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THE ROLE OF SPINAL SEROTONERGIC RECEPTORS 5-HT1A AND 5-HT3 IN STRESS-INDUCED URINARY BLADDER HYPERSENSITIVITY by CHELSEA L. CRAWFORD MEREDITH T. ROBBINS, COMMITTEE CHAIR FRANKLIN R. AMTHOR EDWIN C. COOK TIMOTHY J. NESS ROBERT E. SORGE 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 2014 Copyright by Chelsea L. Crawford 2014 ! ii! THE ROLE OF SPINAL SEROTONERGIC RECEPTORS 5-HT1A AND 5-HT3 IN STRESS-INDUCED URINARY BLADDER HYPERSENSITIVITY CHELSEA L. CRAWFORD BEHAVIORAL NEUROSCIENCE ABSTRACT Disorders in which pain originates from the urinary bladder such as interstitial cystitis (IC) are steadily increasing in prevalence. A common finding among patients with IC is the comorbidity with stress or anxiety disorders. In rats, footshock stress alone is sufficient to elicit bladder hypersensitivity. Serotonin (5-hydroxytryptamine; 5-HT) has been established as a mediator in anxiety and pain separately, but little is known about the role of 5-HT in stress-induced visceral hypersensitivity. The current set of studies addresses three main concerns: (1) the impact of spinal 5-HT1A and 5-HT3 receptor blockade with WAY-100635 (10 µg) and ondansetron (10 µg), respectively, on visceromotor reflex (VMR) responses to urinary bladder distension (UBD) of rats exposed to chronic footshock stress, (2) the impact of chronic footshock stress on spinal and cerebrospinal fluid 5-HT, 5-HIAA, and 5-HIAA/5-HT concentrations, and (3) the impact of spinal 5-HT3 receptor blockade with ondansetron (100 µg ) on dorsal horn neuronal responses to UBD. Experiments used to test these concerns utilized Lewis rats, a strain which has not been used in studies of stress-induced bladder hypersensitivity. A significant increase in abdominal EMG responses to UBD was observed in rats exposed to chronic footshock stress compared to sham stress. Intrathecal WAY-100635 or ondansetron had no significant effect on abdominal EMG responses to UBD in stressed or non-stressed rats. Chronic footshock stress did not significantly alter spinal or CSF concentrations of 5-HT, 5-HIAA, or 5-HIAA/5-HT. Spinal application of ondansetron ! iii! did not significantly alter neuronal responses to UBD in stressed or non-stressed rats. In light of these findings, preliminary results demonstrate that spinal non-specific 5-HT receptor blockade with methysergide (30 µg) significantly augmented the VMR response to UBD. Therefore, these results indicate that 5-HT is involved in the facilitation of stress-induced bladder hypersensitivity, but this facilitation does not rely exclusively on activation of either spinal 5-HT1A or 5-HT3 receptors. Keywords: bladder, visceral pain, stress, serotonin, interstitial cystitis ! iv! DEDICATION To my husband, Scott, who has always shown me unwavering love and support throughout this occasionally “stressful” life. And to our two beautiful children, Elliotte and Dylan, to whom I looked to for the inspiration and determination to accomplish this goal. ! v! ACKNOWLEDGEMENTS I would like to thank God for providing such a mysterious and wonderful world that I have the great pleasure of trying to figure out. I want to thank my family, both born of and married into, for always supporting and encouraging my educational endeavors. They helped me set forth my goals and made me confident that I could achieve them. I would especially like to thank Dr. Meredith Robbins for mentoring me both professionally and personally while working on this dissertation and throughout my graduate training. I would also like to thank the other members of my dissertation committee, Dr. Franklin Amthor, Dr. Edwin Cook, Dr. Timothy Ness, and Dr. Robert Sorge, for their contribution of suggestions and critiques throughout this process. Few people have made such a lasting impression on my career and life as the three men of science I am about to mention. Nettles Moore ignited my passion for questioning everything in our physical world. Dr. Jim Daniels helped to cultivate this passion by constantly encouraging and checking in on me to be sure that I was using my skills to contribute to the scientific body of knowledge. Dr. Alan Randich challenged and cheered me on from my lofty dream of entering graduate school through the completion of my dissertation. Each of these men took the time to personally acknowledge my strengths and abilities and to provide the positive reinforcement needed to succeed in academia. ! vi! I finally want to thank all of my friends, past and present, who have shown support and stood by me through the highs and lows and all of the life changes that have occurred during this time in graduate school. I am eternally grateful. ! vii! TABLE OF CONTENTS Page ABSTRACT .............................................................................................................. iii DEDICATION .......................................................................................................... v ACKNOWLEDGEMENTS ...................................................................................... vi LIST OF TABLES .................................................................................................... xi LIST OF FIGURES ................................................................................................... xii LIST OF ABBREVIATIONS ................................................................................... xiii CHAPTER 1 INTRODUCTION ......................................................................................... 1 1.1 The Clinical Problem ............................................................................. 1 1.2 Purpose .................................................................................................. 3 1.3 Hypotheses and Specific Aims .............................................................. 3 2 BACKGROUND ........................................................................................... 6 2.1 Pain: Physiology and Modulation .......................................................... 6 2.1.1 Transmission of Pain ................................................................... 6 2.1.2 Descending Modulation of Pain .................................................. 8 2.1.3 Diffuse Noxious Inhibitory Control ............................................ 11 2.2 Stress: Physiology and Role in Pain Perception ..................................... 13 2.2.1 Physiology of Stress Response .................................................... 15 2.2.2 Models of Laboratory Stress ....................................................... 16 2.2.3 Stress as an Analgesic ................................................................. 18 2.2.4 Stress as an Exacerbator or Pain .................................................. 18 2.3 Serotonin: Physiological Function and Role in Stress and Pain Systems ......................................................................... 19 2.3.1 Serotonergic Receptors/Function ................................................ 19 2.3.2 Relationship of Serotonin and Stress ........................................... 25 2.3.3 Serotonergic Pain Modulation ..................................................... 26 2.4 Urinary Bladder: Physiology and Interaction with Stress and Serotonin ....................................................................... 29 ! viii! 2.4.1 Bladder Innervation ..................................................................... 29 2.4.2 Models of Bladder Nociception................................................... 30 2.4.3 Role of Serotonin in Bladder Function ........................................ 32 2.5 Spinal Neuronal Characterization........................................................... 33 3 METHODS .................................................................................................... 36 3.1 3.2 3.3 3.4 3.5 3.6 3.7 3.8 Selection of Species ................................................................................ 36 Anesthesia............................................................................................... 37 Footshock Protocol ................................................................................. 37 Urinary Bladder Distension .................................................................... 38 Receptor Antagonists.............................................................................. 38 Implantation of Intrathecal Catheters ..................................................... 39 Assessment of VMR Response .............................................................. 39 Enzyme-Linked Immunosorbent Assay ................................................. 41 3.8.1 Tissue Collection ......................................................................... 41 3.8.2 Serotonin...................................................................................... 41 3.8.3 5-HIAA ........................................................................................ 42 3.8.4 Corticosterone.............................................................................. 43 3.9 Spinal Electrophysiology........................................................................ 43 3.9.1 Surgical Preparation .................................................................... 43 3.9.2 Quantification of Neuronal Responses ........................................ 44 3.9.3 Neuronal Characterization ........................................................... 44 3.10 Statistical Analyses ............................................................................... 45 3.10.1 Statistical Significance ................................................................ 45 3.10.2 VMR Response ............................................................................ 45 3.10.3 Enyme-Linked Immunosorbent Assay ........................................ 46 3.10.4 Spinal Electrophysiology............................................................. 46 4 METHODOLOGICAL DEVELOPMENT ................................................... 48 4.1 Strain Selection ....................................................................................... 48 4.2 Effect of Intrathecal Catheter ................................................................. 51 4.3 Effect of Serotonin on Chronic Footshock-Induced Bladder Pain ......... 53 5 RESULTS ...................................................................................................... 60 5.1 Specific Aim 1 ........................................................................................ 60 5.1.1 Purpose ........................................................................................ 60 5.1.2 Effect of Footshock ..................................................................... 60 5.1.3 Effect of WAY 100635 ............................................................... 61 5.1.4 Effect of Ondansetron.................................................................. 61 5.2 Specific Aim 2 ........................................................................................ 62 5.2.1 Purpose ........................................................................................ 62 5.2.2 Enyme-Linked Immunosorbent Assay ........................................ 62 5.3 Specific Aim 3 ........................................................................................ 63 ! ix! 5.3.1 5.3.2 5.3.3 5.3.4 5.3.5 6 Purpose ........................................................................................ 63 Effect of Footshock ..................................................................... 64 Effect of Ondansetron.................................................................. 64 Percent of Baseline Comparisons ................................................ 64 Neuronal Characterization ........................................................... 66 DISCUSSION................................................................................................ 77 6.1 Summary of Results ............................................................................... 77 6.2 Integration of Findings with Current Literature ..................................... 78 6.2.1 The Comorbidity of Chronic Stress and Pain .............................. 78 6.2.2 Serotonin in Stress-Induced Hyperalgesia................................... 80 6.2.3 Dorsal Horn Neuronal Activity in Bladder Pain Models ............ 82 6.3 Discussion of Results ............................................................................. 84 6.3.1 Specific Aim 1 ............................................................................. 84 6.3.2 Specific Aim 2 ............................................................................. 87 6.3.3 Specific Aim 3 ............................................................................. 89 6.4 Strengths ................................................................................................. 91 6.5 Limitations .............................................................................................. 91 6.6 Future Directions .................................................................................... 92 6.7 Conclusions ............................................................................................ 94 LIST OF REFERENCES .......................................................................................... 96 APPENDIX A IACUC NOTICE OF APPROVAL ................................................................ 121 B IACUC NOTICE OF APPROVAL FOR PROTOCOL MODIFICATION ..................................................................... 123 ! x! LIST OF TABLES Table Page 4.1 Effect of intrathecal catheter on visceromotor reflex responses......................... 59 5.1 Effect of footshock on spinal and CSF 5-HT, 5-HIAA, and 5-HIAA/5-HT concentrations ...................................................................... 76 ! xi! LIST OF FIGURES Figure Page 4.1 Effect of rat strain on visceromotor reflex responses ......................................... 55 4.2 Effect of rat strain on serum corticosterone levels ............................................. 56 4.3 Effect of intrathecal catheter on visceromotor reflex responses......................... 57 4.4 Effect of non-specific serontonergic receptor blockade on visceromotor reflex responses ....................................................................... 58 5.1 Effect of footshock on visceromotor reflex responses ....................................... 67 5.2 Effect of WAY-100635 on visceromotor reflex responses ................................ 68 5.3 Effect of ondansetron on visceromotor reflex responses ................................... 69 5.4 Effect of footshock on spinal and CSF serotonin concentrations....................... 70 5.5 Effect of footshock on spinal and CSF 5-HIAA concentrations ........................ 71 5.6 Effect of footshock on spinal and CSF 5-HIAA/5-HT concentrations .............. 72 5.7 Effect of footshock on dorsal horn neuronal activity ......................................... 73 5.8 Effect of ondansetron on dorsal horn neuronal activity ..................................... 74 5.9 Electrode depth from spinal cord dorsum .......................................................... 75 ! xii! LIST OF ABBREVIATIONS 5-HIAA 5-indoleacetic acid 5-HT 5-hydroxytryptamine 5-HTP 5-hydroxytryptaphan 8-OH-DPAT 8-Hydroxy-N,N-dipropyl-2-aminotetralin ACC anterior cingulate cortex ACTH adrenocorticotropic hormone ANOVA analysis of variance BPS bladder pain syndrome cAMP cyclic adenosine monophosphate CFS chronic footshock CNS central nervous system CRF corticotropin releasing factor CRD colorectal distension CSF cerebrospinal fluid DNIC diffuse noxious inhibitory control DOI 2,5-dimethoxy-4-iodoamphetamine E epinephrine ELISA Enzyme-Linked Immunosorbant Assay EMG electromyograph ! xiii! GABA γ-aminobutyric acid GI gastrointestinal HNCS heterotopic noxious conditioning stimuli HPA hypothalamic-pituitary-adrenal IC interstitial cystitis LC locus coeruleus LS lumbosacral LSD lysergic acid diethylamide l-STT lateral spinothalamic tract MDMA 3,4-methylenedioxy-N-methylamphetamine NE norepinephrine NFS no footshock NGC nucleus reticularis gigantocellularis NMDA N-methyl-D-aspartate NS nociceptive specific ODS ondansetron PAG periaqueductal gray PBS painful bladder syndrome PKA protein kinase A POMC pro-opiomelanocortin PVN paraventricular nucleus RNA-i ribonucleic acid interference RVM rostroventral medulla ! xiv! SAL saline SD Sprague Dawley sh-RNA short hairpin ribonucleic acid SIA stress-induced analgesia SIH stress-induced hyperalgesia SNL spinal nerve ligation SRD subnucleus reticularis dorsalis SRT spinoreticular tract SSRI selective serotonin reuptake inhibitor STT spinothalamic tract UBD urinary bladder distension v-STT ventral spinothalamic tract VI variable interval VMR visceromotor reflex VP vasopressin WAY WAY 100635 WDR wide dynamic range ! xv! CHAPTER 1 INTRODUCTION 1.1 The Clinical Problem Interstitial cystitis (IC) is clinically defined as “an unpleasant sensation (pain, pressure, discomfort) perceived to be related to the urinary bladder, associated with lower urinary tract symptoms of more than six weeks duration, in the absence of infection or other identifiable causes” (Hanno & Dmochowski, 2009). Pain is the hallmark feature of IC. It may be experienced in the suprapubic region, throughout the pelvis, as well as the lower back and abdomen, and it may be associated with bladder filling, voiding, or both (FitzGerald, Kenton, & Brubaker, 2005; Tincello & Walker, 2005; Warren et al., 2008). In addition, patients describe a sense of an urgent need to urinate and/or an increased frequency of urination that is distinguished from overactive bladder syndrome (Diggs et al., 2007; Hanno, Burks, et al., 2011). The patient population disproportionately favors women, with reports of worsening symptoms during menstruation (Powell-Boone et al., 2005). Similar diagnoses include painful bladder syndrome (PBS), which is characterized as suprapubic pain that is related to bladder filling, and bladder pain syndrome (BPS), defined as chronic pelvic pain, pressure, or discomfort that is related to the bladder and has accompanying symptoms of urgency, frequency, or both (Hanno, Nordling, & Fall, 2011). Patients generally fall into one of two categories: Type I- those with submucosal petechial hemorrhages (glomerulations) and Type II- those with 1 Hunner’s ulcers with or without glomerulations (Buffington, 2004). There is much overlap in the definitions of IC, PBS, and BPS, and they are often used interchangeably. For simplicity, all references to these bladder syndromes will be referred to as IC throughout this document. The impact of IC carries a heavy burden on our society. The National Institute for Diabetes, Digestive, and Kidney Diseases reported in 2000 that IC accounted for 4.1 million outpatient and clinic visits. The prevalence of IC has only grown since then. In 2009, between 3.4% and 7.8% of women in the United States were reported to be affected (Berry et al., 2011). While the etiology of IC is largely unknown, a critical finding among IC patients is the correlation of stress and anxiety with exacerbation of symptoms. In laboratory experiments, acute or chronic stress has been shown to increase the response to visceral pain. Similarly, stress leads to exacerbation of symptoms in painful conditions of visceral organs such as IC (Klausner & Steers, 2004; Larauche et al., 2008). In a population-based study of over 2300 participants, patients with IC were over 4 times more likely to have an anxiety disorder than matched controls (Chung, Liu, Lin, & Chung, 2014). Stress or anxiety is said to have a nocebo effect- that is, the anxiety increases the perception of pain. Physicians often try to decrease pain perception through behaviors that decrease anxiety (Erickson et al., 2009). In fact, stress reduction is in Tier 1 of clinical guidelines aimed to treat IC (Hanno, Burks, et al., 2011). Treatment options for patients are lacking, and treatments in current use are only efficacious in subpopulations of IC patients. While descending modulation of bladder pain has been the subject of numerous studies (especially involving the opioid system 2 (Heinricher, Tavares, Leith, & Lumb, 2009; Porreca, Ossipov, & Gebhart, 2002; Ren & Dubner, 2002; Stamford, 1995), fewer investigations have examined the role of modulation involving monoamine systems, and no reports exist on serotonergic involvement in stress-induced bladder hypersensitivity. 