SEROTONERGIC SIGNALING AT 5-HT 3 RECEPTORS IN SERTOTONIN TRANSPORTER (SERT)
KNOCKOUT (KO) RAT, A SEX SPECIFIC ANIMAL MODEL OF VISCERAL HYPERSENSITIVTY
By
Nadine Chahine El-Ayache

A DISSERTATION
Submitted to
Michigan State University
in partial fulfillment of the requirements
for the degree of
Pharmacology and Toxicology-Doctor of Philosophy
2017

ABSTRACT
SEROTONERGIC SIGNALING AT 5-HT 3 RECEPTORS IN SERTOTONIN TRANSPORTER (SERT)
KNOCKOUT (KO) RAT, A SEX SPECIFIC ANIMAL MODEL OF VISCERAL HYPERSENSITIVTY
By
Nadine Chahine EL-Ayache
The irritable bowel syndrome (IBS) is a functional gastrointestinal motor and visceral
sensation disorder that is more common in women compared to men. 5-Hydroxytryptamine (5HT, serotonin) signaling is disrupted in some IBS patients possibly due to polymorphic variations
in the gene encoding the serotonin transporter (SERT) which result in increased extracellular 5HT availability. Female SERT knockout (KO) rats exhibit visceral hypersensitivity to colonic
distention that mimics colonic hypersensitivity known to occur in female IBS patients. Studies
performed herein focused on understanding the role of 5-HT3 receptor signaling in SERT KO
rats. The visceromotor response (VMR) to colorectal distension (CRD) was determined following
inhibition of peripheral and central 5-HT3 receptors in SERT KO and WT rats. In female SERT KO
rats spinal 5-HT3 receptor inhibition with alosetron caused an increase in VMR to CRD. In Male
SERT KO rats activation of spinal 5-HT3 receptors increased VMR to CRD. Depletion of
descending serotonergic input from the brainstem reversed the effects of 5-HT 3 receptor
inhibition in SERT KO female rats and activation in SERT KO male rats. Based on these studies, I
concluded that this sex specific response observed in SERT KO rats is due to differential pattern
of 5-HT3 receptor expression in male and female SERT KO rats.
The effects of ovarian hormone on VMR to CRD were also investigated in SERT KO and WT
female rats. Ovariectomy and estrogen receptor (ER) antagonist studies were performed in
SERT KO and WT female rats. VMR to CRD was enhanced in the proestrus phase of the estrous

cycle in SERT KO but not WT female rats. Ovariectomy increased discomfort to CRD in SERT KO
female rats in a time dependent manner. VMR to CRD increased on day 7 post-ovariectomy and
lasted for up to 3 weeks. Intrathecal administration of ICI 182 780 (ER-α and ER-β) antagonist
increased VMR to CRD in SERT KO female rats. The G-protein coupled ER (GPR30) antagonist,
G15, did not affect VMR to CRD in SERT KO and WT female rats. These studies suggest that in
SERT KO female rats classical ERs (ERα and ERβ) play an antinociceptive role in the presence of
serotonergic dysfunction.

This dissertation is dedicated to my dear uncle Eddie Chaaban.
Thank you for being my role model.

iv

ACKNOWLEDGEMENTS

I am forever thankful for my advisor Dr. James Galligan for his mentorship and support
throughout the years. Dr. Galligan helped me develop my scientific skills and equipped me with
the knowledge required to become a successful researcher. He also served as a professional
role model of mine. He lead by example and demonstrated the importance of patience and
kindness when interacting with colleagues, staff and students. I know that these personal and
professional skills will serve me well throughout my career and I hope to pass these qualities to
others in the future.
I would also like to thank my committee members for their guidance during the past 5
years. Last but not least, I want to extend my gratitude for Mrs. Bethany Heinlen and Dr. Justin
McCormick for always being there for me throughout difficult times.

