stereotaxic surgery for bilateral cannulae implantation. Four days after surgery, drug (15 μg/0.5 μl) was applied to the CeA and EPM testing was performed 15 min later. % of open arm time (upper panel) and ethological behaviors (lower panel) were assessed. Sample sizes ranged from 5-6/group. There was no significant difference between CORT and CHOL in % time spent on the open arm. * indicates a significant effect of acute CeA stimulation (p <.05). 100

115 Figure 6.4. Histological representation of CeA microinjection sites for Specific Aim 3 (Figure 6.1). Rats were administered bilateral stereotaxic microinjections of corticosterone (15 μg/0.5 μl, triangles) or cholesterol (circles) to the CeA and UBDevoked EMG responses were measured 30 min later. The dashed lines represent cannulae tracks. 101

116 Figure 6.5. Histological representation of CeA drug injection sites for Specific Aim 4 (Figures 6.2 and 6.3). Rats were administered bilateral stereotaxic microinjections of corticosterone (15 μg/0.5 μl, triangles) or cholesterol (circles) to the CeA. Anxiety-like behavior was assessed 15 min later. Blood was drawn prior to the day of testing and again15 min following removal from the EPM for measurement of plasma corticosterone concentration. The dashed lines represent cannulae tracks. 102

117 Table 6-1. Statistical analyses of UBD-evoked EMG responses (pressures of mmhg) following acute application of corticosterone or cholesterol to the CeA. * indicates p <.05 main effects of drug treatment (DRUG) and distension pressure (UBD). Holm s correction was used to maintain family-wise α in simple contrast analyses. Repeated-measures ANOVA Simple Contrast Analyses Corticosterone vs. Cholesterol Visceromotor Response df F p mmhg DRUG (1,10) * Corticosterone > Cholesterol NS UBD (1,10) * DRUG UBD (1,10)

118 Table 6-2. Statistical analyses of plasma corticosterone concentration (pg/ml) following acute stereotaxic delivery of corticosterone (15 μg.0.5 μl) or cholesterol to the CeA. There were no significant differences between drug treatment groups at baseline or following EPM testing. ANOVA Corticosterone vs. Cholesterol Plasma Corticosterone Concentration df F p DRUG (1,22) TIME (1,22) DRUG TIME (1,22)

119 Table 6-3. Statistical analyses of the % open arm time, and frequency of stretch-attend posture (SAP), freezing, and rearing behavior during EPM testing following acute stereotaxic delivery of corticosterone (15 μg/0.5 μl) or cholesterol to the CeA. ANOVA Corticosterone vs. Cholesterol df t p % Open Arm Time NS Stretch-Attend Posture NS Freezing Corticosterone > Cholesterol p <.05 Rearing NS 105

120 CHAPTER 7 RESULTS SPECIFIC AIM Purpose The purpose of Specific Aim 5 was to determine whether modulation of urological nociceptive responses by the CeA involved a spinal mechanism of action. Rats underwent bilateral stereotaxic administration to the CeA of either corticosterone or cholesterol (Section 3.9). UBD (60 mmhg, 20 sec, 3 min intertrial interval, 120 min) was administered to half of the rats in each drug treatment condition, and the remaining half were anesthetized but not administered UBD. 7.2 Research Hypotheses and Findings Specific Aim 5 tested the hypothesis that acute chemical stimulation of the CeA with corticosterone will enhance c-fos expression in the spinal cord in response to UBD. The results from this Specific Aim are presented Figure 7.1. As predicted, there was a significant main effect of UBD on spinal c-fos expression [F(1,12)=8.552, p=0.013]. The hypothesis that corticosterone treatment would increase c-fos expression was based on preliminary results (Section 4.3.3) showing that acute stereotaxic delivery of corticosterone to the CeA enhanced urological nociceptive responses. It was presumed that this finding might result from descending facilitation of spinal dorsal horn neuronal activity. Analyses indicated that there was a significant main effect of drug [F(1,12)=5.235, p=0.041], but in contrast to the hypothesis, corticosterone application 106

121 decreased c-fos expression. The drug UBD interaction was not significant [F(1,12)=0.206, p=.658]. After correction for multiple comparisons, post-hoc analyses revealed that CHOL- UBD expressed significantly greater spinal c-fos than CORT-NO UBD [F(1,12)=13.585, p<.01]. There were non-significant trends toward greater c-fos expression in CHOL- UBD relative to CHOL-NO UBD [F(1,12)=5.708, p=.034] and CORT-UBD [F(1,12)=3.760, p=.076]. Histological representation of microinjection sites for this Specific Aim is presented in Figure Summary Related to Specific Aim 5 The goal of Specific Aim 5 was met with the demonstration of a statistically significant effect of both UBD and acute CeA stimulation on spinal c-fos expression. The UBD-evoked enhancement of c-fos expression was anticipated. Although the effect of CeA stimulation on c-fos (a decrease) is not what was expected (an increase), a role for a spinal mechanism is clearly demonstrated. 107

122 Figure 7.1. Group mean integrated density of c-fos immunoreactivity (upper panel) and Western blots of spinal cord c-fos and β-actin immunoreactivity (lower panel) in rats following acute application of corticosterone (CORT) or cholesterol (CHOL) to the CeA. Rats were administered bilateral stereotaxic microinjections of CORT (15 μg/0.5 μl) or CHOL to the CeA. Half of the rats in each drug treatment group were administered UBD (60 mmhg, 20 sec, 3 min intertrial interval, 120 min) and the remaining were anesthetized without UBD. Sample sizes equaled 4/group. 108

123 Figure 7.2. Histological representation of CeA drug microinjection sites for Specific Aim 5 (Figure 7.1). Rats were administered bilateral stereotaxic microinjections of corticosterone (15 μg/0.5 μl) or cholesterol to the CeA and were administered UBD (60 mmhg, 20 sec, 3 min intertrial interval, 120 min) or no UBD. Spinal cord tissue was harvested and processed for Western blotting of c-fos. The dashed lines represent cannulae tracks. 109