THE ROLE OF SPINAL SEROTONERGIC RECEPTORS 5-HT1A … · Serotonin (5-hydroxytryptamine; 5-HT) has been established as a mediator in anxiety and pain separately, but little is known

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

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Copyright by Chelsea L. Crawford

2014

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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

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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

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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.

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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.

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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.

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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

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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 Selection of Species ................................................................................ 36 3.2 Anesthesia ............................................................................................... 37 3.3 Footshock Protocol ................................................................................. 37 3.4 Urinary Bladder Distension .................................................................... 38 3.5 Receptor Antagonists .............................................................................. 38 3.6 Implantation of Intrathecal Catheters ..................................................... 39 3.7 Assessment of VMR Response .............................................................. 39 3.8 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

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5.3.1 Purpose ........................................................................................ 63 5.3.2 Effect of Footshock ..................................................................... 64 5.3.3 Effect of Ondansetron .................................................................. 64 5.3.4 Percent of Baseline Comparisons ................................................ 64 5.3.5 Neuronal Characterization ........................................................... 66

6 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

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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

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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

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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

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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

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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

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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

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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

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(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 stress-

induced 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.

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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 5-

HT3, 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:

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• 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?

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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.

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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

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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

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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).

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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).

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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 γ-

aminobutyric 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 5-

HT (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 on-

cell 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,

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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, 5-

hydroxytryptophan (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, &

13

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, &

14

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

15

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).

16

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

17

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

18

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

19

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

20

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).

21

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.

22

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 5-

HT2A 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

23

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-N-

methylamphetamine (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).

24

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

25

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 5-

HT7 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

26

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 5-

HT3 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, Antkiewicz-

Michaluk, & 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

27

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 hairpin-

RNA (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 5-

HT receptors is activated. While there are at least 15 known 5-HT receptor subtypes, 5-

HT1A 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

28

(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,N-

dipropyl-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

29

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

30

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

31

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).

32

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

33

the focus of 5-HT receptor studies of bladder function in the literature Specifically, 5-

HT1A 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

34

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

35

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).

36

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.

37

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 0.75-

0.8% 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-

38

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

39

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 atlanto-

occiptial 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 &

40

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 inter-

trial 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).

41

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.

42

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

43

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

44

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

45

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).

46

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

47

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. non-

footshock) on the ratio of WDR to NS neurons was assessed separately for Type II

neurons using Fisher’s Exact Test.

48

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

49

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

50

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,

51

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).

52

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 intrathecally-

implanted 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

53

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 UBD-

evoked 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-footshock-

methysergide (n=7), non-footshock-saline (n=7).

Figure 4.4 shows that abdominal EMG responses to UBD in chronic footshock-

stressed 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

54

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.

55

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.

Sprague Dawley

Bladder Pressure (mmHg)

10 20 30 40 50 60

Gro

up M

ean

EM

G

0

1

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3

4

5

6FootshockNo Footshock

Lewis

Bladder Pressure (mmHg)

10 20 30 40 50 60

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up M

ean

EM

G

0

1

2

3

4

5

6FootshockNo Footshock

A

B

*$

*$

*$ *$

56

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

CFS NFS

Cor

ticos

tero

ne (n

g/m

l)

0

20

40

60

80

100

120

140

*$

57

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.

Bladder Pressure (mmHg)

10 20 30 40 50 60

Gro

up M

ean

EMG

0

1

2

3

4Footshock- No CatheterNo Footshock- No CatheterFootshock- CatheterNo Footshock- Catheter

*$

#$

*$

**$ **$

#$

#$#$

58

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

Bladder Pressure (mmHg)10 20 30 40 50 60

Gro

up M

ean

EM

G

0

1

2

3

4

5MethysergideSaline

No Footshock

Bladder Pressure (mmHg)10 20 30 40 50 60

Gro

up M

ean

EM

G

0

1

2

3

4

5MethysergideSaline

B

A

*$

*$

*$*$

59

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 p-value Catheter (1,23) 5.997 0.022 Stress (1,23) 15.877 0.001 Catheter x Stress (1,23) 3.707 0.067 UBD x Catheter (5,115) 4.471 0.001 UBD x Stress (5,115) 13.422 <0.001 UBD x Catheter x Stress (5,115) 3.527 0.005

60

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 footshock-

ondansetron (n=9), non-footshock-saline (n=8), non-footshock-WAY 100635 (n=8), non-

footshock-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.

61

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:

62

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.

63

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-footshock-

saline (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.

64

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

65

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 non-

footshock 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; NFS-

ODS: 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).

66

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.