v

TABLE OF CONTENTS

LIST OF TABLES .............................................................................................................................. viii
LIST OF FIGURES .............................................................................................................................. ix
Chapter 1 General Introduction .................................................................................................... 1
1.1 Pain transmission .................................................................................................................. 1
1.1.1 Anatomical organization of pain pathways originating from the colon......................... 3
1.1.2 Neurochemical signaling along the pain pathway ......................................................... 9
1.1.3 Serotonergic signaling in pain transmission ................................................................. 12
1.2 Irritable Bowel Syndrome.................................................................................................... 15
1.3 Visceral Hypersensitivity ..................................................................................................... 17
1.4 Serotonin signaling dysfunction in IBS ................................................................................ 18
1.5 Sex differences in IBS .......................................................................................................... 20
1.6 Serotonin transporter knockout (SERT KO) rat, sex specific animal model of VHS ............ 21
1.7 Hypothesis and specific aims .............................................................................................. 23
BIBLIOGRAPHY ........................................................................................................................... 24
Chapter 2 Spinal 5-HT3 receptor inhibition increases visceral hypersensitivity in SERT KO
female rats. ................................................................................................................................... 30
2.1 Abstract ............................................................................................................................... 30
2.2 Introduction......................................................................................................................... 31
2.3 Methods ............................................................................................................................. 40
2.3.1 Animals ......................................................................................................................... 40
2.3.2 Surgery ......................................................................................................................... 40
2.3.3 Visceromotor response (VMR) to colorectal distension (CRD) and volume pressure
loop data acquisition. ............................................................................................................ 41
2.3.4 Intrathecal injection..................................................................................................... 42
2.3.5 Cerebrospinal fluid (CSF) fluid collection ..................................................................... 43
2.3.6 High-performance liquid chromatography (HPLC) ....................................................... 43
2.3.7 Immunohistochemistry ................................................................................................ 43
2.3.8 Statistics ........................................................................................................................ 44
2.3.9 Time-line of studies performed .................................................................................... 45
2.4 Results ................................................................................................................................. 47
2.5 Discussion ............................................................................................................................ 65
BIBLIOGRAPHY ........................................................................................................................... 73
Chapter 3 Classical estrogen receptors play an antinociceptive role in SERT KO female rats ... 79
3.1 Abstract ............................................................................................................................... 79
3.2 Introduction......................................................................................................................... 80
vi

3.3 Methods .............................................................................................................................. 83
3.3.1 Animals ......................................................................................................................... 83
3.3.2 Surgery .......................................................................................................................... 84
3.3.3 Visceromotor response (VMR) to colorectal distension (CRD) .................................... 85
3.3.4 Intrathecal injection ..................................................................................................... 86
3.3.5 Vaginal lavage and cytology ......................................................................................... 86
3.3.6 Statistics ........................................................................................................................ 86
3.3.7 Time-line of studies performed .................................................................................... 87
3.4 Results ................................................................................................................................. 88
3.5 Discussion ............................................................................................................................ 92
BIBLIOGRAPHY ........................................................................................................................... 99
Chapter 4 General discussion .................................................................................................... 104
4.1 Ovarian hormones, serotonin and visceral hypersensitivity ............................................ 104
4.2 Technical considerations ................................................................................................... 108
4.2.1 Visceromotor response (VMR) to colorectal distension (CRD) .................................. 108
4.2.2 5,7-DHT mechanism of action. ................................................................................... 110
4.2.3 Ovariectomy limitations ............................................................................................. 113
4.3 Clinical significance and future directions ........................................................................ 114
BIBLIOGRAPHY ......................................................................................................................... 116

vii

LIST OF TABLES

Table 2.1 Primary and secondary antibodies used for IHC ........................................................... 44
Table 2.2 Summary of results from 5-HT3 receptor antagonist studies ...................................... 67
Table 2.3 Summary of 5-HT3 receptor antagonist and agonist studies ....................................... 71