1.2 Purpose Stress and anxiety appear to contribute largely to the pain experienced by patients who suffer from IC. However, only a small fraction of IC-related research is devoted to studying the impact of stress on bladder hypersensitivity. Reports in the literature point to 5-hydroxytryptamine (serotonin; 5-HT) as a key modulator in chronic pain (Bardin, 2011; Cirillo, Vanden Berghe, & Tack, 2011; Crowell, 2004; Phillips & Clauw, 2011), though there remains a gap in our knowledge of 5-HT involvement in IC or stressinduced bladder hypersensitivity. The purpose of the current set of studies is to begin a line of research to elucidate the role of 5-HT in stress-induced bladder hypersensitivity. This research will aim to address two main questions: (1) What is the effect of 5-HT on stress-induced bladder hypersensitivity, and (2) what is the effect of stress on levels and metabolism of 5-HT? 1.3 Hypotheses and Specific Aims The general hypothesis proposed in this thesis is as follows: Chronic stress leads to exacerbation of urinary bladder hypersensitivity, which is facilitated by an upregulation of serotonergic activity in the spinal cord dorsal horn. 3 The following sub-hypotheses expound on this hypothesis: 1. Activation of spinal serotonergic receptors, 5-HT1A and 5-HT3, facilitates the chronic stress-induced increase in the visceromotor reflex (VMR) response to urinary bladder distension (UBD). 2. Chronic stress leads to an increase in spinal and cerebrospinal fluid (CSF) content of 5-HT as well as an increase in the rate of 5-HT turnover. 3. Activation of spinal receptors, 5-HT1A and 5-HT3, amplifies activity of Type II dorsal horn neurons in response to UBD. Specific Aims formed to address these hypotheses are as follows: Specific Aim 1: To determine the role of spinal serotonergic receptors, 5-HT1A and 5HT3, in chronic footshock stress-induced augmentation of VMR responses to UBD. Data collected in this Aim will address the following question: • What are the effects of spinal administration of specific 5-HT receptor antagonists on stress-induced exacerbation of reflex responses to UBD? Specific Aim 2: To quantitatively determine whether chronic footshock stress increases 5-HT levels in the lumbosacral spinal cord and CSF. Data collected in this Aim will address the following questions: • What are normal levels of 5-HT in the CSF and lumbosacral spinal cord? • How does chronic footshock stress affect the 5-HT levels and rate of turnover? Specific Aim 3: To determine the role of serotonergic receptors, 5-HT1A and 5-HT3, in the response of spinal dorsal horn neurons to UBD in chronically stressed rats. Data collected in this Aim will address the following questions: 4 • What is the effect of chronic footshock stress on response properties of Type II spinal dorsal horn neurons? • What are the effects of specific serotonergic receptor antagonists on the responses of Type II spinal dorsal horn neurons to bladder distension in chronically stressed rats with intact nervous systems? 5 CHAPTER 2 BACKGROUND 2.1 Pain: Physiology and Modulation 2.1.1 Transmission of Pain Pain receptors, or nociceptors, are specialized peripheral nerve endings that respond to noxious stimuli, including temperature extremes, intense pressure, and chemicals. Thus, nociceptors are of three main types: thermal, mechanical, and chemical. If a nociceptor responds to more than one type of noxious stimulus, it is referred to as a polymodal nociceptor. For example, mechanoheat nociceptors respond to painful thermal and mechanical stimulation. Silent nociceptors are those which are generally insensitive to intense stimuli but which become responsive under conditions of inflammation or injury. Transmission of nociceptive signals, or action potentials, occurs along axons of these sensory neurons. A-δ fibers are medium diameter, myelinated primary afferents that are rapidly-conducting. Nociceptive information transmitted along A-δ fibers is therefore perceived quickly, and the pain is well-localized and often described as instant and sharp. C-fibers are small diameter, unmyelinated primary afferents that conduct action potentials more slowly. As a consequence, pain is perceived a second or more after the stimulus is applied and gradually increases in intensity. It is more generalized and is often described as dull, achy, throbbing, and burning. 6 These afferent nerve fibers synapse onto the dorsal root ganglia of the spinal cord, where their signals can be transmitted further to other spinal regions or to the brain. Specifically in the dorsal horn of the spinal cord, two classes of pain-signaling neurons exist. Nociceptive specific (NS) neurons elicit action potentials only in response to painful stimuli; thus NS cells receive input exclusively from A-δ and C-fibers. In contrast, wide dynamic range (WDR) neurons are responsive to noxious and non-noxious stimuli. WDR neurons also synapse with large diameter, highly myelinated A-β fibers, in addition to A-δ and C-fibers. A-β fibers generally convey information about light touch or other innocuous peripheral stimulation. Consequently, WDR neurons are more sensitive to stimulus intensity than their NS counterparts (Dickenson & Sullivan, 1987). Following integration in the dorsal horn, nociceptive information is transmitted to supraspinal sites via ascending pain pathways. The spinothalamic tract (STT) is the major ascending pain pathway, with projections leading to the thalamus. The STT has two main divisions: the lateral or neospinothalamic tract (l-STT) and the ventral or paleospinothalamic tract (v-STT). The l-STT projects to posterior thalamic nuclei and is thought to be involved in spatial and temporal discrimination of pain and touch (Price & Dubner, 1977). The v-STT projects to medial and intralaminar thalamic nuclei and is considered integral to aversive motivational processing (Holloway, Fox, & Iggo, 1978; Kerr, 1975; Price, Hayes, Ruda, & Dubner, 1978). The spinoreticular tract (SRT) has common projections terminating in the reticular formation. The majority of SRT projections encode information pertaining to innocuous stimuli, although the nucleus reticularis gigantocellularis (NGC) of the reticular formation is an important nucleus for nociceptive processing (Collins & Randt, 1960). The reticular formation has further 7 projections to the thalamus, hypothalamus, and limbic structures (Casey, 1969). Cortical areas involved in pain processing include a distribution which is considered the “pain matrix” (Ingvar, 1999). These areas include the anterior cingulate cortex (ACC), insula, frontal cortex, amygdala, and somatosensory cortices I and II (Ingvar, 1999; Jones, 1992; Peyron, Laurent, & Garcia-Larrea, 2000; Talbot et al., 1991). Somatosensory cortices are thought to process information relating to location and intensity (Bushnell et al., 1999; Kanda et al., 2000), and the ACC is said to process the affective component of nociception (Rainville, Duncan, Price, Carrier, & Bushnell, 1997). Finally, the insula is thought to be involved in encoding information pertaining to intensity, laterality, and affective components of pain (Brooks, Nurmikko, Bimson, Singh, & Roberts, 2002; Coghill, Sang, Maisog, & Iadarola, 1999; Singer et al., 2004). Cutaneous pain and visceral pain are, to an extent, processed separately in the central nervous system (CNS). This is important for the strong emotional responses that can be evoked by visceral pain (Traub, Silva, Gebhart, & Solodkin, 1996). The brain’s response to pain is generally thought to be through spinal inhibition of afferent signals produced mostly by endogenous opioids and, to a lesser degree, other neurotransmitters. 2.1.2 Descending Modulation of Pain Descending modulation of pain can be inhibitory or facilitatory in action (Zhuo & Gebhart, 1990, 1992, 1997). Initial reports of descending inhibition resulted from stimulation of the periaquaductal gray (PAG), which produced antinociception sufficient to perform laparoscopic surgery in rats (Reynolds, 1969). Motor responses were unaffected, and responses to nociceptive stimuli returned following the termination of 8 electrical stimulation (Reynolds, 1969). It was later discovered that microinjections of opiates, including morphine, into the PAG produced similar analgesia that was reversed by naloxone (Cheng, Fields, & Heinricher, 1986; Kosterlitz & Hughes, 1977; Llewelyn, Azami, & Roberts, 1986). Both electrical stimulation and drug administration into the PAG showed tolerance and cross-tolerance to each other, demonstrating endogenous opioid pain control (Hughes, 1975; Mayer & Hayes, 1975). Stimulation-produced analgesia from the PAG can be blocked completely or partially by lesioning sites in the rostroventral medulla (RVM), indicating the RVM as the output center in descending pain controls (Porreca et al., 2002). Stimulation of the RVM itself produces inhibition, facilitation, or intensity-dependent biphasic effects on nociceptive processing (Millan, 1999; Urban & Gebhart, 1999). Lower intensities of electrical stimulation produce facilitation, whereas higher intensities produce more inhibition (Zhuo & Gebhart, 1997). These findings were reproduced using glutamate stimulation of the RVM (Zhuo, Sengupta, & Gebhart, 2002). Furthermore, electrical stimulation of the nucleus raphe magnus (NRM) decreased the responsiveness of dorsal horn neurons to noxious heat applied to the tail of the rat (Llewelyn et al., 1986). Other sites within the RVM that produce stimulation-induced pain inhibition, facilitation, or both include the NGC, NGC pars α, nucleus raphe obscuris, and nucleus raphe pallidus. In studies of bladder hypersensitivity, stimulation of the RVM produced predominant inhibition of the VMR to UBD (Randich, Mebane, DeBerry, & Ness, 2008). Stimulation of the RVM also produced site- and intensity-dependent facilitation of the VMR to UBD but to a lesser extent than inhibition (Randich, Mebane, et al., 2008). 9 During electrophysiological recording in the RVM, Fields et al. (1983), found that populations of neurons responded differentially to the tail flick reflex, a nociceptive test in rodents that measures the time to withdraw the tail from a noxious radiant heat source (D'amour & Smith, 1941). Some neurons abruptly stopped firing prior to withdrawal of the tail from the heat source, while others abruptly or gradually started firing just before the tail withdrawal. These were named on-cells and off-cells, respectively. Another subset of neurons demonstrated no change in response to tail flick and were termed neutral-cells (Fields, Bry, et al., 1983). All on-cells are excited by noxious pinch of the skin, while most off-cells are inhibited by pinch (Fields, Bry et al. 1983). Micro-injection of morphine or cannabinoid CB1 receptor agonist, WIN-55,212-2, into the RVM extinguished on-cell firing, off-cell cessation, and tail withdrawal in the tail flick test (Meng & Johansen, 2004). Both on- and off-cells are excited by electrical stimulation of the PAG (Fields, Vanegas, Hentall, & Zorman, 1983). Heinricher et al. (1989), discovered through simultaneous recordings of RVM neurons that neurons of the same class (i.e. on- or off-) are all active at the same time, and these periods of activity alternate with the opposing class. This is one line of evidence that supports the conclusion that on- and off-cells play reciprocal roles, and that off-cells are inhibitory of on-cells. Further support is that off-cells discontinue their activity just prior to withdrawal reflexes such as the tail flick and the onset of activity of thalamic neurons in response to noxious stimulation (Hernández, López, & Vanegas, 1989). In addition, only off-cells become active upon systemic or PAG administration of morphine, while morphine causes on-cells to become quiescent (Barbaro, Heinricher, & Fields, 1986; Cheng et al., 1986; Fields, Vanegas, et al., 1983). 10 As far as which neurotransmitters are involved in on-/off-cell function, less is known. It has been suggested that the off-cell pause is influenced by activation of γaminobutyric acid (GABA)-A receptors (Heinricher & Kaplan, 1991). On-cells are GABA-containing and most likely inhibit off-cells during noxious stimulation (Heinricher & Tortorici, 1994). The RVM is rich in serotonergic cells, but it is unclear whether any of these are on- or off-cells (Geranton, Fratto, Tochiki, & Hunt, 2008; Ossipov, Dussor, & Porreca, 2010; Porreca et al., 2002; Rahman, Suzuki, Webber, Hunt, & Dickenson, 2006; Suzuki, Morcuende, Webber, Hunt, & Dickenson, 2002; Suzuki, Rygh, & Dickenson, 2004). Interestingly, about 50% of neutral-cells stain positive for 5HT (Potrebic, Fields, & Mason, 1994). Other reports indicate a potential role for norepinephrine (NE) in modulating on- and off-cell activity. NE-containing fibers and terminals have been identified throughout the RVM (Dahlstrom & Fuxe, 1964; Fuxe, Hökfelt, & Ungerstedt, 1969). Microinjection of the α2 receptor agonist, clonidine, resulted in an increase of the tail flick latency and produced long-lasting inhibition of oncell firing (Fields, Heinricher, & Mason, 1991). 2.1.3 Diffuse Noxious Inhibitory Control Diffuse noxious inhibitory control (DNIC) describes processes in which a hyperalgesic response to a noxious stimulus is inhibited by the application of another noxious stimulus to a spatially-distinct receptive field (Dickenson, Rivot, Chaouch, Besson, & Le Bars, 1981). DNIC is mediated through a spino-bulbo-spinal pathway with ascending transmission from the ventrolateral spinal cord to supraspinal areas and descending through the dorsolateral funiculi to the spinal cord dorsal horn (Bouhassira, 11 Chitour, Villanueva, & Le Bars, 1993; Le Bars & Villanueva, 1988; Roby-Brami, Bussel, Willer, & Le Bars, 1987). DNIC relies on an intact nervous system, and is therefore presumed to be mediated by supraspinal structures (Le Bars, Dickenson, & Besson, 1979; Morton, Maisch, & Zimmermann, 1987). Lesions of brainstem nuclei such as PAG, RVM, locus coeruleus (LC), and parabrachial nucleus have no effect on DNIC (Bouhassira, Bing, & Le Bars, 1990; Bouhassira et al., 1993). However, DNIC is abolished by lesioning the subnucleus reticularis dorsalis (SRD; (Bouhassira, Villanueva, Bing, & le Bars, 1992; Villanueva, Bouhassira, & Le Bars, 1996). This finding, along with others that the SRD is favorably activated by nociceptive stimuli and has descending projections to the spinal cord dorsal horn, suggests that the SRD is a crucial structure for DNIC processing (Bernard, Villanueva, Carroue, & Le Bars, 1990; Villanueva et al., 1996). Peripherally, responses are inhibited by noxious stimulation of A-δ or C-fibers (Villanueva & Le Bars, 1985). Centrally, DNIC is dependent on an ascending and descending loop through ventro- and dorso-lateral funiculi (Villanueva, Chitour, & Le Bars, 1986). Pharmacological mediation of DNIC is still under investigation, although some reports indicate involvement of serotonergic and opioid systems. Nociceptive inhibition produced by DNIC is significantly reduced by systemic blockade of 5-HT receptors and is augmented after administration of the 5-HT precursor, 5hydroxytryptophan (5-HTP) (Chitour, Dickenson, & Le Bars, 1982). Opioidergic modulation of DNIC is less clear. Low dose morphine is said to decrease inhibition brought on by DNIC, which is reversed by naloxone (Le Bars, Villanueva, Bouhassira, & 12 Willer, 1992; Willer, Le Bars, & De Broucker, 1990). On the other hand, administration of naloxone alone has no effect on DNIC (Edwards, Ness, & Fillingim, 2004). Dysfunction of DNIC has been observed in populations of patients with functional disorders such as irritable bowel syndrome and fibromyalgia (Lautenbacher & Rollman, 1997; Wilder-Smith, Schindler, Lovblad, Redmond, & Nirkko, 2004). A dysfunction of DNIC has also been reported in patients with IC (Ness, Lloyd, & Fillingim, 2014). Alterations in DNIC function suggest an imbalance in nociceptive facilitation and inhibition. 2.2 Stress: Physiology and Role in Pain Perception 2.2.1 Physiology of Stress Response 2.2.1.1 Hypothalamic-Pituitary-Adrenal Axis. The body’s reaction to stress is mediated in part by the hypothalamic-pituitary-adrenal (HPA) axis. The role of the HPA axis is ultimately to increase glucocorticoid secretion, which, in turn, increases catecholamine secretion. The anatomical pathway of the HPA axis is from the medial parvocellular region of the paraventricular nucleus (PVN) of the hypothalamus to the corticotrope cells of the anterior pituitary to the zona fasciculata cells of the adrenal cortex, which send negative feedback information to the hypothalamus and anterior pituitary. During times of stress, neurosecretory cells from the PVN secrete corticotropin-releasing factor (CRF), a 41 amino acid peptide hormone, onto capillaries of the median eminence that penetrate the anterior pituitary. In the anterior pituitary, binding of CRF to the CRF type 1 receptor (CRFR1) stimulates the release of adrenocorticotropic hormone (ACTH; 39 amino acids; (Aguilera, Rabadan-Diehl, & 13 Nikodemova, 2001). Vasopressin (VP), a 9 amino acid peptide hormone, has also been shown to stimulate the release of ACTH from the anterior pituitary. CRF$increases$ transcription$of$proopiomelanocortin$(POMC),$a$precursor$to$ACTH,$as$well$as$ increases$cyclic adenosine monophosphate/protein kinase A (cAMP/PKA)$to$increase$ ACTH$release$from$cells.$ In$addition,$VP,$acting$at$VP1$receptors$causes$the$release$ of$ACTH$from$multiple$cells$(Levin, Blum, & Roberts, 1989). ACTH released from the anterior pituitary binds to ACTH receptors on the surface of adrenocoritical cells of the zona fasciculata. Binding of the receptor leads to activation of PKA, which phosphorylates cholesterol esterase and steroidogenic acute regulatory protein, both of which lead to synthesis of the glucocorticoid, cortisol in humans and corticosterone in rodents and many non-mammals. Cortisol is involved in numerous physiological activities, including: stimulation of gluconeogenesis in the liver to increase blood glucose, stimulation of glycogenesis to increase glycogen in the liver, promotion of protein catabolism in skeletal muscle to provide an energy source (amino acids) for gluconeogenesis, promotion of fat catabolism also to provide an energy source (glycerol) for gluconeogenesis, induction of phenylethanolamine-N-methyltransferase (PNMT) to synthesize catecholamines, and suppression of the immune response. Completing the HPA cycle, glucocorticoids inhibit the release of CRF from the hypothalamus (Sakakura, Yoshioka, Kobayashi, & Takebe, 1981). Glucocorticoids, such as cortisol or corticosterone, released from the adrenal cortex exert negative feedback on CRF and ACTH. Two classes of glucocorticoid receptors are located throughout the brain. Type I, cortisol/corticosterone receptors, are found primarily in limbic neurons, while Type II receptors are found throughout the 14 hypothalamus (McEwen, 1994). Type I receptors respond mainly to emotional and environmental stimuli, while Type II receptors have a strong affiliation with the negative feedback effects of cortisol (Sapolsky, Krey, & McEwen, 1986) CRF is also present in neurons outside of the hypothalamus in brainstem, midbrain, limbic areas, cerebral cortex, and spinal cord (De Souza et al., 1985). CRF is regulated by factors such as pain, emotion, and blood pressure. In particular, an increase in blood pressure exerts an inhibitory response on CFR secretion, while a decrease in blood pressure results in higher levels of CRF secretion (Ganong, 1988). CRF is also stimulated by NE and E, acetylcholine, and serotonin. CRF secretion is inhibited by neurotransmitters such as GABA, opioids, ACTH, and glucocorticoids (Calogero, Bernardini, Gold, & Chrousos, 1988). . 