67

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

Bladder Pressure (mmHg)

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Ondansetron

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A

B

C

*$

*$*$

*$

68

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

Bladder Pressure (mmHg)

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A

69

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

Bladder Pressure (mmHg)

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70

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.

5-HT Spinal Cord

CFS NFS

5-H

T (n

g/m

l)

0

50

100

150

200

Col 1 vs Col 2 - Col 14 Col 1 vs Col 15 - Col 29 5-HT CSF

CFS NFS

5-H

T (n

g/m

l)

0

50

100

150

200

B

A

71

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.

5-HIAA Spinal Cord

CFS NFS

5-H

IAA

(ng/

ml)

0

2000

4000

6000

8000

10000

12000

14000

5-HIAA CSF

CFS NFS

5-H

IAA

(ng/

ml)

0

2000

4000

6000

8000

10000

12000

14000

B

A

72

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.

5-HIAA/5-HT Spinal Cord

CFS NFS

5-H

T (n

g/m

l)

0

10

20

30

40

50

5-HIAA/5-HT CSF

CFS NFS

5-H

IAA

/5-H

T (n

g/m

l)

0

100

200

300

400

500

B

A

73

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.

Gro

up M

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500FootshockNo Footshock

Saline

Saline

Ondansetron

Ondansetron

A B

C D

74

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.

Bladder Pressure (mmHg)

20 40 60

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No$Footshock

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Chronic$Footshock

Chronic$Footshock

A B

C D

75

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

CFS NFS

Dis

tanc

e fr

om D

orsu

m (

mm

)

0.0

0.2

0.4

0.6

0.8

1.0

1.2

1.4

1.6

1.8

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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.

Independent Samples t-test 5-HT, 5-HIAA, 5-HIAA/5-HT Concentrations

Chronic Footshock vs. No Footshock ELISA Tissue df t p-value 5-HT CSF 28 1.087 0.286 5-HT LS 26 -0.458 0.651 5-HIAA CSF 28 -1.922 0.075 5-HIAA LS 26 -1.891 0.07 5-HIAA/5-HT CSF 28 -0.891 0.380 5-HIAA/5-HT LS 26 -1.391 0.177

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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 stress-

induced 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.

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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

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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

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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 (Vachon-

Presseau 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

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(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 stress-

induced 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

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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.

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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

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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 non-

significant 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

85

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 5-

HT7. Spinal nerve ligation (a model of neuropathic pain) produces an upregulation of 5-

HT2A 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

86

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, López-

Santillán, 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 5-

HT7 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

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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 stress-

induced 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,

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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 5-

HT1A 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.

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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

90

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

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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.

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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 5-

HT 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

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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 5-

HT1A, 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.

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Next, to gain a more complete understanding of the spinal physiology of stress-

induced 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 inflammation-

induced 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

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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. There is a facilitatory role of 5-HT

is stress-induced bladder hypersensititivy, but it is not being driven by either 5-HT1A or

5-HT3 receptor activity alone. In light of these findings, it remains unclear which

component of the descending serotonergic system is involved in stress-induced bladder

hyperalgesia.

96

$$$$

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APPENDIX A

INSTITUTIONAL ANIMAL CARE AND USE COMMITTEE (IACUC)

NOTICE OF APPROVAL

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THE UNIVERSITY OF ALABAMA AT BIRMINGHAM Institutional Animal Care and Use Committee (IACUC)

Institutional Animal Care and Use Committee Mailing Address:

CH19 Suite 403 CH19 Suite 403 933 19th Street South 1530 3RD AVE S

205.934.7692 BIRMINGHAM AL 35294-0019 FAX 205.934.1188

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APPENDIX$B$$

INSTITUTIONAL ANIMAL CARE AND USE COMMITTEE (IACUC)

NOTICE OF APPROVAL FOR PROTOCOL MODIFICATION

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DATE: April 10, 2013

TO: MEREDITH T. ROBBINS, Ph.D.BMR2-270(205) 975-9684

FROM:

Robert A. Kesterson, Ph.D., ChairInstitutional Animal Care and Use Committee (IACUC)

SUBJECT: Title: Stress-Induced Bladder Hyperalgesia: A Potential MediatorSponsor: NIHAnimal 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 Use Category Number In CategoryRats A Zero - Procedural

modification onlyRats B Zero - Procedural

modification onlyRats 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

Mailing Address:CH19 Suite 4031530 3rd Ave SBirmingham AL 35294-0019

THE UNIVERSITY OF ALABAMA AT BIRMINGHAM

Institutional Animal Care and Use Committee (IACUC)

Notice of Approval for Protocol Modification