viii

LIST OF FIGURES

Figure 1.1 Ascending pain pathway ................................................................................................ 2
Figure 1.2 Extrinsic sensory innervation of the gastrointestinal tract............................................ 4
Figure 1.3 Extrinsic afferent ending types innervating the colon wall ........................................... 6
Figure 1.4 Descending innervation of the dorsal horn of the spinal cord ...................................... 8
Figure 1.5 Organization of the grey matter of the spinal cord ..................................................... 11
Figure 1.6 5-HT receptors types.................................................................................................... 13
Figure 1.7 5-HT homeostasis in the gut ........................................................................................ 14
Figure 1.8 Location of stop codon in SERT protein ....................................................................... 22
Figure 2.1 5-HT3 receptor expression along the pain pathway ................................................... 33
Figure 2.2 Spino-bulbo-spinal loop in the dorsal horn of the spinal cord .................................... 37
Figure 2.3 Time-line for studies of subcutaneous drug administration ....................................... 45
Figure 2.4 Ramosetron and alosetron dose response and intrathecal study time-lines ............. 46
Figure 2.5 Intracerebroventricular study timeline ....................................................................... 46
Figure 2.6 5, 7-DHT experiment time-line .................................................................................... 47
Figure 2.7 Subcutaneous administration of 5-HT3 receptor inhibitor increased VMR to CRD in
SERT KO but not WT female rats .................................................................................................. 48

ix

Figure 2.8 Subcutaneous administration of the 5-HT3 receptor inhibitor, alosetron, increased
VMR to CRD in WT but not SERT knockout (KO) male rats. ......................................................... 49
Figure 2.9 Volume-Pressure loop analysis .................................................................................... 50
Figure 2.10 Alosetron dose-response in SERT KO female rats ..................................................... 51
Figure 2.11 Dose-response curve of s.c. ramosetron in SERT KO female rats ............................. 53
Figure 2.12 Intrathecal administration of alosetron increased VMR to CRD in serotonin
transporter (SERT) knockout (KO) but not WT female rats. ......................................................... 54
Figure 2.13 Inhibition of spinal 5-HT3 receptors did not affect VMR to CRD in SERT KO or WT
male rats ....................................................................................................................................... 55
Figure 2.14 Intracerebroventricular administration of alosetron didn’t affect VMR to CRD in
SERT KO and WT rats .................................................................................................................... 56
Figure 2.15 Activation of 5-HT3 receptor increased VMR to CRD in male SERT KO rat ............... 57
Figure 2.16 5,7 DHT (100 μg, i.t.) treatment decreased 5-HT levels in the lumbar and sacral
spinal cord of KO and WT rats. ..................................................................................................... 59
Figure 2.17 Norepinephrine levels are not affected by 5, 7-DHT ................................................. 60
Figure 2.18 Treatment with 5,7-DHT prevented the increased VMR to CRD in SERT KO female
following alosetron treatment...................................................................................................... 61
Figure 2.19 Treatment with 5,7-DHT prevented the increase in the VMR to CRD in SERT KO male
rats following SR 57227 treatment ............................................................................................... 62
Figure 2.20 5-HT3A subunit and GABA expression in the superficial dorsal horn of the spinal
cord in SERT KO rats ...................................................................................................................... 63
Figure 2.21 5-HT3A subunit and GABA expression in the superficial dorsal horn of the spinal
cord in WT rats .............................................................................................................................. 64
Figure 2.22 Differential 5-HT3 receptor exppression in female and male SERT KO rats............. 72
x

Figure 3.1 Timeline of ovariectomy studies.................................................................................. 87
Figure 3.2 Estrogen receptor antagonist experiment timeline .................................................... 87
Figure 3.3 SERT KO rats exhibit a sex dependent increase in VMR to CRD. ................................. 88
Figure 3.4 The increase VMR to CRD is estrous cycle dependent ................................................ 89
Figure 3.5 Ovariectomy increased VMR to CRD in SERT KO female rats in a time dependent
manner .......................................................................................................................................... 90
Figure 3.6 Ovariectomy did not affect VMR to CRD in WT female rats........................................ 91
Figure 3.7 The effect of intrathecal administration of ER receptor antagonists .......................... 92
Figure 3.8 Fluctuation of ovarian hormones during the estrus cycle ........................................... 94
Figure 4.1 Inhibitory and excitatory 5-HT signaling in the dorsal horn of the spinal cord ......... 108
Figure 4.2 Effects of morphine on VMR to CRD.......................................................................... 111
Figure 4.3 Effect of clonidine on VMR to CRD in WT male rats .................................................. 112