2.2.1.2 LC-NE/Sympatho-Medullary Systems. The LC-NE/sympathetic systems represent the principal autonomic response to external stress. The LC is the primary source of NE-producing neurons in the brain with effects of arousal and increased anxiety (Shimizu, Katoh, Hida, & Satoh, 1979). Sympathetic activation results in release of NE from the adrenal medulla, and is typically advantageous to the organism in a stressful situation. Sympatho-medullary NE and E generally stimulate the regulation of catecholamine biosynthesis (Landsberg & Weiss, 1976; McCarty, 1983). This is demonstrated in the studies showing that acute stress results in an increase in secretion of sympatho-medullary NE and E, while chronic stress results in increased catecholamine synthesis and catecholamine concentrations in rat blood (Dobrakovova et al., 1984; Tilders & Berkenbosch, 1986). 15 2.2.2 Models of Laboratory Stress Stress may be categorized as physical or psychological. Physical (extereoceptive; systemic) stress is induced by stimuli that produce actual physical perturbations which offset homeostasis. Psychological (interoceptive; neurogenic; processive) stress is elicited by stimuli that compromises the organism’s perceived or anticipated state (Dayas, Buller, Crane, Xu, & Day, 2001; Herman & Cullinan, 1997). Stressors may be further classified as acute or chronic depending on the temporal presentation of the stimuli. An acute stressor is presented one time, while a chronic stressor is usually presented for 7-10 days consecutively. Purely physical stressors include the following: cold water immersion, in which rats are placed in a tank of cold water without the ability to escape for 15 min; cold environment, in which rats are placed in a 4°C cold room for 15 min; hemorrhage, in which a total of around 12 ml/kg of blood is removed; and immune challenge, in which a pro-inflammatory cytokine such as interleukin-1β is injected to induce an inflammatory response (Dayas et al., 2001; Jaggi et al., 2011). Purely psychological stressors include the following: maternal separation, in which rat pups are separated from their dams each day for around 3 h; predatory stress, in which an animal is exposed to its natural predator or the scent of the predator; social defeat, in which an “intruder” rat is placed into a cage of “resident” rats until the intruder displays subordinate postures; water avoidance, in which rats are placed on a small platform surrounded by water on all sides; and noise stress, in which the animal is exposed to aversive auditory stimuli such as white noise (Campos et al., 2013; Dayas et al., 2001; Jaggi et al., 2011). Both physical and psychological stressors have been shown to 16 activate neurons in the PVN, while physical stress preferentially activates the central amygdala and psychological stress preferentially activates the medial amygdala (Dayas et al., 2001). A subset of stress models are not consistently labeled as physical or psychological, and thus they are labeled as mixed physical-psychological. Mixed stressors include the following: forced swim, in which rats are placed in a cylindrical tank filled with water to require swimming; restraint stress, in which animals are placed in a conical or cylindrical tube or their limbs are taped to a surgical board to immobilize the animal; and footshock stress, in which animals are placed on an electrified floor-grid that delivers mild electrical shock at variable intervals (VI; (Dayas et al., 2001; Jaggi et al., 2011; Kant, Mougey, Pennington, & Meyerhoff, 1983). The current experimental procedures employ chronic footshock to induce stress in the rat. Footshock is considered a mixed paradigm due to the physical component of electrical shock and the psychological component of unpredictability in the timing of the shocks. Stress paradigms typically must demonstrate activation of the HPA axis to ensure that the stimulus is indeed inducing a stress response (Bodnar, Glusman, Brutus, Spiaggia, & Kelly, 1979; Bonaz & Tache, 1994; Kant et al., 1983). Kant et al. (1983) demonstrated that footshocks ranging from 0.2-3.2 mA delivered to rats on a VI schedule produced increased plasma stress-related hormones, including corticosterone and beta-endorphin. 2.2.3 Stress as an Analgesic Historically, acute stress elicits a fight or flight response. In this situation, nociceptive processes are blunted, allowing the organism to respond in the most adaptive 17 manner. Indeed, if an animal had to stop to attend to an injured limb while in pursuit, it would most likely fall prey to its predator. The first studies of stress-induced analgesia (SIA) reported an increase in pain threshold to a cutaneous noxious stimulus after inescapable footshock that was not always reversible by naloxone administration (Hayes, Price, Bennett, Wilcox, & Mayer, 1978). Neurotransmitter systems involved in SIA typically include glutamate, GABA, glycine, vasopressin, oxytocin, opioids, and cannabinoids (Butler & Finn, 2009; Marek, Mogil, Sternberg, Panocka, & Liebeskind, 1992). The HPA axis also plays a role in SIA; hypophysectomized rats demonstrated a reversal of cold water swim SIA (Bodnar et al., 1979). 2.2.4 Stress as an Exacerbator of Pain In contrast to SIA, stress-induced hyperalgesia (SIH) occurs in response to stress that is particularly unpredictable and uncontrollable. In this case, pain is augmented instead of inhibited. Indeed, stress has been shown to increase nociceptive responses in animal models of visceral pain. Clinically, stress leads to exacerbation of symptoms in irritable bowel syndrome, IC, and other chronic visceral pain disorders (Dickhaus et al., 2003; Klausner et al., 2005; Larauche, Kiank, & Tache, 2009). Chronic footshock stress induces urinary bladder hypersensitivity characterized by an increase in the VMR response to UBD (Robbins & Ness, 2008). Chronic water avoidance stress also induced hyperresponsive electromyographic (EMG) activity to UBD (Robbins, DeBerry, & Ness, 2007). In colorectal pain studies, chronic water avoidance stress enhanced EMG responses to colorectal distension (CRD; (Bradesi et al., 2005; Hong et al., 2009; Wang et al., 2013). Furthermore, using cerebral blood flow mapping, stressed rats showed greater 18 activation of insular cortex, amygdala, and hypothalamus and less activation in the prelimbic area of prefrontal cortex evoked by CRD. Stressed rats lacked the negative correlation found between the prelimbic area and the amygdala, which was found in sham rats (Wang et al., 2013). Chronic variant stress using the paradigm of alternating cold, restraint, water avoidance, or forced swim daily for 9 days produced colorectal hypersensitivity (Winston, Xu, & Sarna, 2010). It would appear that clinical populations and animal models alike have an increased sensitivity to chronic stress resulting in enhanced visceral nociception. 2.3 Serotonin: Physiological Function and Role in Stress and Pain Systems 2.3.1 Serotonergic Receptors/Function 2.3.1.1 Overview. Seven receptor families comprise the receptors with affinity for the monoamine, 5-HT. This section will discuss the location, function, and mechanism of action of each receptor subtype. Nevertheless, this is far from being an exhaustive list, as that would extend beyond the scope of this report (for review see Barnes & Sharpe, 1999). The seven families are categorized by mechanism of action and/or G-protein coupling. The 5-HT1 receptors are coupled to the Gi/Go protein, and activation leads to a decrease in cAMP production. 5-HT2 receptors are coupled to the Gq11 protein. Activation of 5-HT2 leads to an increase in inositol triphosphate and diacyl glycerol, enhancing protein phosphorylation and excitatory neurotransmission. The 5-HT3 receptor is the only ionotropic receptor of the 5-HT super family of receptors, and its activation leads to an excitatory neuronal response. Receptors 5-HT4, 5-HT6, and 5-HT7 19 are Gs-protein coupled, and produce an excitatory response by increasing levels of cAMP. The 5-HT5 receptors are coupled to the Gi/Go protein. Activation leads to an inhibitory response via a decrease in cAMP. 2.3.1.2 5-HT1A. The 5-HT1A receptor is the most widespread, encompassing areas in the cortex, hippocampus, amygdala, raphe nucleus, basal ganglia, and thalamus, and it has a diverse range of functions (Glennon et al., 2000; Ito, Halldin, & Farde, 1999). Activation of 5-HT1A receptors in the RVM produces a decrease in blood pressure and heart rate via peripheral vasodilation. Vagal nerve stimulation through 5-HT1A activation also contributes to these vascular effects (Dabire, 1991). Mood disorders such as anxiety and depression show a reduction with receptor activation (Kennett, Dourish, & Curzon, 1987; Parks, Robinson, Sibille, Shenk, & Toth, 1998). In addition, antiemetic and analgesic effects are observed with activation of 5-HT1A receptors in the dorsal raphe nucleus that are colocalized with neurokinin 1 receptors, modulating the release of substance P (Baker et al., 1991). 5-HT1A activation enhances dopamine release within the medial prefrontal cortex, striatum, and hippocampus (Bantick, De Vries, & Grasby, 2005; Li, Ichikawa, Dai, & Meltzer, 2004). Conversely, stimulation inhibits the release of glutamate and acetylcholine, temporarily impairing learning and memory (Ogren et al., 2008). On the endocrine level, activation induces the secretion of HPA-axis hormones, including cortisol/corticosterone and ACTH, as well as oxytocin, prolactin, and β-endorphin (Koenig, Gudelsky, & Meltzer, 1987; Lorens & Van de Kar, 1987; Pitchot, Wauthy, Legros, & Ansseau, 2004). 20 In addition to the multitude of physiological effects of 5-HT1A receptor activation, its role as an autoreceptor is equally as important. In this respect, 5-HT1A receptors located on 5-HT-releasing neurons inhibit further release of 5-HT. The action of 5-HT1A autoreceptors is reported to be a large contributing factor in the therapeutic lag of selective serotonin reuptake inhibitors (SSRI). The autoreceptors must become desensitized to allow an increase in extracellular 5-HT (Hjorth et al., 2000). 2.3.1.3 5-HT1B. Receptors of the 5-HT1B class are located throughout the brain, mainly in frontal cortex, basal ganglia, striatum, and hippocampus, as well as within blood vessels (Hen, 1993; Maroteaux et al., 1992; Martin et al., 1997). In the CNS, activation is attributed to diminished release of dopamine and glutamate (Huang, Yeh, Wu, & Hsu, 2013). The more prominent role of 5-HT1B receptors appears to be in vasoconstriction. Agonists of 5-HT1B include various triptan drugs and ergotamine which exert powerful vasoconstricting effects used in the treatment of migraine headaches. 2.3.1.4 5-HT1D. 5-HT1D receptors are predominantly expressed in basal ganglia but also in blood vessels (Cubero et al., 2011; Domenech, Beleta, & Palacios, 1997; Longmore et al., 1997). Activation of central 5-HT1D receptors presynaptically inhibits synaptic transmission, contributing to modulation of locomotion (Schwartz, Gerachshenko, & Alford, 2005). Like the 5-HT1B agonists, those of 5-HT1D are also involved in the mitigation of migraine pain via vasoconstriction. 21 2.3.1.5 5-HT1E. Putative 5-HT1E receptors are located in the frontal cortex, hippocampus, and olfactory bulb (Bai et al., 2004). While their function has yet to be elucidated, theories include involvement in the improvement of learning and memory (Brudeli et al., 2010). 2.3.1.6 5-HT1F. Most closely related to 5-HT1E, the 5-HT1F receptor shares a 70% sequence homology (Barnes & Sharp, 1999). 5-HT1F receptors are located in the dorsal raphe, hippocampus, cortex, striatum, thalamus, and hypothalamus (Cubero et al., 2011). 5-HT1F is targeted by sumatriptan, suggesting a possible role in migraine treatment (Hamon & Bourgoin, 2000; Waeber & Moskowitz, 1995). 2.3.1.7 5-HT2A. The 5-HT2A receptor is highly expressed in cortical brain regions, especially in layer V pyramidal cells (Aghajanian & Marek, 1999). Receptors are also observed in blood vessels, and particularly in platelets, where activation promotes platelet aggregation and vasoconstriction (Gomez-Gil et al., 2002). The 5HT2A subtype is highly associated with the effects of psychedelic drugs such as lysergic acid diethylamide (LSD), mescaline, and psilocybin (McCorvy, Harland, Maglathlin, & Nichols, 2011). Antagonism of 5-HT2A is the target of many antipsychotic medications (Seeman, 2002). 2.3.1.8 5-HT2B. 5-HT2B receptors are located in cerebellum, lateral septum, hypothalamus, and medial amygdala (Duxon et al., 1997). These receptors play a pivotal role in cardiovascular function. The receptor is critical for proper cardiac development as 22 demonstrated by a knock-out mouse model (Nebigil, Etienne, Messaddeq, & Maroteaux, 2003). In the periphery, activation of vascular 5-HT2B receptors stimulates pulmonary vasoconstriction (Launay et al., 2002). There is a strong implication of 5-HT2B receptor activation in valvular heart disease induced by the use of the weight-loss drug fenfluramine (Connolly et al., 1997). In the CNS, activation of receptors may induce psychedelic behavioral effects with drugs such as 3,4-methylenedioxy-Nmethylamphetamine (MDMA; (Doly et al., 2008). 2.3.1.9 5-HT2C. One of the most ubiquitous 5-HT receptors, 5-HT2C is located in the brain in the prefrontal cortex, striatum, nucleus accumbens, hippocampus, hypothalamus, and amygdala (Pompeiano, Palacios, & Mengod, 1994). In the periphery, receptors have been shown in blood vessels, platelets, gastro-intestinal (GI) tract, and smooth muscle. 5-HT2C receptors perform an important function in the regulation of mood, feeding, and reproduction (Heisler et al., 2007). Mood regulation is generally achieved through antagonism of 5-HT2C receptors (Berg, Harvey, Spampinato, & Clarke, 2005; Ni & Miledi, 1997). 5-HT2C activation by SSRIs also contributes to the therapeutic lag since the receptors must downregulate over the first 1-2 week period to achieve the desired antidepressive effects (Berg et al., 2005). Many drugs of addiction, including cocaine, opiates, caffeine, and nicotine, exert antagonistic properties on 5-HT2C receptors, increasing the release of dopamine, therefore contributing to drug reinforcement (Bubar & Cunningham, 2006). In the paraventricular nucleus, activation of 5-HT2C receptors is responsible for the increased release of CRF and vasopressin (Heisler et al., 2007). 23 2.3.1.10 5-HT3. Expression of the 5-HT3 receptor has been shown extensively throughout the central and peripheral nervous system (Yakel & Jackson, 1988). The major function of the 5-HT3 receptor is its mediation of emesis, specifically by enhancing the emetic effects of radiation and chemotherapy. Consequently, antagonists of 5-HT3 receptors are used for their antiemetic effects (Thompson, 2013). 2.3.1.11 5-HT4. 5-HT4 receptors are located within the GI tract, peripheral nervous system, and brain areas, including basal ganglia, raphe, pontine nuclei, and thalamus (Varnas, Halldin, Pike, & Hall, 2003). 5-HT4 activation increases GI motility by stimulating acetylcholine release (Martin & Humphrey, 1994). Additionally, it has been hypothesized that 5-HT4 activation enhances memory performance via increased synaptic transmission within the CNS (Barnes & Sharp, 1999). 2.3.1.12 5-HT5A. Little is known about the location and function of 5-HT5A receptors, although expression has been demonstrated on astrocytes and specific motor neurons (Carson, Thomas, Danielson, & Sutcliffe, 1996; Xu et al., 2007). Regulation of sleep has been reported through 5HT5A agonism via valerenic acid (Dietz, Mahady, Pauli, & Farnsworth, 2005). 2.3.1.13 5-HT6. The 5-HT6 receptor is restricted to, yet widely distributed throughout, the brain (Gerard et al., 1997; Kohen et al., 1996; Woolley, Marsden, & Fone, 2004). Antagonism of the receptor increases the release of excitatory 24 neurotransmitters, including glutamate, acetylcholine, dopamine, and NE (Dawson, Nguyen, & Li, 2000, 2001; Lacroix, Dawson, Hagan, & Heidbreder, 2004). Antagonists provide the greatest therapeutic value, ranging from improvement in learning and memory to the treatment of obesity (Heal, Smith, Fisas, Codony, & Buschmann, 2008; King, Marsden, & Fone, 2008). In contrast, activation of 5-HT6 receptors enhances GABA signaling, and 5-HT6 agonists have improved mood disorders (Schechter et al., 2008). These receptors may be a critical component in the mechanism of SSRI therapy (Carr, Schechter, & Lucki, 2011). 2.3.1.14 5-HT7. The 5-HT7 receptor is expressed in the brain, GI tract, and blood vessels (Bard et al., 1993). Blockade of the 5-HT7 receptor produces antipsychotic and antidepressive effects (Mullins, Gianutsos, & Eison, 1999; Roth et al., 1994). The 5HT7 receptor has also been implicated in sleep, circadian rhythms, and thermoregulation to a lesser degree (for review, see (Hedlund, 2009). 2.3.2 Relationship of Serotonin and Stress Chronic stress induces changes in many neuroendocrine systems, including the monoamines. In rats, exposure to 1h of restraint-stress for 24 days resulted in increased brain levels of 5-HT (Adell & Artigas, 1998), and increased levels of tryptophan hydroxylase were observed in the RVM of rats exposed to 3 weeks of restraint stress (Imbe, Iwai-Liao, & Senba, 2006). Chronic exposure to an elevated open platform resulted in a sustained increase in the release and turnover of 5-HT in rats (Storey et al., 2006). These examples indicate that chronic stress leads to an increase in 5-HT 25 production, release, and turnover in the CNS. Typically lower levels of 5-HT are associated with individuals who are severely depressed or have committed suicide. It may be the case that especially in the early stages of chronic stress, 5-HT production and activity are increased as a coping mechanism. Furthermore, 5-HT facilitates stress-induced effects in rats via 5-HT1A and 5HT3 receptor activation. In a model of stress-induced colorectal pain, treatment with the 5-HT3 antagonist, alosetron, abolished visceral hypersensitivity (Bradesi et al., 2007). Chronic stress-induced decrease in sucrose consumption was ameliorated with administration of the 5-HT1A antagonist, WAY 100635 (Papp, Nalepa, AntkiewiczMichaluk, & Sanchez, 2002). Activation of serotonergic receptors facilitate the VMR response to UBD in rats with zymosan-inflamed bladders (Randich, Shaffer, Ball, & Mebane, 2008). Intrathecal administration of the non-selective 5-HT receptor antagonist, methysergide; 5-HT1A-specific antagonist, WAY 100635; or 5-HT3-specific antagonist, ondansetron, resulted in a decrease in the VMR response to UBD in rats pretreated with intravesicle zymosan. These data further support a link between stress, visceral hypersensitivity and the 5-HT1A and 5-HT3 receptors. 