xi

Chapter 1
General Introduction
1.1 Pain transmission
Sensory information is transmitted from visceral organs to the brain along a three
neuronal network as shown in Figure 1.1. Once a stimulus is applied to the peripheral terminals
of primary afferent neurons, cell depolarization occurs. This leads to the release of excitatory
neurotransmitters at central terminals of the primary afferent neurons located in the dorsal
horn of the spinal cord. In turn, the excitatory neurotransmitters will activate second order
neurons which relay the signal to the thalamus along the spinothalamic tract (STT) and dorsal
column (DC) (Palecek, Paleckova et al. 2002) and excitatory interneurons. STT and
spinoreticulothalamic tract collaterals relay sensory information to subcortical structures that
are critical in pain modulation (Chen, Li et al. 2001). From the thalamus, third order neurons
project to different areas of the brain for pain perception. This ascending pain signal is subject
to extensive modulation in the dorsal horn of the spinal cord where the primary and secondary
order neurons synapse (Millan 2002; Blackshaw, Brookes et al. 2007). Descending modulation
can either enhance or dampen pain transmission. A detailed explanation of anatomical
organization and neurochemical signaling in visceral pain transmission is discussed in this
chapter.

1

Figure 1.1 Ascending pain pathway. Upon activation by a stimulus, primary afferent neurons
transmit sensory information to the dorsal horn where they synapse on second order projecting
neurons. Second order neurons carry the information to the thalamus via the dorsal column
(DC) and spinothalamic tract (STT). From the thalamus, third order neurons carry the sensory
information to various brain regions for pain perception.

2

1.1.1 Anatomical organization of pain pathways originating from the colon
The gastrointestinal (GI) tract is innervated by an intrinsic network of neurons housed
within the GI tract known as the enteric nervous system (ENS). The ENS controls motor and
intrinsic sensory function of the GI tract to regulate nutrient absorption, secretion, and motility.
Cells bodies of the ENS neurons are located in the myenteric and submucosal plexuses. The GI
tract is also innervated by extrinsic neurons that modulate gut function and transmit sensory
information from the gut to the central nervous system (CNS). (Blackshaw, Brookes et al. 2007).
Two types of extrinsic primary afferent neurons innervate the GI tract including vagal
afferents and spinal afferents. The peripheral terminals of vagal afferents innervate the
proximal GI tract (esophagus, stomach, small intestine and proximal colon). Cell bodies of the
vagal afferents are located in the nodose ganglia and their central terminals are found in the
nucleus tractus solitarious (NTS) in the brainstem. Spinal afferents innervate the entire length
of the GI tract and are divided into three groups. The thoracolumbar, lumbosacral and sacral
spinal afferents innervate the proximal, middle and distal GI tract, respectively (Figure 1.2). The
cell bodies of spinal afferents are found in the dorsal root ganglia and their central terminals
are located in the superficial lamina of the dorsal horn in the spinal cord (Furness, Rivera et al.).