2.3.4 Serotonergic Pain Modulation The role of 5-HT in nociceptive signaling is complicated; there is evidence that it may facilitate or inhibit pain. Most 5-HT-producing cells that have projections to the spinal cord dorsal horn are located in the rostral ventromedulla (RVM). Electrical 26 stimulation of the nucleus raphe magnus of the RVM increased latency in the tail withdrawal to noxious heat. This effect was blocked by intrathecal administration of the 5-HT receptor antagonist, methysergide (Hammond & Yaksh, 1984), demonstrating an inhibitory role for 5HT in nociceptive processing. Wei et al. (2010) selectively knocked down 5-HT-producing cells in the RVM using ribonucleic acid-interference (RNAi). Recombinant plasmids encoding for tryptophan hydroxylase 2 (Tph-2) short hairpinRNA (shRNA) were microinjected into the RVM of adult rats. Using this model, rats were then tested for their response to the formalin test (Tjolsen, Berge, Hunskaar, Rosland, & Hole, 1992), a nociceptive test that evaluates acute (phase 1) and persistent (phase 2) pain. During phase 1, there was no difference in nocifensive responses between RVM 5-HT-depleted rats and controls. On the other hand, pain behaviors were significantly reduced during phase 2 in RVM 5-HT-depleted rats, suggesting a facilitatory role of 5-HT, particularly in persistent pain (Wei et al., 2010). Also utilizing RVM 5-HT-depletion, Wei et al. (2010) further assessed persistent pain in the spinal nerve ligation (SNL) model of neuropathic pain. In control rats, SNL produced thermal and mechanical hyperalgesia 14 d before and after gene transfer. RVM 5-HT-depleted rats showed a significant reduction in thermal and mechanical hyperalgesia up to 7 d after gene transfer. Whether 5-HT is facilitatory or inhibitory may depend on which of the myriad 5HT receptors is activated. While there are at least 15 known 5-HT receptor subtypes, 5HT1A and 5-HT3 are predominant in the spinal cord and have a more defined role in nociceptive processing (Hochman, 2001). Activation of 5-HT1A has been shown to facilitate pain signals in the dorsal horn, possibly as an indirect or secondary mechanism 27 (Ali, Wu, Kozlov, & Barasi, 1994; Zhang et al., 2001). To illustrate this point, Song, et al. (2007), utilized intrathecal administration of the 5-HT1A agonist, 8-Hydroxy-N,Ndipropyl-2-aminotetralin (8-OH-DPAT), which produced inhibition of mu opioid receptor internalization, a proxy for determining mu opioid receptor activation. This was reversed with the co-administration of 8-OH-DPAT and the 5-HT1A antagonist, WAY 100135, suggesting 5-HT1A-mediated inhibition of opioid release in the spinal cord. Intrathecal 8-OH-DPAT also increased the responsiveness of WDR neurons to electrical stimulation (Zhang et al., 2001). 5-HT3 receptors are located on fibers and primary afferent terminals (Kidd et al., 1993) and have been implicated to a greater extent in nociceptive augmentation (Asante & Dickenson, 2010; Suzuki et al., 2004). Their activation leads to the release of substance P, calcitonin gene related peptide, and neurokinin A, all of which are involved in inflammation and nociceptive processing (Inoue, Hashimoto, Hide, Nishio, & Nakata, 1997; Saria, Javorsky, Humpel, & Gamse, 1990). In the formalin test, intrathecal administration of the 5-HT3 receptor antagonist, ondansetron, attenuated hyperexcitability of dorsal horn neurons during the late phase (Svensson, Tran, Fitzsimmons, Yaksh, & Hua, 2006). Ondansetron also attenuated neuronal responses to mechanical stimuli in rats with spinal nerve ligation (Suzuki et al., 2004). Mice with null 5-HT3 receptors via knock-out of the A subunit of the 5-HT3 receptor did not differ in the nociceptive behavioral responses to phase 1 of the formalin test but displayed a significant reduction of pain behavior during phase 2 (Zeitz et al., 2002). Furthermore, activity of spinal dorsal horn neurons during the formalin test was significantly attenuated during phase 2 in knock-out mice compared to wild-type (Zeitz et al., 2002). These 28 findings implicate a facilitatory role of 5-HT in spinal pain transmission, especially under persistent pain conditions. 2.4 Urinary Bladder: Physiology and Interaction with Stress and Serotonin 2.4.1%%Bladder%Innervation% The bladder is innervated by 3 major nerves: the hypogastric, pelvic, and pudendal, which extend from the thoracolumbar and lumbosacral spinal cord segments (De Groat, 1986, 1998). Afferent innervation of the bladder is primarily through the hypogastric and pelvic nerves with cell bodies located in thoracolumbar and lumbosacral dorsal root ganglia, respectively. Information related to bladder filling is generally transmitted via the hypogastric and pelvic nerve, whereas messages encoding a full bladder are transmitted via the pelvic and pudendal nerve (Andersson, 2002). Bladder afferents terminate in laminae I, V, and VII in the spinal cord dorsal horn, where they form synapses with second-order neurons (Morgan, Nadelhaft, & de Groat, 1981; Thor, Morgan, Nadelhaft, Houston, & De Groat, 1989). Sensory information from the bladder travels through Aδ-fibers and C-fibers (Habler, Janig, & Koltzenburg, 1990, 1993). Both, to some degree, transmit information regarding bladder filling, but under conditions of noxious stimulation, such as over-filling, injury, or inflammation, C-fibers represent the majority of transmission fibers (de Groat, 1998). Control of micturition is influenced by spinal and bulbospinal reflexes. Sympathetic spinal reflexes inhibit bladder contractions and increase urethral muscle tone during bladder filling. When the bladder reaches its urine capacity, a parasympathetic spinobulbospinal reflex activating the pontine micturition center causes the bladder to 29 contract and relaxes urethral muscle, allowing voiding of urine to take place (de Groat, 1998). 2.4.2 Models of Bladder Nociception In humans with IC, symptoms and pathologies are not consistent across the board. For example, cystoscopy of IC patients may reveal submucosal petechial hemorrhages (glomerulations), Hunner’s fissures (ulcers), or a combination of both. However, only a small percentage of patients have Hunner’s ulcers, and these patients tend to experience greater severity of symptoms (Tomaszewski et al., 2001; Warren, Jackson, Langenberg, Meyers, & Xu, 2004). Recent data from women undergoing tubal ligation revealed that glomerulations may also be present in bladders of women without IC (Hanno, Burks, et al., 2011; Payne, Joyce, Wise, & Clemens, 2007). Examination of biopsy tissue may or may not reveal inflammation, and bladders may appear completely normal (Waxman, Sulak, & Kuehl, 1998b). Bearing this in mind, it is difficult to produce an animal model that truly mimics the general population of IC patients. The most common animal models of bladder pain typically involve administration of an agent into the bladders of rats, rabbits, and monkeys to induce an inflammatory response (Ghoniem, Shaaban, & Clarke, 1995; Kato, Kitada, Longhurst, Wein, & Levin, 1990; Shimizu, Kawashima, & Hosoki, 1999). These irritants include acetic acid, acetone, croton oil, lipopolysaccharide, mustard oil, ovalbumin, turpentine, and zymosan (Ghoniem et al., 1995; Kato et al., 1990; McMahon & Abel, 1987; Randich, Uzzell, Cannon, & Ness, 2006; Shimizu et al., 1999; Westropp & Buffington, 2002). Changes observed in these models include an increased presence of inflammatory cells such as 30 mast cells and neutrophils, decreased bladder capacity, decreased voiding volume, bladder hyperreflexia, and plasma extravasation (Elgebaly et al., 1992; McMahon & Abel, 1987; McMahon, Dmitrieva, & Koltzenburg, 1995). These models produce urodynamic effects, similar to what is observed in IC patients, such as decreased bladder capacity and decreased voiding volume (Kuo, Chang, & Hsu, 1992). Biopsies of bladder tissue frequently reveal submucosal inflammation with infiltrates (Rosamilia, Igawa, & Higashi, 2003). Evaluating bladder nociception is most commonly and reliably performed by measuring the VMR response to bladder distension (Ness, Lewis-Sides, & Castroman, 2001; Su, Riedel, Leon, & Laping, 2008b). The bladder is distended in a graded manner (10-60 mmHg) by air administration via a transurethral catheter, and electrodes are placed in the external oblique musculature of the rat to record abdominal contractions to distensions. UBD produces increases in abdominal EMG activity, heart rate, and arterial blood pressure in female rats (Ness et al., 2001). Intravesical instillation of the yeast cell wall component, zymosan, produces an augmentation of these responses to UBD (Randich et al., 2006). Bladder histology in rats with zymosan-induced inflammation was similar to studies in humans with IC demonstrating essentially normal bladder histology in that there were no gross changes to bladder thickness, fibrosis, or mast cells (DeBerry, Randich, Shaffer, Robbins, & Ness, 2010). Furthermore, rats with bladders inflamed with zymosan showed an increased VMR response to intravesical ice-water testing-- a parallel finding to a human study in which IC patients reported increased pain to intravesical ice-water compared to healthy controls (Mukerji et al., 2006; Randich, Mebane, & Ness, 2009). 31 A critical finding among human IC patients is the correlation of stress and anxiety with exacerbation of symptoms. Rothrock et al. (2001) reported greater levels of daily stress and bladder symptoms in IC patients vs. matched controls who were asked to keep a one-month diary of symptoms and stress levels. A positive correlation was also observed between disease severity and symptoms of disease/stress levels in IC patients. Patients with IC had higher urinary cortisol levels compared to controls (Lutgendorf et al., 2002). Stress-induced bladder hypersensitivity can be produced in animals using a chronic footshock paradigm. Seven consecutive days of chronic intermittent footshock produces a reliable increase in the magnitude of the EMG response to UBD (Robbins & Ness, 2008). This stress-induced hypersensitivity model may be of greater clinical relevance due to the comorbidity of anxiety-related disorders in IC patients (Chung et al., 2014) and the difficulty of diagnosis based on histological observations (Waxman, Sulak, & Kuehl, 1998a). 2.4.3 Role of Serotonin in Bladder Function Serotonin influence on bladder activity is not straightforward; most reports conclude that the site of action of serotonergic modulation takes place within the CNS. Intravenous and intrathecal serotonergic agonists generally facilitate sympathetic activity and suppress parasympathetic activity (Espey, Downie, & Fine, 1992; Thor, Hisamitsu, & de Groat, 1990; Thor, Katofiasc, Danuser, Springer, & Schaus, 2002). The raphe nuclei send serotonergic projections to the lumbosacral spinal cord, where multiple 5-HT receptor subtypes have been localized, including 5-HT1A, 5-HT1B, 5-HT1D, 5-HT2A, and 5-HT3 receptors. 5-HT1A and 5-HT3 receptors are predominantly expressed and are 32 the focus of 5-HT receptor studies of bladder function in the literature Specifically, 5HT1A receptors are believed to have an excitatory influence on micturition at supraspinal and spinal levels (Ramage, 2006). 5-HT1A receptor activation by intrathecal agonists increased bladder contractions (Kakizaki, Yoshiyama, Koyanagi, & De Groat, 2001) and decreased micturition pressure and urine volume voiding threshold in rats (Conley, Williams, Ford, & Ramage, 2001). The 5-HT1A receptor antagonist, WAY 100635, given intravenously increased bladder capacity in anesthetized and unanesthetized rats in a dose-dependent manner (Conley et al., 2001; Testa et al., 1999). Mediation of bladder function via 5-HT3 receptors is not as well characterized and differs between studies in cats and rats. In the cat, intrathecal blockade by the 5-HT3 receptor antagonist, zatosetron, resulted in decreased volume threshold for micturition (Espey & Downie, 1995). Antagonists also produced an augmentation in pelvic nerve-evoked activity in the thoracolumbar spinal cord (Espey, Du, & Downie, 1998). In rats, neither intravenous or intracerebroventricular administration of agonists nor antagonists to 5-HT3 receptors produced any change to bladder contractility or capacity (Ishizuka et al., 2002; Testa et al., 2001). 2.5 Spinal Neuronal Characterization Neurons of the spinal cord dorsal horn that respond to mechanical stimulation of the bladder have been systematically characterized and can be divided into two distinct populations, Type I and Type II (Ness & Castroman, 2001). This nomenclature is analogous to that used in models of colorectal distension, which label neurons as Abrupt and Sustained, respectively (Ji, Murphy, & Traub, 2003; Ness & Gebhart, 1989). While 33 both neuronal populations respond in a graded manner to increasing intravesical pressure (Ness & Castroman, 2001), there are differences between Type I and Type II neurons. The first distinction is in the response to counter-irritation, also referred to as a heterotopic noxious conditioning stimulus (HNCS), such as a painful pinch or noxious heat applied to a dermatome distal to the recording site. Type I neuronal activity is inhibited by HNCS, while Type II neuronal activity is not inhibited by similar stimuli (Ness & Castroman, 2001). Another way these two neuronal populations differ is in their response to analgesics. Intravenous morphine and lidocaine produced a dose-dependent inhibition of UBD-evoked activity in Type II neurons, but no effect of these drugs was found in Type I neurons (Ness & Castroman, 2001). In rats receiving morphine, intravenous naloxone reversed the inhibitory effects of morphine Type II neurons are thought to send excitatory pain signals to higher-order brainstem nuclei, while Type I neurons send inhibitory messages involved in pain perception. These two neuronal types can be further characterized by their responses to zymosan-induced bladder inflammation. Spontaneous and UBD-evoked activity of Type I neurons was either decreased or unaffected following inflammation, while Type II neurons showed increased spontaneous and evoked activity (Ness, Castroman, & Randich, 2009). In addition to Type I and Type II classification, neurons that respond to bladder distension can also be categorized based on cutaneous receptive field properties. Class 2 or WDR neurons respond to noxious and non-noxious cutaneous stimuli, while Class 3 or NS neurons respond only to noxious stimuli. Class 1 neurons respond to non-noxious 34 stimulation only. One electrophysiological study reported that of the Type I neurons, 88% were found to be Class 2 and 12% were Class 3. Of the Type II neurons, 60% were Class 2 and 40% were Class 3 (Ness & Castroman, 2001). 35 CHAPTER 3 METHODS 3.1 Selection of Species Female virgin rats were used in all experiments. Rats were chosen based on their extensive use in behavioral, neurochemical, neurophysiological, and pharmacological studies of visceral pain, as well as their similarities shared with primates (Willis, 1985). Females were chosen because of the disproportionate bias toward women in the patient population of IC with the end result of this research to be instrumental in the translation from bench to bedside (Hanno, Burks, et al., 2011). Sprague Dawley rats weighing 200-250 g were used in methodological development experiments. Lewis rats weighing 150-200 g were used in all other experiments, including methodological development. All rats were obtained from Harlan (Prattville, AL, USA). Rats were allowed ad libitum access to food and water in temperature-controlled quarters. The light-dark cycle for all rats was 06:00-18:00. A one-week habituation period was allowed from the date of delivery to our animal facility to the commencement of any experimental procedures. All studies were approved by the University of Alabama at Birmingham Institutional Animal Care and Use Committee and conformed to the guidelines of the International Association for the Study of Pain. 36 3.2 Anesthesia Rats were anesthetized throughout all surgical and pretreatment procedures. Rats received an inhalation induction of anesthesia using a tight-fitting mask, which delivered an isoflurane/oxygen mixture with levels of isoflurane at 5%. All surgical procedures (venous and tracheal cannulae, intravesical catheters, incisions, and injections) occurred under 3% isoflurane anesthesia. In spinal electrophysiological experiments, intratracheal, intravenous, and intravesical cannulae were placed, at which point the animal was moved to an artificial ventilator maintained at 62 bpm and placed on a heating pad to maintain body temperature at ~37C. After electrode placement, anesthesia was reduced to 0.750.8% isoflurane for the duration of neuronal recording. In the VMR experiments anesthesia was maintained at 1-1.25% isoflurane. This level of anesthesia is sufficient to prevent spontaneous movement, but still allows for flexion-withdrawal reflexes in response to noxious stimuli. 3.3 Footshock Protocol Two groups of rats were established. The experimental group was termed chronic footshock and the control group was termed non-footshock. Rats in both groups were placed inside of operant conditioning boxes contained within sound and light attenuating chambers. In the chronic footshock group, shocks of 1.0 mA intensity (1 sec duration) were delivered via a parallel rod floor 30 times over a 15 min period on a variable interval schedule which consisted of randomized inter-shock intervals ranging from 1 sec to 3 min. Footshock treatments occurred once per day for 7 days. Rats in the non- 37 footshock group were placed in the chambers for 15 min once per day for 7 days, but did not receive footshock. 3.4 Urinary Bladder Distension A 22-gauge polytetrafluoroethylene angiocather was transurethrally placed in the bladder and held by a tight suture around the distal urethral orifice. Intravesical pressure was monitored continuously by an inline, pneumatically-linked, low-volume pressure transducer and was controlled using a pressure control device (Anderson 1987). Bladders were distended in a phasic manner with constant-pressure air for 20 sec durations via the urethral catheter. In the VMR experiments, baseline responses to three 60 mmHg distensions were followed by ascending graded distensions (10-60 mmHg). In electrophysiology experiments, baseline neuronal responses (consisting of at least 3 trials) were recorded using phasic distending pressures of 60 mmHg, followed by responses to ascending graded distensions at pressures of 20, 40, and 60 mmHg. The intertrial interval was 3 min. 3.5 Receptor Antagonists Receptor antagonists, WAY 100635 and ondansetron, were obtained from Sigma (St. Louis, MO, USA). All drugs were dissolved in 15 µl physiological saline (0.9% NaCl in ultrapure water [Millipore,$Billerica, MA, USA]), and the saline vehicle was used in control experiments. In VMR experiments, 10 µg of receptor antagonists were administered. In electrophysiology experiments, ondansetron was administered at a dose of 100 µg. Compounds were chosen based on their selectivity for 5-HT receptors. WAY 38 100635 selectively blocks the 5HT1A receptor, and ondansetron selectively blocks the 5HT3 receptor. Drug doses were chosen based on a previous study in which 10 µg of either WAY 100635 or ondansetron significantly inhibited VMR responses to UBD in rats with zymosan-induced bladder inflammation (Randich, Shaffer, et al., 2008). 3.6 Implantation of Intrathecal Catheters Under deep anesthesia (isoflurane 3%), rats were secured in a stereotaxic device. Catheters consisted of polyethylene (PE) 10 tubing (BD Intramedic, NJ, USA). A loose knot was formed in the tubing and secured using dental cement. The catheters were trimmed 7.8 cm below the knot. Using a #11 scalpel blade, a 1 cm midline incision was made to expose the atlanto-occipital membrane. A small slit was made in the atlantoocciptial membrane and the catheter inserted and threaded caudally to the L6-S1 spinal cord region. Sutures were placed around the knot in the catheter in the muscle covering the atlanto-occipital membrane to prevent catheter migration. At the end of the testing procedure, drug administration at the target location in the spinal cord was verified in a subset of rats with Evan’s blue injection through the intrathecal catheter. 3.7 Assessment of VMR The VMR is defined as a reflex contraction of abdominal muscles in response to visceral stimulation, particularly noxious stimulation. This reflex indicates a noxious response, and this model of visceral pain measurement has been characterized and reproduced by many investigators in models of bladder and colorectal pain (Castroman & 39 Ness, 2001; Ness & Gebhart, 1988; Ness et al., 2001; Su, Riedel, Leon, & Laping, 2008a). Following footshock or non-footshock on the seventh day, animals were immediately anesthetized with isoflurane and a transurethral catheter placed for bladder distension (section 3.4). Platinum or silver wire electrodes (two recording and one ground electrode) were inserted into the left external oblique muscle through an incision in the abdominal skin for differential amplification of EMG activity. Anesthesia was lowered to between 1-1.25 % isoflurane, after which time the animal was tested for a hind paw withdrawal reflex. If no reflex was observed, the animal was allowed more time to acclimate to the anesthesia level before initiation of testing. The 5-HT receptor antagonists, ondanestron or WAY 100635, or saline vehicle was administered via intrathecal catheter. Responses to UBD were recorded 10 min following ondansetron and 15 min following WAY 100635 or saline administration. The bladder was distended via air pressure according to the following sequence: 3- 20 sec 60 mmHg distensions in order to overcome initial sensitivity and establish stable EMG responses. Immediately following baseline recordings, responses to graded distensions were obtained. The intertrial interval was 3 min. Abdominal EMG activity was amplified using a Grass P511 amplifier (Grass Technologies, Warwick, RI, USA) and captured and recorded using a CED 1401 interface and Spike 2 software. The amplifier settings were as follows: EMG amplification factor=200; low frequency filter=10Hz; high frequency filter=3kHz; sample rate=10kHz. The VMR response was defined as: (rectified EMG activity during UBD - rectified baseline EMG prior to UBD) / (rectified baseline EMG). 40 3.8 Enzyme-Linked Immunosorbant Assay (ELISA) 3.8.1 Tissue Collection Two groups of rats, chronic footshock and non-footshock, were used to measure 5-HT, 5-HIAA, and corticosterone. After cessation of the footshock protocol, rats were immediately anesthetized using 5% isofluorane. All tissues were collected between 08:00 and 10:00 over the course of four days. A midline incision was made to expose the atlanto-occipital membrane. CSF was collected via needle aspiration by puncturing the atlanto-occipital membrane with a 30 g needle and transferred to a 0.5 µl centrifuge tube. The aspirate was frozen on dry ice. Blood was collected by cardiac puncture using a 20 g needle. Serum was obtained by allowing the blood to clot at room temperature (RT) for 90 min and then centrifuging for 15 min at 2000 x g. The supernatant was transferred to 1.5 µl centrifuge tubes and frozen at -80°C. Animals were then decapitated and the spinal cord removed by hydraulic extrusion. Lumbosacral segments (5 mm) were isolated and dissected, placed into 1.5 µl centrifuge tubes, and frozen on dry ice. Spinal cord tissue was homogenized in 0.01 M phosphate buffered saline with 1% protease inhibitor cocktail (Thermo Scientific, Rockford, IL, USA). The homogenates were centrifuged for 15 min at 16000 x g at 4°C. The supernatant was removed and total protein measured using the Pierce BCA Protein Assay Reagent Kit (Rockford, IL, USA). All samples remained frozen at -80°C until processing for ELISA. 41 3.8.2 Serotonin The Serotonin (Research) ELISA (ALPCO Diagnostics, Salem, NH, USA) was performed using 48 µg of protein per sample for spinal cord tissue and 2 µl of CSF per sample. Samples, standards, and controls were acylated for 30 min at RT. The acylated samples, standards, and controls were then transferred in duplicate to a microtiter plate and incubated with rabbit 5-HT antiserum for 24 h at 4°C. The plate was washed and anti-rabbit IgG conjugated with peroxidase was added and allowed to incubate for 30 min at RT. The plate was washed again and the substrate, tetramethylbenzidine (TMB), was added. The plate was incubated in the dark for 25 min at RT. Stop solution (0.25 M sulphuric acid) was added to each well and the optical density was read at 450 nm using a Fluostar Omega plate reader (BMG Labtech, Offenburg, Germany). The concentrations of samples were multiplied by a correction factor of 50. 3.8.3 5-Hydroxyindoleacetic Acid (5-HIAA) The 5-HIAA ELISA (ALPCO Diagnostics, Salem, NH, USA) was performed using 96 µg of protein for spinal cord samples and 5 µl of CSF per sample. Standards, samples, and controls were diluted, methylated for 20 min at RT, and diluted further. The methylated standards, samples, and controls were pipetted in duplicate onto a microtiter plate. Rabbit anti-5-HIAA was added to each well and the plate was incubated for 1 h at RT. The plate was washed and peroxidase-conjugated anti-rabbit IgG was added, and the plate was again incubated for 1 h at RT. The plate was washed again and the substrate, TMB, was added. After a 25 min incubation in the dark at RT, 0.25 M sulphuric acid was added to stop the reaction. The optical density was read at 450 nm 42 using a Fluostar Omega plate reader (BMG Labtech, Offenburg, Germany). 3.8.4 Corticosterone Serum obtained from chronic footshock and non-footshock rats was analyzed for corticosterone content (EIA kit from Cayman Chemical Company, Ann Arbor, MI, USA). 10 µl of serum was added to 200 µl of diluent, and then 50 µl of the diluted sample from each animal and corticosterone standards were added to a rabbit anti-sheep IgG coated microtiter plate in duplicate. Corticosterone conjugated to acetylcholine esterase and sheep anti-corticosterone were added to the plate and allowed to incubate for 2 h at RT. The plate was washed and Ellman’s reagent was added. The plate was covered and incubated in the dark for 75 min at RT at which point the optical density was read at 412 nm using a Fluostar Omega plate reader (BMG Labtech, Offenburg, Germany). 3.9 Spinal Electrophysiology 3.9.1 Surgical Preparation The external jugular vein was cannulated using PE10 tubing. Tracheal and transurethral cannulae were placed for artificial respiration and UBD, respectively. A laminectomy was performed to expose the L6-S2 spinal cord region. Rats were suspended by vertebral clamps placed rostral and caudal to the laminectomy for stabilization. The dura mater was cut and removed to expose the spinal cord. Physiological saline-saturated gauze was applied to the spinal cord to prevent damage 43 due to drying. Tungsten microelectrodes (Microprobes, Gaithersburg, MD, USA) were placed into the dorsal spinal cord 0-1.0 mm lateral to midline and 0.1-1.2 mm deep for single-unit extracellular recordings and rats were paralyzed using pancuronium bromide (0.2 mg/h, intravenous) to prevent electrode movement. UBD was used as the search stimulus, and neurons that were consistently excited by UBD were isolated for use for the remainder of the experiment. 3.9.2 Quantification Of Neuronal Responses Neuronal responses to UBD were monitored on an oscilloscope and quantified by discrimination from background and converted to uniform pulses. The responses were saved on a computer as peristimulus-time histograms. Spontaneous activity was determined as the average rate of action potentials in the 10 sec period prior to onset of UBD. Total activity was determined as the rate of action potentials in the 20 sec period during the UBD stimulus. Evoked activity was calculated as the difference between the total activity and spontaneous activity. 3.9.3 Neuronal Characterization Neurons responding to UBD were characterized based on their responses to UBD and somatic mechanical stimuli and their receptive fields were mapped. Initially neurons were characterized based on their responses to noxious cutaneous (heterotopic noxious conditioning stimuli; HNCS) input to upper thoracic and cervical dermatomes. Spontaneous activity was recorded 10 sec before and 10 sec after applying a pinch using an alligator clip to apply a constant and consistent noxious pressure to the skin overlying 44 the scapulae. Neurons were classified as Type I if their response to UBD was inhibited >20% by HNCS or Type II if their response was excited, unaffected, or inhibited <20% by HNCS. Only Type II neurons were tested further. Type II neurons responding to weak tactile stimuli, such as brushing of the skin with a cotton-tipped applicator, as well as intense mechanical stimuli, such as pinching of the skin or hindpaw with an alligator clip, were classified as WDR. Those responding only to intense mechanical stimulation were classified as NS. Responses to UBD as described above were then recorded for individual neurons. All responses were recorded and saved on a computer for quantification and analysis. After determining baseline responses to visceral and somatic stimuli, 10 µl of the receptor antagonist or saline vehicle was delivered directly on top of the exposed spinal cord. After 15 min, responses to UBD and cutaneous stimuli were again recorded for individual neurons as described above. Upon termination of neuronal recording, rats were euthanized by overdose of inhaled anesthetic (isoflurane) and cardiac puncture. 3.10 Statistical Analyses 3.10.1 Statistical Significance All comparisons were considered significantly different at p≤0.05. All post-hoc tests were performed using Holm’s correction to maintain a family-wise α=0.05 (Holm, 1979). 45 3.10.2 Visceromotor Reflex Response Evoked abdominal EMG responses were analyzed using a repeated-measures analysis of variance (ANOVA), comparing stress (chronic footshock vs. no footshock), drug (saline, ondansetron, or WAY 100635), and distending pressures (10-60 mmHg). Separate ANOVAs were performed to answer separate questions. The first ANOVA assessed the effect of stress on EMG activity in the presence of the selective antagonist (chronic footshock vs. no footshock, all antagonists). The second ANOVA assessed EMG activity after the selective antagonist only in chronic footshock-treated animals (antagonist vs. saline, all chronic footshock). The third ANOVA assessed EMG activity after the selective antagonist only in non-footshock treated animals (antagonist vs. saline, all no footshock). 3.10.3 Enzyme-Linked Immunosorbant Assay Two-tailed, independent samples t-tests were used to compare the values of spinal cord corticosterone, 5-HT, 5-HIAA, and 5-HIAA/5-HT levels, separately, between rats exposed to chronic footshock stress vs. sham-stress. 3.10.4 Spinal Electrophysiology 3.10.4.1 Graded UBD. Lumbosacral dorsal horn neuronal activity in response to UBD was analyzed using a repeated-measures ANOVA comparing stress (chronic footshock vs. no footshock), drug (saline or ondansetron), and distending pressures (20, 40, and 60 mmHg). Separate ANOVAs were performed to answer separate questions. The first ANOVA assessed the effect of stress on neuronal responses in the presence of 46 the selective antagonist (chronic footshock vs. no footshock, all antagonists). The second ANOVA assessed neuronal activity after the selective antagonist only in chronic footshock-treated animals (ondansetron vs. saline, all chronic footshock). The third ANOVA assessed neuronal activity after the selective antagonist in only non-footshock treated animals (ondansetron vs. saline, all no footshock). 3.10.4.2 Baseline UBD. Three baseline measures of UBD-evoked dorsal horn neuronal activity were averaged and compared using an independent samples t-test. Comparisons were made of chronic footshock vs. no footshock in saline or ondansetron treated rats separately. Comparisons were also made of saline vs. ondansetron in footshock or non-footshock treated rats separately. 3.10.4.3 Neuronal distribution. The effect of stress (chronic footshock vs. nonfootshock) on the ratio of WDR to NS neurons was assessed separately for Type II neurons using Fisher’s Exact Test. 47 CHAPTER 4 METHODOLOGICAL DEVELOPMENT 4.1 Strain Selection Sprague Dawley rats are one of the most widely used strains in scientific and medical research. They are an outbred strain derived from the Wistar rat. One notable characteristic is their ease of handling. Phenotypically, they appear more docile and resilient to stress and anxiety. Originally, Sprague Dawley rats were chosen for the experiments contained within this thesis. Sprague Dawley rats had been used in all of the preliminary supporting studies utilizing the footshock-induced urinary bladder hypersensitivity model (Marek et al., 1992; Robbins & Ness, 2008). However, since stress-induced hypersensitivity carries a substantial degree of variability, it seemed worthwhile to determine if another strain may be more suitable to this line of research. Lewis rats were chosen as a candidate because they have been documented to exhibit a higher degree of anxiety (Ramos, Berton, Mormede, & Chaouloff, 1997). Lewis rats display high levels of avoidance on the elevated plus maze and in the central area of the open field test. Diazepam administration significantly increased the amount of time spent in the open arm of the elevated plus maze, demonstrating that the anxiety behaviors observed can be pharmacologically manipulated (Ramos et al., 1997). Lewis rats are often used in research relating to deficits in immune function, such as experimental allergic encephalitis and rheumatoid arthritis. This is due to the fact that an 48 inadequate HPA response to stress can lead to an increased allostatic load (McEwen & Stellar, 1993). Indeed, the Lewis rat displays a hypoactive HPA axis (Sternberg et al., 1989). The disregulation of HPA activity leads to increases in pro-inflammatory cytokines (McEwen, 1998). The Lewis rat appeared to be a sound choice due to its susceptibility to stress and pain-related disorders. Prior to our studies, the Lewis rat had never been tested as a model of stress-induced bladder hypersensitivity. The VMR responses to UBD in Sprague Dawley and Lewis rats were measured and compared in response to chronic footshock stress. For each strain two groups were established, chronic footshock and non-footshock. Sample sizes are as follows: Sprague Dawley-chronic footshock (n=5), Sprague Dawley-non-footshock (n=5), Lewis-chronic footshock (n=6), Lewis-non-footshock (n=6). The footshock protocol was carried out as described in section 3.3. Following footshock, the abdominal EMG responses to UBD were recorded in each rat in ascending pressures from 10-60 mmHg as previously described in section 3.7. The rats did not have intrathecal catheters placed, and no drugs or vehicle were administered during any time of pretreatment or testing. A main effect of stress was observed in abdominal EMG responses to UBD following chronic footshock or no footshock in the Lewis strain of rats but not in the Sprague Dawley strain (Lewis: F(1,10)=9.413 p<0.05; SD: F(1,8)=0.014, p=0.907). Post-hoc analyses revealed significantly greater responses following chronic footshock at UBD pressures of 30-60 mmHg in Lewis rats (p<0.05). In a separate analysis examining the effect of strain, there was no difference in abdominal EMG responses to UBD when comparing the strain of rat in only chronic footshock treated rats (F(1,9)=0.082, p=0.781). However, among non-footshock animals, there was a main effect of strain 49 such that VMR responses were significantly lower in Lewis rats compared to Sprague Dawley (F(1,10)=9.75, p<0.05). Post-hoc analyses showed significant differences at UBD pressures of 50-60 mmHg (p<0.05). These results are presented in Figure 4.1. In a subset of Lewis rats, serum corticosterone was measured (section 3.8.4) in rats undergoing chronic footshock or no footshock (section 3.3) to determine the validity of induction of the HPA axis by chronic footshock in Lewis rats. Sample sizes were as follows: chronic footshock (n=11) and non-footshock (n=14). Figure 4.2 illustrates that corticosterone was significantly elevated in rats that underwent chronic footshock versus non-footshock (t(23)=7.78, p<0.001) These results indicate that the Lewis strain is a more robust and reliable model of footshock stress-induced bladder hypersensitivity. The Lewis rats have a lower baseline (i.e. non-stress condition) EMG response to graded UBD than do the Sprague Dawley rats. This lower baseline activity makes any deviations in the direction of increased sensitivity to noxious stimulation more easily detectable. Stress-induced alterations can be achieved in Sprague Dawley rats, as many reports indicate, including those of our own laboratory (Black, Ness, & Robbins, 2009; Martenson, Cetas, & Heinricher, 2009; Robbins & Ness, 2008). However, the footshock-induced bladder hypersensitivity in Sprague Dawley rats is often associated with a higher degree of variability. The phenomenon may or may not be observed, and when it is, a larger group of animals is usually required. Clearly, footshock stress induced a marked increase in corticosterone release in Lewis rats, indicating a strong HPA-axis stress response. While an increase was also seen previously in Sprague Dawley rats, Lewis rats had an increase of almost three-fold, 50 while the increase in Sprague Dawley rats was closer to two-fold (Marek et al., 1992). These results, coupled with the aforementioned stress-induced change in VMR response, show that the Lewis strain is the superior choice for the experiments contained within this thesis, as well as for future experiments utilizing the footshock stress-induced bladder pain model. 4.2 Effect of Intrathecal Catheter Intrathecal drug delivery is a widely accepted method of administering drugs into the subarachnoid space clinically in humans and also in animal models in scientific research. However, in the context of research, placement of the catheter itself should be viewed as an independent variable. Insertion of the catheter causes changes in intraspinal pressure and presumably an enhanced inflammatory response to the foreign material. A search of the literature was unable to cast light on the effect of intrathecal catheter placement on responses to noxious stimulation and specifically, visceromotor responses. The following study addresses the role of intrathecal catheter introduction on the VMR response to UBD in animals with stress-induced urinary bladder hypersensitivy. One group of rats had an intrathecal catheter placed into the subarachnoid space at the lumbosacral region (section 3.6) while another group of rats was left intact. Both groups were subdivided into chronic footshock and non-footshock groups and received treatments per the footshock protocol (section 3.3). VMR responses to UBD were recorded (section 3.7). All catheters contained physiological saline. Sample sizes were as follows: chronic footshock-no catheter (n=6), no footshock-no catheter (n=6), chronic footshock-catheter (n=9), and no footshock-catheter (n=8). 