3

Figure 1.2 Extrinsic sensory innervation of the gastrointestinal tract. Sensory information from
the gastrointestinal tract is carried to the central nervous system via the vagus and spinal
nerves. The vagus nerve innervates the proximal gastrointestinal tract and transmits nonnoxious stimuli. On the other hand, spinal nerves innervate the entire gastrointestinal tract and
travel alongside sympathetic neurons. CG: celiac ganglion, GSN: greater splanchnic nerve, IMG:
inferior mesenteric ganglion, LuSN: lumbosacral nerve, PG: pelvic ganglion, PN: pelvic nerve,
SMG: superior mesenteric ganglia.
The receptive (peripheral) terminals of extrinsic afferent neurons are classified
according to their response to different types of stimuli and intensities (Brookes, Spencer et al.
2013; Gebhart and Bielefeldt 2016) . The peripheral terminals include: enteric intraganglionic
laminar afferents ending (IGLEs), vascular afferents, mucosal, muscular-mucosal and
intramuscular afferents (Figure 1.3). All types of afferent endings respond to probing and thus
must express mechanoreceptors. Only mucosal and muscular-mucosal afferents respond to
stroking of the mucosa. IGLEs, muscular-mucosal and intramuscular afferents respond to
stretch. Vascular nerve ending respond to high threshold but not low threshold probing
4

suggesting that they convey noxious stimuli. A subset of vascular afferents is called silent or
mechanically insensitive afferents (MIA) because they lack mechanosensitivity unless sensitized
by capsaicin or inflammatory mediators in different experimental settings (Furness, Rivera et al.
; Blackshaw, Brookes et al. 2007; Brookes, Spencer et al. 2013).
Vascular afferents innervate blood vessels located in the mesentery and submucosa of
the GI tract and send collaterals to myenteric and submucosal ganglia, mucosa and muscle
layers. Vascular afferents are exclusively spinal and are believed to be the predominant type of
neurons capable of transmitting noxious stimuli to the spinal cord. They exhibit varicose
branching axons (VBAs) and can be activated both mechanically and chemically (Furness, Rivera
et al. ; Su and Gebhart 1998).

5

Figure 1.3 Extrinsic afferent ending types innervating the colon wall. The colon wall is
innervated by extrinsic primary afferent neurons at various locations. The muscularis mucosa is
innervated by muscular-mucosal nerve endings. Intermuscular afferent ending innervate the
longitudinal and circular muscles in the colon. Mucosal afferent endings extend to the villi to
innervate the mucosa. Intraganglionic laminar ending (IGLEs) are found in the myenteric plexus
in close proximity to enteric neurons. Vascular endings terminate on blood vessels in the
mucosa as well as blood vessels in the mesentery and serosa (not depicted). Viscerofugal
afferents are not technically extrinsic since their cell bodies are found in the myenteric plexus,
however they project to the spinal cord.
Studies conducted in endothelin-3 knockout mice which lack enteric nerves in the distal
colon suggest that high threshold vascular nerve endings are not the only type of nerve ending
capable of transmitting noxious stimuli. Rather, low threshold, wide dynamic range muscular
and muscular-mucosal nerve endings play an important role in pain transmission (Zagorodnyuk,
Kyloh et al. 2011). This theory is in line with the fact that low threshold stimulation can cause

6

abdominal pain in patients with visceral hypersensitivity due to inflammation or other
pathological states (Gebhart 1999).
Regardless of the type of afferent nerve being stimulated, when sympathetic axons
(where thoracolumbar and lumbosacral spinal afferent are located) supplying the GI tract have
been severed, pain transmission is prevented indicating that spinal afferents covey noxious
signals to the CNS (Furness, Rivera et al.) .
Noxious sensation is carried to the spinal cord via c-fibers and Aδ fibers. Both have small
diameter cell bodies and are unmyelinated (c-fibers) or lightly myelinated (Aδ) fibers. The
central terminals of these afferents terminate on ascending projecting neurons or excitatory
interneurons in the layers of the dorsal horn. Descending cortical and subcortical structures
send direct and indirect projections to the dorsal horn of the spinal cord to modulate pain
transmission as shown in Figure 1.4. Descending pain modulation can either decrease
(descending inhibition) or increase (descending facilitation) pain transmission depending on the
neurotransmitter released and type/location of the receptors modulated (Millan 2002).