51 The findings in Table 4.1 indicate a significant main effect of intrathecal catheter and stress and significant interactions of UBD x catheter, UBD x stress, and UBD x catheter x stress. Rats in the chronic footshock group without intrathecal catheters had significantly greater abdominal EMG responses to UBD compared to rats with footshock and catheters, no footshock and catheters, and no footshock and no catheters. Post-hoc analyses revealed a significant effect of stress in rats with catheters and no catheters and a significant effect of catheter in stressed rats at UBD pressures of 50-60 mmHg (p<0.05). These results are illustrated in Figure 4.3. These findings suggest that the simple placement of an intrathecal catheter may be a confounding variable since it produces a significant decrease in the magnitude of the EMG response to UBD. Although a significant reduction in the EMG response is only seen in chronically stressed rats when testing for pharmacological manipulations, this finding may mask the effect of a drug. Specifically, if the expected outcome of drug administration is to mitigate the stress-induced increase in VMR response, it may prove more challenging to obtain a significant effect since both the drug and the administration of vehicle would produce a similar diminishing effect. The effect of the catheter alone must be taken into account when interpreting data from animals with intrathecallyimplanted catheters. It would prove useful to further examine if there are histological and biochemical changes that occur due to catheter implantation. In the experiments described in Specific Aim 1, the choice was made to use intrathecal catheters because it is the most efficient way to deliver a drug into the subarachnoid space of a rat. Furthermore, the method has been used for decades in 52 pharmacological animal research (Gustafsson, Post, Edvardsen, & Ramsay, 1985; Svensson, Persson, Fitzsimmons, & Yaksh, 2013; Yaksh & Rudy, 1976). 4.3 Effect of Serotonin on Chronic Footshock-Induced Bladder Pain Prior to undertaking the current experiments involving specific serotonergic receptor antagonists, it needed to be determined if serotonin in general had an influence in stress-induced bladder hypersensitivity. To achieve this, effects of the non-specific serotonergic receptor antagonist, methysergide (Sigma, St. Louis, MO, USA), on UBDevoked VMRs was examined. Inthrathecal administration was chosen to determine if the receptor effects were acting at the spinal cord level. Two groups of rats were established, chronic footshock and non-footshock. The footshock protocol was carried out as previously described. On the day of testing, rats were implanted with intrathecal catheters (section 3.6) and prepped for VMR recording (Section 3.7). Half of the rats received intrathecal methysergide (30 µg in 15 µl saline) and half received saline vehicle (15 µl) 15 min prior to UBD testing. EMG responses to UBD at pressures from 10-60 mmHg were recorded. Sample sizes were as follows: chronic footshock-methysergide (n=9), chronic footshock-saline (n=7), non-footshockmethysergide (n=7), non-footshock-saline (n=7). Figure 4.4 shows that abdominal EMG responses to UBD in chronic footshockstressed rats were significantly diminished following intrathecal administration of methysergide compared to saline vehicle (significant main effect of drug: F(1,14)=6.409, p<0.05). Post-hoc tests showed significant group differences at UBD pressures of 30-60 53 mmHg (p<0.05). There was no effect of the drug in non-footshock rats (F(1,11)=0.053, p=0.822). Spinal administration of the non-specific 5-HT receptor antagonist, methysergide, significantly attenuated VMR responses to UBD in stressed rats, implicating a facilitatory role of 5-HT in stress-induced bladder hypersensitivity. This is similar to a previous report by Randich et al. (2008) in which methysergide significantly reduced VMR responses in a model of inflammation-induced bladder pain. Since methysergide is a non-specific antagonist, this warrants further investigation with drugs that target specific 5-HT receptors. 54 Figure 4.1. Group mean abdominal EMG activity at UBD pressures of 10-60 mmHg in Sprague Dawley and Lewis rat strains. Rats were exposed to footshock (closed circles) or no footshock (open circles) for 15 min daily for 7 days. Sprague Dawley rats (A) showed no difference in the magnitude of the EMG response between chronic footshock and no footshock groups, while chronic footshock significantly increased EMG responses compared to no footshock in Lewis rats (B). * indicates significantly higher EMG responses in the chronic footshock group (p<0.05). N=5-6/group. A Sprague Dawley 6 Footshock No Footshock Group Mean EMG 5 4 3 2 1 0 10 20 30 40 50 60 Bladder Pressure (mmHg) B Lewis 6 Footshock No Footshock Group Mean EMG 5 4 *$ *$ 50 60 3 *$ 2 *$ 1 0 10 20 30 40 Bladder Pressure (mmHg) 55 Figure 4.2. Serum corticosterone concentration in Lewis rats following chronic footshock or no footshock. Rats were exposed to footshock (black bar) or no footshock (gray bar) for 15 min daily for 7 days. Footshock significantly increased levels of corticosterone compared to no footshock (* p<0.001). N=11-14/group. Corticosterone 140 Corticosterone (ng/ml) 120 *$ 100 80 60 40 20 0 CFS NFS 56 Figure 4.3. Group mean EMG responses to graded UBD at pressures of 10-60 mmHg in rats with and without intrathecal catheters. Rats were exposed to footshock or no footshock for 15 min daily for 7 days. * and ** indicate that EMG responses from rats with chronic footshock with no catheter are significantly higher than responses with no footshock-no catheter, chronic footshock-catheter, and no footshock-catheter (p<0.05 and 0.001, respectively). # indicates significantly higher EMG responses with chronic footshock compared to no footshock (p<0.05). N=6-9/group. 4 **$ **$ Footshock- No Catheter No Footshock- No Catheter Footshock- Catheter No Footshock- Catheter #$ #$ Group Mean EMG 3 *$ 2 *$ #$ #$ 1 0 10 20 30 40 Bladder Pressure (mmHg) 57 50 60 Figure 4.4. Group mean EMG responses to graded UBD at pressures of 10-60 mmHg in rats given intrathecal methysergide or saline. Rats were exposed to footshock or no footshock for 15 min daily for 7 days. Panel A shows decreased EMG magnitude with intrathecal methysergide with prior chronic footshock (* p<0.05). Panel B indicates no change in EMG after methysergide with no footshock. N=7-9/group. Chronic Footshock A 5 Group Mean EMG Methysergide Saline *$ *$ 4 *$ 3 *$ 2 1 0 10 20 30 40 50 60 Bladder Pressure (mmHg) B No Footshock 5 Group Mean EMG Methysergide Saline 4 3 2 1 0 10 20 30 40 50 Bladder Pressure (mmHg) 58 60 Table 4.1. Statistical analyses of EMG responses to graded UBD in rats with or without intrathecal catheter implantation. Rats were exposed to 15 min of daily chronic footshock stress or no footshock for 7 days. Repeated Measures ANOVA Chronic Footshock vs. No Footshock Catheter vs. No Catheter Mean EMG df F Catheter (1,23) 5.997 Stress (1,23) 15.877 Catheter x Stress (1,23) 3.707 UBD x Catheter (5,115) 4.471 UBD x Stress (5,115) 13.422 UBD x Catheter x Stress (5,115) 3.527 59 p-value 0.022 0.001 0.067 0.001 <0.001 0.005 CHAPTER 5 RESULTS 5.1 Specific Aim 1 5.1.1 Purpose The purpose of Specific Aim 1 was to test the effects of blocking specific serotonin receptors, 5-HT1A and 5-HT3, on the stress-induced enhancement of the VMR response to UBD. Rats were exposed to the chronic footshock or no footshock treatments for 7 days. All animals were implanted with intrathecal catheters that extended to the lumbosacral region of the spinal cord and were given WAY 100635, ondansetron, or saline vehicle. VMR responses were recorded in the presence of each drug in both the stress and no stress groups. Sample sizes are as follows: chronic footshock-saline (n=9), chronic footshock-WAY 100635 (n=9), chronic footshockondansetron (n=9), non-footshock-saline (n=8), non-footshock-WAY 100635 (n=8), nonfootshock-ondansetron (n=8). In all instances where ANOVA was used to compare differences in mean EMG recordings, tests were conducted to determine normality of distribution, homogeneity of variance, and sphericity and were concluded to not be in violation of these assumptions. 60 5.1.2 Effect of Footshock ANOVA revealed that chronic footshock treatment significantly increased the abdominal EMG recordings in response to graded UBD compared to no footshock in rats given intrathecal saline (main effect of stress: F(1,15)=4.897, p<0.05; interaction of UBD x stress: F(5,75)=3.994, p<0.01). Post-hoc tests revealed significant group differences at UBD pressures of 20 and 40-60 mmHg (p<0.05). There were no significant effects of footshock stress in rats with intrathecal WAY 100635 or ondansetron (WAY: F(1,15)=1.093, p=0.312; ODS: F(1,16)=0.350, p=0.563). These results are presented in Figure 5.1. 5.1.3 Effect of WAY 100635 In addition to examining the effect of footshock stress in animals with intrathecal saline or 5-HT receptor antagonist, the same data presented in 5.1.2 was analyzed as an effect of drug in separate groups of rats that had similar stress treatments. This section addresses the effect of WAY 100635, while the following section addresses the effect of ondansetron. Spinal administration of WAY 100635 did not significantly alter the magnitude of EMG responses to bladder distension in either the chronic footshock or the non-footshock condition (CFS: F(1,16)=0.405, p=0.534; NFS: F(1,14)=1.681, p=0.216; Figure 5.2). 5.1.4 Effect of Ondansetron Ondansetron given intrathecally had no effect on EMG responses of rats following chronic footshock or no footshock (CFS: F(1,16)=1.404, p=0.253; NFS: 61 F(1,15)=1.301, p=0.272). However, ANOVA revealed a significant interaction of UBD x Drug x Footshock (F(5,155)=2.677, p<0.05). These findings are presented in Figure 5.3. 5.2 Specific Aim 2 5.2.1 Purpose The purpose of Specific Aim 2 was to determine whether levels of serotonin and/or its metabolite, 5-HIAA, in CSF or lumbosacral spinal cord were altered by chronic footshock stress. Rats underwent chronic footshock or no footshock treatment, and then spinal cord and CSF samples were tested using ELISA for 5-HT and H-HIAA. These two concentrations were then used to calculate the ratio of 5-HIAA to 5-HT, which gives a better understanding of serotonin turnover due to 5-HIAA being the major metabolite of serotonin. 5.2.2 Enzyme-Linked Immunosorbent Assay The results from Specific Aim 2 are presented in Table 5.1 and Figures 5.4-5.6. Concentrations of 5-HT and 5-HIAA are not significantly changed with chronic footshock stress compared to sham stress in either lumbosacral spinal cord or CSF. The ratio of 5-HIAA to 5-HT is not significantly different in footshock or non-footshock treated rats in both spinal cord and CSF samples. 62 5.3 Specific Aim 3 5.3.1 Purpose The purpose of Specific Aim 3 was to determine the effect of 5-HT3 receptor blockade on the activity of spinal dorsal horn neurons in response to UBD in rats with stress-induced bladder hypersensitivity. Recordings were obtained from neurons that responded by increasing their activity to mechanical bladder stimulation. Furthermore, these neurons had to exhibit an increase or no change in the rate of firing (i.e., not inhibited) in response to a counter-stimulus (see section 3.9.3). Neuronal activity was recorded in response to graded UBD in both chronically stressed and non-stressed rats and in the presence of spinal ondansetron or saline vehicle. Dorsal horn neurons were further classified by their receptive field properties. Sample sizes were as follows: chronic footshock-saline (n=7), chronic footshock-ondansetron (n=8), non-footshocksaline (n=8), non-footshock-ondansetron (n=8). Where ANOVA was used to determine differences in neuronal responses as an effect of stress or drug, tests were conducted to determine normality of distribution, homogeneity of variance, and sphericity. These assumptions were met in all comparisons except that there was a violation of sphericity in the comparison of saline vs. ondansetron in stressed rats. In this particular comparison, there were no clear differences in neuronal responses between the drug and vehicle groups, therefore no correction of sphericity was made since it would not have affected the outcome of the ANOVA. 63 5.3.2 Effect of Footshock Prior to recording neuronal responses to graded UBD, a stable baseline response was established using a 60 mmHg UBD stimulus. No more than a 10% change was allowed to occur in 3 sequential trials in order to be considered “stable”. No significant difference was detected in the mean baseline neuronal responses between chronically stressed or non-stressed rats administered ondansetron or saline (ODS: t(14)=0.151, p=0.883; SAL: t(13)=1.939, p=0.075). Following spinal administration of ondansetron or saline, there was no significant main effect of chronic footshock stress on dorsal horn neuron excitability in response to graded UBD (ODS: F(1,14)=0.334, p=0.572; SAL: F(1,13)=1.09, p=0.316). These results are depicted in Figure 5.7. 5.3.3 Effect of Ondansetron Figure 5.8 shows that the administration of ondansetron did not decrease neuronal activity in response to UBD in chronically stressed rats (F(1,13)=0.636, p=0.439). Ondansetron also did not change neuronal responses in non-stressed rats (F(1,14)=0.058, p=0.813). Mean baseline neuronal activity trended toward a significant increase in ondansetron treated rats compared to saline treated rats in the chronic footshock but not the non-footshock group (CFS: t(13)=2.008, p=0.066; NFS: t(14)=0.175, p=0.863). 5.3.4 Percent of Baseline Comparisons In the previous two sections, the effects of footshock and of ondansetron were analyzed using group mean neuronal discharges to compare neuronal responses. Furthermore, the analyses utilized a between-subjects design. The neuronal response 64 data from Specific Aim 3 was also analyzed as a percentage of baseline and using a within-subjects design. Specifically, baseline and graded UBD responses were recorded before and after spinal drug administration. Graded neuronal responses were normalized as a percentage of the pre-drug baseline response average. These alternative analyses were conducted as a means to reduced variability within comparison groups. 5.3.4.1 Effect of Footshock. The pre-drug baseline neuronal responses from chronic footshock rats and non-footshock rats were compared. An independent samples t test did not reveal a difference in baseline responses between footshock and nonfootshock groups (t(29)=0.057, p=0.995). A repeated measures ANOVA did not detect an effect of stress (F(1,29)=0.803, p=0.378). 5.3.4.2 Effect of Drug. Pre- and post-drug responses were compared in rats with chronic footshock and no footshock. Baseline responses were compared in each treatment group before and after drug administration. There were no detectable differences observed in baseline neuronal responses after administration of ondansetron or saline vehicle in either stressed or non-stressed rats (CFS-ODS: t(14)=0.230, p=0.821; NFS-ODS: t(9)=0.419, p=0.685; CFS-SAL: t(11)=-0.388, p=0.705; NFS-SAL: t(13)=1.202, p=0.250). Furthermore, a repeated measures ANOVA was conducted on the responses to graded bladder distension in each group. No effects of ondansetron or saline were found in stressed or non-stressed rats (CFS-ODS: F(1,13)=4.160, p=0.62; NFSODS: F(1,14)=0.048, p=0.830; CFS-SAL: F(1,12)=0.639, p=0.440; NFS-SAL: F(1,14)=2.244, p=0.156). 65 5.3.5 Neuronal Characterization According to predetermined guidelines, only Type II neurons were chosen for experiments in Specific Aim 3; that is, the neurons responded to counter-stimulation by an increase or no change in activity. All but one neuron retained this characteristic after spinal ondansetron or saline in both the stressed and non-stressed groups. Nearly all of the neurons from rats in the chronic footshock group were classified as WDR (87.5%) compared to NS (12.5%), while neurons from rats in the non-footshock group were mostly NS (62.5%; WDR: 37.5%). A Fisher’s exact test indicated that, indeed, neurons$ from$rats$exposed$to$footshock$were$more$likely$to$be$WDR,$and$those$from$rats$in$ the$nonNstress$group$were$more$likely$to$be$NS$(p<0.05). The depth from the spinal cord dorsum was recorded for most neurons and is presented in Figure 5.9. 66 Figure 5.1. Group mean EMG responses to graded UBD at pressures of 10-60 mmHg. Rats were exposed to footshock (closed circles) or no footshock (open circles) for 15 min daily for 7 days and administered either intrathecal saline, WAY 100635, or ondasetron. Panel A shows chronic footshock significantly increased EMG responses in rats with intrathecal saline at pressures 40-60 mmHg (*p<0.05). EMG responses were not different between chronic footshock and no footshock with intrathecal WAY 100635 (B) or ondansetron (C). N=8-9/group. Saline A 2.0 Footshock No Footshock *$ Group Mean EMG 1.5 *$ 1.0 *$ 0.5 *$ 0.0 10 20 30 40 50 60 50 60 50 60 Bladder Pressure (mmHg) WAY 100635 B 2.0 Footshock No Footshock Group Mean EMG 1.5 1.0 0.5 0.0 10 20 30 40 Bladder Pressure (mmHg) C Ondansetron 2.0 Footshock No Footshock Group Mean EMG 1.5 1.0 0.5 0.0 10 20 30 40 Bladder Pressure (mmHg) 67 Figure 5.2. Group mean EMG responses to graded UBD at pressures of 10-60 mmHg. In panel A, rats were exposed to footshock for 15 min daily for 7 days and administered either intrathecal WAY 100635 or saline. Panel B shows responses from rats exposed to no footshock for 15 min daily for 7 days and administered either intrathecal WAY 100635 or saline. There was no difference in EMG magnitude between saline and WAY 100635 treated rats following chronic footshock or no footshock. N=8-9/group. Chronic Footshock A 2.0 WAY 100635 Saline Group Mean EMG 1.5 1.0 0.5 0.0 10 20 30 40 50 60 50 60 Bladder Pressure (mmHg) B No Footshock 2.0 WAY 100635 Saline Group Mean EMG 1.5 1.0 0.5 0.0 10 20 30 40 Bladder Pressure (mmHg) 68 Figure 5.3. Group mean EMG responses to graded UBD at pressures of 10-60 mmHg. In Panel A, rats were exposed to footshock for 15 min daily for 7 days and administered either intrathecal ondansetron or saline. Panel B shows responses from rats exposed to no footshock for 15 min daily for 7 days and administered either intrathecal ondansetron or saline. There was no difference in EMG magnitude between saline and ondansetron treated rats following chronic footshock or no footshock. N=8-9/group. Chronic Footshock A 2.0 Ondansetron Saline Group Mean EMG 1.5 1.0 0.5 0.0 10 20 30 40 50 60 50 60 Bladder Pressure (mmHg) No Footshock B 2.0 Ondansetron Saline Group Mean EMG 1.5 1.0 0.5 0.0 10 20 30 40 Bladder Pressure (mmHg) 69 Figure 5.4. Serotonin levels in lumbosacral spinal cord and cerebrospinal fluid. Rats were exposed to footshock (black bars) or no footshock (gray bars) for 15 min daily for 7 days. Panel A shows serotonin levels in lumbosacral spinal cord. Panel B shows serotonin levels in CSF. There were no differences in serotonin levels between rats with chronic footshock and no footshock in either tissue. N=13-15/group. A 5-HT Spinal Cord 200 5-HT (ng/ml) 150 100 50 0 CFS B Col 1 vs Col 2 - Col 14 Col 1 vs Col 15 - Col 29 NFS 5-HT CSF 200 5-HT (ng/ml) 150 100 50 0 CFS NFS 70 Figure 5.5. 5-HIAA levels in lumbosacral spinal cord and cerebrospinal fluid. Rats were exposed to footshock (black bars) or no footshock (gray bars) for 15 min daily for 7 days. Panel A shows 5-HIAA levels in lumbosacral spinal cord. Panel B shows 5-HIAA levels in CSF. There were no differences in 5-HIAA levels in either tissue; however, there was a trend of decreased 5-HIAA in chronically stressed compared to non-stressed rats in both spinal cord (p=0.07) and CSF (p=0.075). N=13-15/group. A 5-HIAA Spinal Cord 14000 12000 5-HIAA (ng/ml) 10000 8000 6000 4000 2000 0 CFS B NFS 5-HIAA CSF 14000 12000 5-HIAA (ng/ml) 10000 8000 6000 4000 2000 0 CFS NFS 71 Figure 5.6. Ratio of 5-HIAA to 5-HT levels in lumbosacral spinal cord and cerebrospinal fluid. Rats were exposed to footshock (black bars) or no footshock (gray bars) for 15 min daily for 7 days. Panel A shows 5-HIAA/5-HT levels in lumbosacral spinal cord. Panel B shows 5-HIAA/5-HT levels in CSF. There were no differences in 5-HIAA/5-HT levels between rats with chronic footshock or no footshock in either tissue. N=13-15/group. A 5-HIAA/5-HT Spinal Cord 50 5-HT (ng/ml) 40 30 20 10 0 CFS B NFS 5-HIAA/5-HT CSF 500 5-HIAA/5-HT (ng/ml) 400 300 200 100 0 CFS NFS 72 Figure 5.7. Group mean evoked discharges from dorsal horn neurons in rats with spinally administered saline or ondansetron. Rats were exposed to footshock or no footshock for 15 min daily for 7 days. Panels A and B show responses of Type II neurons to UBD pressures of 20, 40, and 60 mmHg. No significant difference was observed in neuronal responses with saline (A) or ondansetron (B). Panels C and D show mean baseline responses of Type II neurons to UBD at 60. A trend of increased baseline activity was seen in rats with chronic footshock (black bars) treated with saline (C) but not in ondansetron-treated rats (D). N=7-8/group. Saline A Ondansetron B 500 500 Footshock No Footshock Footshock No Footshock 400 Group Mean Discharges Group Mean Discharges 400 300 200 100 300 200 100 0 0 20 40 60 20 Bladder Pressure (mmHg) Saline C Ondansetron D 400 400 Footshock No Footshock Footshock No Footshock 300 Group Mean Discharges Group Mean Discharges 40 Bladder Pressure (mmHg) 200 100 0 300 200 100 0 73 60 Figure 5.8. Group mean evoked discharges from dorsal horn neurons. Rats were exposed to footshock or no footshock for 15 min daily for 7 days, and neuronal activity was recorded after spinal administration of saline or ondansetron. Panels A and B show responses of Type II neurons to UBD pressures of 20, 40, and 60 mmHg. No significant difference was observed between neuronal responses in rats with saline versus ondansetron after footshock (A) or no footshock (B). Panels C and D show mean baseline responses of Type II neurons to UBD at 60. A trend of decreased baseline activity was seen in rats after chronic footshock (C) treated with ondansetron (black bars) compared to saline (gray bars). No difference in baseline activity was observed after no footshock (D). N=7-8/group. A B Chronic$ Footshock 500 No$Footshock 500 Saline Ondansetron Saline Ondansetron 400 Group Mean Discharges Group Mean Discharges 400 300 200 300 200 100 100 0 0 20 40 20 60 C D Chronic$ Footshock 400 No$Footshock 400 Saline Ondansetron Saline Ondansetron 300 Group Mean Discharges Group Mean Discharges 40 Bladder Pressure (mmHg) Bladder Pressure (mmHg) 200 100 0 300 200 100 0 74 60 Figure 5.9. Electrode depth from spinal cord dorsum (mm). Closed circles indicate neurons sampled from rats in the chronic footshock group (depth range: 0.3-1.45 mm). Open circles indicate neurons sampled from rats in the non-footshock group (depth range: 0.16-1.55 mm). Neuronal Depths 1.8 Distance from Dorsum (mm) 1.6 1.4 1.2 1.0 0.8 0.6 0.4 0.2 0.0 CFS NFS 75 Table 5.1. Statistical analyses of 5-HT, 5-HIAA, and 5-HIAA/5-HT concentrations in CSF and lumbosacral spinal cord (LS). Tissues were extracted immediately following chronic footshock or no footshock. ELISA 5-HT 5-HT 5-HIAA 5-HIAA 5-HIAA/5-HT 5-HIAA/5-HT Independent Samples t-test 5-HT, 5-HIAA, 5-HIAA/5-HT Concentrations Chronic Footshock vs. No Footshock Tissue df t CSF 28 1.087 LS 26 -0.458 CSF 28 -1.922 LS 26 -1.891 CSF 28 -0.891 LS 26 -1.391 76 p-value 0.286 0.651 0.075 0.07 0.380 0.177 CHAPTER 6 DISCUSSION 6.1 Summary of Results The purpose of the current set of experiments was to further elucidate the mechanism by which chronic stress potentiates urinary bladder hypersensitivity, specifically with regard to the role of 5-HT. The experiments contained within this thesis utilized a different strain of rat than has been previously used in models of bladder pain. The first set of experiments established the Lewis rat as a model animal for stressinduced bladder hypersensitivity. These experiments also tested the effects of two 5-HT receptor antagonists on the VMR response to UBD in chronically stressed rats. No change in abdominal EMG activity was observed with intrathecal WAY 100635 or ondansetron in either stressed or non-stressed rats. The second set of experiments tested whether chronic footshock stress altered the levels of 5-HT, 5-HIAA, or 5-HIAA/5-HT. Surprisingly, there was no increase found in neurotransmitter, metabolite, or the ratio of the two with chronic stress. If anything, there was a slight decrease in 5-HIAA following footshock stress. The final set of experiments tested the role of 5-HT3 receptors in the responses of spinal dorsal horn neurons to UBD in chronically stressed rats. The results found did not support the hypothesis that an alteration in Type II neurons was involved in stress-induced bladder hypersensitivity. Furthermore, blockade of spinal 5-HT3 receptors had no bearing on the activity of Type II neurons. 77 6.2 Integration of Findings with Current Literature 6.2.1 The Comorbidity of Stress and Chronic Pain Pain and stress are similar to the chicken and the egg. It is often hard to discern which was the precipitating factor. Mayer et al. (2001) described the development and maintenance of irritable bowel syndrome in terms of risk, trigger, and perpetuating factors. Risk factors include a genetic predisposition; a pathological, or life-threatening, stressor; or an early life stressor, such as neglect or abuse. Trigger factors could be a physical event, such as a surgery or infection, or a psychosocial stressor. Regardless of the origin, the common thread is that the pain and the anxiety about the pain or other symptoms perpetuate each other, creating a stress-symptom cycle. This schema can be applied not only to irritable bowel syndrome but also to many chronic pain diseases/syndromes, including IC. There is an increasing awareness of the relationship between chronic pain and stress or anxiety disorders. A large, randomized study including approximately 6,000 participants conducted using in-home interviews concluded that 35% of people with chronic pain had a comorbid anxiety disorder, compared to 18% of people in the general population (McWilliams, Cox, & Enns, 2003). Specific to IC, Rothrock and colleagues (2001) had 45 patients and 31 healthy matched controls record a daily diary reporting urinary frequency, urgency, pain, and stress levels. All participants were also required to complete a Symptom Severity Scale and Perceived Stress Scale. IC patients had significantly higher reports of stress, both in their diary recordings and on the Perceived Stress Scale compared to controls. Disease severity was also correlated with a higher stress level. In a separate survey-based study, women with lower urinary tract symptoms 78 reported lower health-related quality of life compared to women without symptoms, and of the women with symptoms, 53% fell into the category of having a clinical anxiety disorder (Coyne et al., 2009). The correlation between chronic pain and stress can be further examined with regard to HPA axis function. In a study examining chronic back pain, salivary cortisol levels were significantly higher in patients compared to controls (Vachon-Presseau et al., 2013). Interestingly, a group of chronic pain clinic patients and healthy controls were assessed using the Perceived Stress Scale and had hair cortisol levels measured. The use of hair samples provides a more long-term account of stress via cortisol levels than plasma or saliva samples. The chronic pain patients had significantly greater levels of cortisol in hair samples compared to controls. Pain patients also rated significantly higher on the Perceived Stress Scale (Van Uum et al., 2008). Patients with fibromyalgia (which like IC, is a chronic pain syndrome with a diagnosis of exclusion) have a dysfunctional HPA axis response. For example, they show an reduced stress response, elevated levels of ACTH, and decreased levels of cortisol and CRF (Lentjes, Griep, Boersma, Romijn, & de Kloet, 1997). Often there is a loss of circadian rhythmicity of cortisol in fibromyalgia patients (Crofford et al., 2004). There is a similar loss of rhythmicity in rheumatoid arthritis patients (Cutolo et al., 2005). The findings included in this thesis support the previous literature that stress exacerbates pain in a model of urinary bladder nociception. As a part of the methodological development, the findings determined that 7 days of footshock stress produces a robust increase in the magnitude of the VMR response to graded UBD in Lewis rats. This finding was replicated in Specific Aim 1 in rats with chronic footshock 79 stress and intrathecal saline. While not performed in the same strain of rat, the current experiments also corroborate previously published findings in Sprague Dawley rats that chronic footshock stress increases the vigor of VMR responses to UBD compared to rats that received sham footshock (Black et al., 2009; Robbins & Ness, 2008). The effect of chronic footshock stress on serum corticosterone levels was also assessed. Lewis rats exhibited significantly higher levels of corticosterone following 7 days of footshock compared to the sham stress controls. This finding is in line with the human data demonstrating increased cortisol levels in chronic pain patients (VachonPresseau et al., 2013; Van Uum et al., 2008). Furthermore, this finding is consistent with the previous report stating that Sprague Dawley rats showed increased plasma corticosterone levels after 7 days of footshock stress compared to sham controls (Marek et al., 1992). 6.2.2 Serotonin in Stress-induced Hyperalgesia While stress-induced hyperalgesia is not a new concept, it has not been a major focus of basic scientific research. Even fewer reports exist in the literature concerning a role of 5-HT in stress-induced hyperalgesia. Alosetron, a 5-HT3 receptor antagonist, became a therapeutic drug of interest in the late 1990’s for its use in irritable bowel syndrome, a syndrome associated with chronic pain and stress (Camilleri et al., 1999; Delvaux, Louvel, Mamet, Campos-Oriola, & Frexinos, 1998; Gershon, 1999; Humphrey, Bountra, Clayton, & Kozlowski, 1999; Mangel & Northcutt, 1999). In animal models of stress-induced colorectal pain, alosetron administration had beneficial outcomes. Colonic hyperalgesia induced by water avoidance stress was reversed with intrathecal alosetron 80 (Bradesi et al., 2007). Alosetron, as well as two other 5-HT3 antagonists, ramosetron and cilansetron, produced a dose-dependent increase in the colonic nociceptive threshold in rats with restraint stress-induced colonic hypersensitivity (Hirata et al., 2008). Though more effort needs to be made in determining the effects of 5-HT3 receptors in stressinduced visceral pain, it appears that the 5-HT3 receptor may play a facilitatory role in colorectal pain. The opposite argument can be made when looking at 5-HT from a broad perspective. Gameiro et al. (2006) established that temporomandibular joint hypersensitivity induced by chronic restraint stress was significantly attenuated with administration of fluoxetine, an SSRI. However, decisive conclusions cannot reasonably be made with such little data currently available. The findings included in Specific Aim 1 neither support nor oppose the current literature reports. Intrathecal ondansetron produced opposite effects in stressed versus non-stressed rats (significant interaction of Stress x Drug x UBD). While the individual comparisons of drug versus saline were not significant, ondansetron slightly decreased VMR responses to UBD in footshock-stressed rats and slightly increased VMR responses in non-stressed rats. These results do not implicate a facilitatory role of 5-HT3 receptor activation in stress-induced bladder hypersensitivity, but they also do not carry enough weight to discredit it. It is possible that alternative results may be found with the use of other 5-HT3 antagonist compounds. It appears to be safer to conclude that 5-HT1A receptors are not involved in chronic stress-induced bladder pain. Intrathecal administration of WAY 100635 in stressed rats produced no changes in EMG activity in response to UBD. Activation or inactivation of 5-HT1A receptors may play more of a role in the maintenance of 5-HT 81 levels. This is discussed further in Section 6.3.2. The experiments discussed here involving blockade of 5-HT1A and 5-HT3 receptors contribute novel findings to the body of research on stress-induced bladder hypersensitivity. 6.2.3 Dorsal Horn Neuronal Activity in Bladder Pain Models The spinal neuronal response to bladder distension was previously described in Section 2.5. Briefly, two populations of neurons were found to respond by increasing their activity to UBD. Type I neurons are inhibited by HNCS, while Type II neurons are either not inhibited or excited by HNCS. A series of experiments further differentiated these two neuron types. Ness and Castroman (2001) demonstrated the analogous comparison of Type I and II neurons with previously characterized “Abrupt” and “Sustained” neurons from colorectal experiments (Ness & Gebhart, 2000). Specifically, Type I neurons have an abrupt cessation of firing after termination of the stimulus, while Type II neurons have a sustained period of activity following stimulus termination. Type I/II neurons are also pharmacologically distinct. Type II neuronal activity is inhibited by intravenous lidocaine or morphine, while Type I activity is not similarly inhibited (Ness & Castroman, 2001). Intravenous N-methyl-D-aspartate (NMDA) receptor antagonists: ketamine, dextromethorphan, and memantine produced inhibition of Type I neuron activity evoked by UBD. The same was not observed in Type II neurons (Castroman & Ness, 2002). The distinction between Type I/II has been observed in two models of bladder pain. In the inflammation-induced bladder pain model, Type II activity was increased after intravesical zymosan instillation either 2 h or 24 h prior to neuronal recordings. 82 Only in the case where zymosan was applied 24 h prior to testing was a decrease in activity observed in Type I neurons (Ness et al., 2009). Chronic footshock stress leads to a significant augmentation of Type II neuronal responses to UBD. This effect was not similarly observed in Type I neurons (Marek et al., 1992). Pertaining to the receptive field subclassifications, more WDR than NS neurons were found in the Type I and Type II population; however, NS neurons were more likely to be Type II (Ness & Castroman, 2001). Robbins et al. (2011) found that NS neurons were more likely to be Type II in both decerebrate and intact rats. In spinally intact rats only, the NS class of neurons was also more likely to be observed in rats that did not receive footshock stress. In the current experiments, only Type II neurons were analyzed based on previous findings that chronic footshock stress showed effects exclusively in Type II (Marek et al., 1992). The increased UBD-evoked activity of neurons from chronically stressed rats was not replicated in the current findings. However, it should be noted that there was a trend of increased activity in stressed rats versus non-stressed rats, but the difference did not reach statistical significance. Similar to reports from Robbins et al. (2011), the findings of Specific Aim 3 determined that NS neurons were more likely to be found in rats that did not receive footshock stress. The current experiments were performed in Lewis rats, rather than Sprague Dawley, so they also present a new line of research with a different strain. Based on the observations in Specific Aim 1 that a greater effect was found on changes in EMG activity in response to UBD with the 5-HT3 antagonist, ondansetron, the choice was made to investigate the role of 5-HT3 receptors in Type II neuronal 83 activity in Specific Aim 3. 5-HT3 receptor blockade did not produce any effect on evoked neuronal responses to graded UBD in stressed or non-stressed rats. These conclusions provide new evidence on the role of spinal 5-HT in stress-induced bladder hypersensitivity. 6.3 Discussion of Results 6.3.1 Specific Aim 1 The hypothesis that chronic footshock stress increases abdominal EMG responses to graded UBD in rats receiving intrathecal saline vehicle was supported in the current findings. This was expected since the methodological study demonstrated a significant effect of stress in Lewis rats, and Robbins et al. (2008) previously demonstrated this same effect in Sprague Dawley rats. These findings support the view that stress alone can induce bladder hypersensitivity. This is important since the etiology of IC is largely unknown and many women present with no histological changes (Waxman et al., 1998a). In the presence of either 5-HT1A or 5-HT3 receptor antagonists, the previously noted augmentation of EMG responses after chronic footshock stress is not seen. This finding demonstrates that these receptors are a component of stress-induced bladder hypersensitivity. In other words, activation of 5-HT1A or 5-HT3 receptors contributes to stress-induced bladder hypersensitivity. Blockade of either receptor subtype disrupts normal manifestation of such hypersensitivity; thus activation seems to be required. Compared to intrathecal administration of saline, WAY 100635 produces a nonsignificant increase in EMG activity in response to UBD in both chronic footshock and non-footshock treated rats. In contrast to the previous results, this can be interpreted as a slightly inhibitory role of 5-HT1A receptors in the VMR response to UBD. This 84 inhibitory role appears even more prominent in the tonic, non-stressed state. Intrathecal ondansetron exerted opposing effects on UBD-evoked VMR responses depending on whether the rat had been previously exposed to chronic stress or sham stress. While there was no main effect of ondansetron, there was a significant interaction of Stress x Drug x UBD. As hypothesized, ondansetron produced a decrease in the VMR response to UBD in chronically stressed rats. This is not a significant effect, but it does implicate a possible facilitatory role of the 5-HT3 receptor. Interestingly, ondansetron produced a non-significant increase in the EMG magnitude of sham-stressed rats similar to that observed with WAY 100635, therefore also suggesting a tonic inhibition produced by the 5-HT3 receptor, particularly in the non-stressed state. These data suggest that under normal conditions, activation of the 5-HT3 receptor may inhibit responses to UBD, but after an inciting event, such as stress, activation of the same receptors may become facilitatory. Since the administration of the non-specific 5-HT receptor antagonist, methysergide, abolishes the stress-induced bladder hypersensitivity effect, there is evidence that 5-HT has a facilitatory role in the phenomenon. It is possible that due to the complex nature of the serotonergic system, more than one receptor subtype is involved. Most likely there is a delicate interplay of a few receptor subtypes that contributes to stress-induced bladder hypersensitivity. Other spinal 5-HT receptors involved in the modulation of pain include 5-HT2A/B/C, 5-HT4, 5-HT5A, 5-HT6, and 5HT7. Spinal nerve ligation (a model of neuropathic pain) produces an upregulation of 5HT2A receptors in the spinal cord dorsal horn, which contributes to dorsal horn hyperexcitability evoked by sciatic nerve C-fiber stimulation (Aira et al., 2012; Aira et 85 al., 2010). The following examples utilize the formalin test, in which formalin is injected into the hindpaw of the rat and three distinct phases of activity can be observed: initial phase of activity (0–10 min, phase 1), a quiescent interphase (10–20 min), and a second phase of activity (20-90 min, phase 2). Both active phases involve ongoing peripheral afferent neural activity, while an inflammatory response contributes only to phase 2. Examination of spinal cord tissue revealed increased 5-HT in the ipsilateral dorsal horn following hindpaw injection of formalin.. Spinal cord tissue used to measure 5-HT content was dissected from rats immediately after mechanical nociceptive testing, which was 6 days following formalin injection. Spinal 5-HT depletion by intrathecal 5,7-DHT prevented formalin-induced hypersensitivity during phase 2 (Godínez-Chaparro, LópezSantillán, Orduña, & Granados-Soto, 2012). Intrathecal administration of 5-HT2B antagonist, RS-127445, prevented formalin-induced nociception in both phases, while on the other hand, the 5-HT2 agonist, 2,5-dimethoxy-4-iodoamphetamine (DOI), increased nociceptive behaviors in the first phase only (Cervantes-Duran et al., 2012). Intrathecal administration of 5-HT inhibited formalin-induced nociception that was reversed by intrathecal 5-HT5A antagonist, SB-699551 (Munoz-Islas et al., 2014). In summary, these studies show that 5-HT2A, 5-HT2B, and 5-HT5A receptors facilitate nociception. In contrast, it has been reported that intrathecal 5-HT4, 5-HT6, or 5-HT7 agonists prevented second phase nociception. The reverse was found after intrathecal 5-HT4, 5-HT6, or 5HT7 antagonists were administered (Godínez-Chaparro et al., 2012). These findings provide evidence of the distinct actions of 5-HT acting at different receptors, adding to the complexity of serotonergic descending modulation of pain. It is likely that the facilitation revealed by intrathecal methysergide is not being driven by one receptor 86 subtype. Rather, there is probably a complex balance between inhibition and facilitation produced by 5-HT at different receptor subtypes, and stress shifts this balance toward facilitation. This complexity could explain why the non-selective antagonist, methysergide, prevented stress-induced bladder hypersensitivity, while selective inhibition of 5-HT1A and 5-HT3 receptors did not. 6.3.2 Specific Aim 2 It was an unanticipated finding that 5-HT levels were not changed after 7 days of footshock stress. Based on one of the methodological development findings that intrathecal methysergide produced a significant decrease in the VMR response to UBD in stressed rats, it was believed that 5-HT was acting in a facilitatory manner in stressinduced bladder hypersensitivity. Therefore, it was expected that chronic stress would lead to an increase in 5-HT in CSF and/or lumbosacral spinal cord. 5-HIAA is the main metabolite of 5-HT; therefore, as 5-HT is utilized, it is expected that an increase of 5-HIAA will be observed. The ratio of 5-HIAA to 5-HT gives an indication of the amount of 5-HT being turned over. Interestingly, there was a less turnover seen in footshock compared to non-footshock treated rats, although it was just a slight decrease and was not statistically significant. This finding was unexpected due to previous reports of increased turnover of 5-HT following stress. For example, chronic variable stress or chronic restraint stress increased the ratio of 5-HIAA/5-HT in the hippocampus (Gamaro, Manoli, Torres, Silveira, & Dalmaz, 2003; Torres, Gamaro, Vasconcellos, Silveira, & Dalmaz, 2002). Acute footshock stress has resulted in increased 5-HT turnover in prefrontal cortex, thalamus, hypothalamus, hippocampus, 87 brainstem, and, in some cases, striatum (Adell, Trullas, & Gelpi, 1988; Dunn, 1988; Malyszko et al., 1994). In most of these cases, 5-HT levels were unchanged after stress, which is why it is important to refer to 5-HT metabolism as a better indicator of activity. While there is no clear-cut difference in the rate of 5-HT turnover, the results of Specific Aim 2 reflect those seen in Specific Aim 1. 5-HT may be utilized more by sham-stressed rats than those receiving footshock stress. This would imply that 5-HT may have a small, tonic inhibitory influence in sham-stressed rats. The fact that the levels change at all shows that stress may be disrupting the typical activity of 5-HT. An absence of a significant alteration in 5-HT levels or rate of turnover after 7 days of stress does not indicate that 5-HT is not involved in stress-induced bladder hypersensitivity. Even a subtle change in 5-HT levels can impact the activation and/or expression of a particular 5-HT receptor. For example, 5-HT diminution generally occurs immediately after a spike in 5-HT release due to 5-HT1A autoreceptor activation. This is the proposed explanation for the therapeutic lag seen in SSRI use (Celada, Puig, Amargos-Bosch, Adell, & Artigas, 2004). Systemic administration of the 5-HT1A agonist, 8-OH-DPAT, resulted in reduced 5-HT release in hippocampus and striatum, measured by in vivo microdialysis (Kreiss & Lucki, 1994). Using in vitro brainstem electrophysiology, Le Poul et al. (1995) demonstrated that after 21 days of continuous 5HT1A receptor stimulation, only an 80% desensitization of autoreceptors was observed; therefore, these receptors maintained the potential to decrease 5-HT release throughout the study. In the current experiments, it is possible that a longer time course of chronic stress needed to be utilized in order to overcome 5-HT autoreceptor negative feedback. 88 Rather than increasing 5-HT levels or turnover, stress could augment facilitation of responses to UBD by altering 5-HT receptor expression or affinity. Quantitative measures of receptor expression were not assessed in the current studies. A lack of alteration in neurotransmitter or metabolite may be explained by a change in receptor expression. It is hypothesized that there would be an upregulation of 5-HT receptors, especially of 5-HT3. Changes in expression may be found on spinal neurons, dorsal root ganglia, and/or primary afferent terminals. 6.3.4 Specific Aim 3 It was surprising to find in the current results that chronic footshock did not increase the activity of Type II neurons in rats with spinally-applied saline. This was expected based on findings of Robbins et al. (1992) that chronic footshock increased Type II neuronal activity. However, those experiments were performed in Sprague Dawley rats; whereas the current experiments were performed in Lewis rats, and neuronal activity in response to UBD in Lewis has not been tested prior to the current studies. The inability to replicate the results across rat strains was possibly also due to a small sample size. Alternatively, this could be a legitimate strain difference, since there was a trend toward increased neuronal activity in the chronic footshock group with saline, and this trend was observed in both graded responses and baseline responses. It is believed that the differentiation of Type I and Type II neurons correlates with facilitatory and inhibitory mechanisms of pain modulation. Type II neurons are typically thought to encode excitatory messages of pain perception, while Type I neurons encode inhibitory messages. This is supported by findings that inflammation- and stress-induced 89 urinary bladder hypersensitivity models show an increased response of Type II neurons to UBD compared to controls and that this increase is not seen in Type I neurons (Marek et al., 1992; Ness et al., 2009). The trend of increased Type II activity in chronically stressed rats most likely also represents an example of facilitation mediated by Type II neurons. Type I neurons were not tested in the current studies. It is possible that responses of Type I neurons decrease after stress in Lewis rats, which would decrease inhibition and therefore contribute to bladder hypersensitivity. It would be important to note the responsiveness of both neuron types to determine the relationship between the two. While searching for UBD-responsive neurons in the dorsal horn, the distinction had to be made between Type I and Type II. While recordings were not obtained from Type I neurons, it is important to note that Type I neurons were more readily found in animals which did not receive footshock, whereas the opposite observation was made in animals which received chronic footshock. The significance of this imbalance is that Type I neurons are inhibited by counter-irritation, such as in the phenomenon of DNIC. In human studies, DNIC is disrupted in patients that suffer from syndromes that are classified by exclusionary diagnoses (aka ‘functional disorders’), such as fibromyalgia and irritable bowel syndrome (Lautenbacher & Rollman, 1997; Wilder-Smith et al., 2004). It is likely that Type I neurons are a key mediator in the DNIC pathway. That fewer Type I neurons were encountered in stressed rats suggests that an impairment of DNIC may exist in stress-induced bladder hypersensitivity. Though the responses of Type II neurons were not altered by chronic stress, the number of Type II neurons could 90 have increased and/or the number of Type I neurons could have decreased, both which would have increased nociceptive signaling from the dorsal horn. 6.4 Strengths The current study made use of an existing animal model of urinary bladder hypersensitivity. However, it did so by introducing and establishing the Lewis strain of rat in chronic footshock stress-induced bladder hypersensitivity. Elevated serum corticosterone validated the use of chronic footshock as a stressor in Lewis rats. While Lewis rats are known to be one of the more anxious strains, evidence of stress-induced bladder nociception is a novel finding. That there is a less variable increase in abdominal EMG responses to UBD in the Lewis strain compared to the Sprague Dawley strain suggests that Lewis rats are a more reliable model of stress-induced bladder hypersensitivity. 6.5 Limitations The most striking limitation to the current set of studies lies with the use of intrathecal catheters to administer spinal drugs. Their use resulted in a diminished EMG response, irrespective of independent variable grouping. The suppression of all EMG responses requires there to be a much greater effect size of chronic stress in the augmentation of EMG response to enable an observable difference. While the effect of footshock was still evident in rats receiving intrathecal saline, it was considerably smaller than rats with no disturbances to the intrathecal space. 91 A further limitation was in the sample size. In some cases, statistical significance may have been achieved by doubling the sample size. However, previous experiments of similar nature have utilized sample sizes of equal proportions to those used in the current studies. Unfortunately, increasing the sample sizes to include two times the N was beyond the scope of this thesis. This is especially true for the electrophysiological experiments, which were labor-intensive but also restricted to a four-hour maximum recording time per neuron, including the time allocated for surgical prep. Neurons that respond to stimulation of the urinary bladder only comprise about 5% of neurons in the lumbosacral dorsal horn. Occasionally, the search for a UBD-responsive neuron would exceed the allotted amount of time, and no data could be obtained for a given animal. Due to the pharmacological nature of the electrophysiology experiments and the irreversibility or inability to wash out a drug, only one neuron was available for use per animal. Given these parameters, consideration had to be taken to minimize the use of laboratory animals. 6.6 Future Directions Based upon the outcomes of the studies contained within this thesis, recommendations have been made to further the likelihood of determining the role of 5HT in stress-induced bladder hypersensitivity. The first set of suggested directions address the issue of a possible lag in 5-HT release and/or accumulation due to 5-HT autoreceptor sensitization, which may be masking bladder sensitivity. A temporal study should be undertaken to determine the number of days of footshock at which the greatest effect on bladder nociception occurs. A suggested design would be to measure the VMR 92 response to UBD after 1, 7, 14, 21, and 28 days of daily footshock stress. In addition to nociceptive quantification, levels of 5-HT, 5-HIAA, and corticosterone should also be measured at each time point. Once the optimal footshock period is determined, then the effect of direct stimulation with 5-HT as well as activation or blockade of receptor subtypes can be tested. It would be efficacious to perform these experiments, as any subsequent experiments would rely on the outcome to have the chance of obtaining the most relevant results. In an effort to address the unexpected findings in Specific Aim 2 that no changes in 5-HT or the ratio of 5-HIAA/5-HT were observed following chronic footshock stress, an alternative study would be to examine the effects of footshock stress on receptor expression. An ideal set of experiments would measure receptor protein and/or mRNA levels. The most obvious tissue to analyze would be the spinal cord, specifically at the thoracolumbar and lumbosacral regions due to their connectivity to the bladder. It would be worthwhile to also examine receptor expression in the dorsal root ganglia of the above mentioned spinal cord regions. The exact location of the 5-HT receptors involved in descending pain modulation at the spinal cord level is unclear. Previously reported 5-HT receptors that modulate pain following intrathecal activation or blockade include 5HT1A, 5-HT2A/B/C, 5-HT3, 5-HT4, 5-HT5A, 5-HT6, and 5-HT7 (Bardin, 2011; Cervantes-Duran, Rocha-Gonzalez, & Granados-Soto, 2013; Godínez-Chaparro et al., 2012; Munoz-Islas et al., 2014; Suwa, Bock, Preusse, Rothenberger, & Manzke, 2014; Wilder-Smith et al., 2004). Based on the outcome of these quantitative experiments, it would also be useful to determine the precise location of receptors that are altered with exposure to chronic stress using immunohistochemistry. 93 Next, to gain a more complete understanding of the spinal physiology of stressinduced bladder hypersensitivity, the activity of Type I spinal dorsal horn neurons should also be assessed. In most chronic pain conditions, there appears to be a disturbance in the balance of facilitatory and inhibitory mechanisms. Discerning the activity patterns of both Type I and Type II neurons and their possible interactions may help to further elucidate this. Lastly, it is well known that painful bladder disorders, such as IC, almost exclusively affect women. This is the indication for using all female subjects in the current studies. When speculating on the explanation for a sex difference that involves females being affected, it is most plausible to look to female sex hormones as a contributing factor. Estrous cycle differences have been documented in inflammationinduced bladder hypersensitivity. Specifically, EMG responses to UBD were significantly increased during metestrus and proestrus phases compared to estrus and diestrus in rats with inflamed bladders. No fluctuations as a result of estrous cycle were observed in non-inflamed rats (Ball, Ness, & Randich, 2010). No data exists on the role of estrous cycle in stress-induced bladder hypersensitivity; therefore, it would be advantageous to determine if differences in nociceptive responses relate to phases of the estrous cycle using the current model. 6.7 Conclusions The studies conducted within this thesis led to several basic conclusions pertaining to the study of stress and 5-HT in relation to bladder pain, which can be further generalized to the understanding of IC. Chronic footshock stress leads to an exacerbation 94 of bladder nociception as evidenced by the VMR response to UBD in Lewis rats. Chronic footshock stress, as it is currently defined, does not alter the level of 5-HT found in the lumbosacral spinal cord or CSF. Chronic footshock stress also does not alter the rate of 5-HT turnover in these tissues. No specific role could be determined for 5-HT3 receptors in rats exposed to chronic footshock stress. 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Journal of Neurophysiology, 87(5), 2225-2236. $ 120 $ APPENDIX A INSTITUTIONAL ANIMAL CARE AND USE COMMITTEE (IACUC) NOTICE OF APPROVAL 121 THE UNIVERSITY OF ALABAMA AT BIRMINGHAM Institutional Animal Care and Use Committee (IACUC) Institutional Animal Care and Use Committee CH19 Suite 403 933 19th Street South 205.934.7692 FAX 205.934.1188 122 Mailing Address: CH19 Suite 403 1530 3RD AVE S BIRMINGHAM AL 35294-0019 $ $ $ $ $ APPENDIX$B$ $ INSTITUTIONAL ANIMAL CARE AND USE COMMITTEE (IACUC) NOTICE OF APPROVAL FOR PROTOCOL MODIFICATION 123 THE UNIVERSITY OF ALABAMA AT BIRMINGHAM Institutional Animal Care and Use Committee (IACUC) Notice of Approval for Protocol Modification DATE: April 10, 2013 TO: MEREDITH T. ROBBINS, Ph.D. BMR2-270 (205) 975-9684 FROM: Robert A. Kesterson, Ph.D., Chair Institutional Animal Care and Use Committee (IACUC) SUBJECT: Title: Stress-Induced Bladder Hyperalgesia: A Potential Mediator Sponsor: NIH Animal Project_Number: 120608853 On April 10, 2013, the University of Alabama at Birmingham Institutional Animal Care and Use Committee (IACUC) reviewed the animal use proposed in the above referenced application. It approved the modification as described: Procedures- chronic catheter implantation. The sponsor for this project may require notification of modification(s) approved by the IACUC but not included in the original grant proposal/experimental plan; please inform the sponsor if necessary. The following species and numbers of animals reflect this modification. Species Rats Use Category A Number In Category Zero - Procedural modification only Rats B Zero - Procedural modification only Rats C Zero - Procedural modification only The IACUC is required to conduct continuing review of approved studies. This study is scheduled for annual review on of before June 26, 2013. Approval from the IACUC must be obtained before implementing any changes or modifications in the approved animal use. Please keep this record for your files. Refer to Animal Protocol Number (APN) 120608853 when ordering animals or in any correspondence with the IACUC or Animal Resources Program (ARP) offices regarding this study. If you have concerns or questions regarding this notice, please call the IACUC office at (205) 934-7692. Institutional Animal Care and Use Committee (IACUC) CH19 Suite 403 933 19th Street South (205) 934-7692 FAX (205) 934-1188 $ 124 Mailing Address: CH19 Suite 403 1530 3rd Ave S Birmingham AL 35294-0019