7

Figure 1.4 Descending innervation of the dorsal horn of the spinal cord. Descending
innervation from cortex and brainstem terminate at various regions in the dorsal horn. For
instance, interneuron activity can either be enhanced or decreased depending on the
neurotransmitter released and the receptor modulated.
Cortical structures such as the anterior cingulate cortex (ACC) increase pain perception
by enhancing descending facilitation. Frontal cortex nuclei also send projections to the
subcortical regions such as nucleus raphe magnus (NRM) which supplies serotonergic input to
the dorsal horn. Serotonergic neurons in the NRM receive input from multiple regions including
direct innervation from deep layers of the dorsal horn (Layer, Keller et al. 2007). NRM neurons
are found in the rostroventral medial medulla (RVM) which is an important region of the brain
stem where ON and OFF cells are located. ON cells enhance pain perception and are inhibited
by opioids, while OFF cells are part of the descending inhibition and are activated by opioids but

8

inhibited by pain. The hypothalamus and parabrachial nucleus (PBN) integrate autonomic and
sensory information to modulate the autonomic response to pain. Nucleus tractus solitarious
(NTS) receives major input from the vagus nerve and spinal afferents of the dorsal horn and
extensively communicates with other subcortical structures such as the periaqueductal grey
(PAG) to influence pain perception. The PAG plays the most pivotal role in modulating pain
processing at the level of the brain (Millan 2002). The PAG modulates the emotional response
to pain due to its extensive input into the amygdala. It also sends projections to other cortical
structures, hypothalamus, RVM, NRM, PBN, NTS and pontine noradrenergic cell groups such as
A5, A6 (locus coeruleus) and A7 (subcoeruleus) (Millan 2002). Noradrenergic nuclei send
projections to the spinal cord and also play an important role in pain modulation at the spinal
level (Llorca-Torralba, Borges et al. 2016).
1.1.2 Neurochemical signaling along the pain pathway
Mechanical distension of the gut wall can activate neurons directly via action of
mechanoreceptors and indirectly by causing release of signaling molecules from specialized
enteroendocrine cells (EEC) such as enterochromaffin cells (EC) (Kirkup, Brunsden et al. 2001;
Blackshaw, Brookes et al. 2007; Gunawardene, Corfe et al. 2011). Serotonin, ATP, and capsaicin
activate cation channels to cause neuronal depolarizations. While inflammatory signaling
molecules such adenosine, thrombin, histamine, bradykinin, prostaglandins, and mast cell
tryptase activate various G-coupled receptors to increase intracellular cAMP to sensitizes
primary afferents (Kirkup, Brunsden et al. 2001). These mediators can be released from various
inflammatory cells including mast cells. Mast cell activation via IgE, IgG, Toll Like Receptors
(TLRs) and CD 161 can lead to the release of inflammatory mediators which will in turn activate
9

receptors such as 5-HT3, PPARs, and TRPV channels on primary afferents (Lee and Lee 2016).
Gut microbiota can affect activity of neurons, EC cells, mast cells and macrophages which
influence gut barrier function and neuronal activation (Bhattarai, Muniz Pedrogo et al. 2017).
Short chain fatty acids, such as butyrate are produced by the gut microbiome secondary to fiber
fermentation, can enhance primary afferent neuron activation via upregulation of ERK1/2
pathways (Xu, Wu et al. 2013).
Once activated, the central terminals of primary afferent neurons will release excitatory
neurotransmitters to activate centrally projecting neurons that are located in Lamina I-III and X
or excitatory interneurons in lamina II in the dorsal horn of the spinal cord as depicted in Figure
1.5. The main excitatory neurotransmitter released by all primary afferent neurons is
glutamate which acts on ionotropic glutamate receptors for fast transmission. A subset of
primary afferents release peptides, such as, substance P and calcitonin-gene related peptide
(CGRP) in addition to glutamate. The expression of different excitatory neurotransmitters
indicates functional differences among nociceptive neurons however the significance of such
differences remains unclear (Iyengar, Ossipov et al. 2017). Eighty percent of centrally projecting
neurons express neurokinin 1 (NK1) receptors suggesting that noxious transmission is mediated
predominantly by substance P expressing primary afferent neurons (Todd 2010).

10

Figure 1.5 Organization of the grey matter of the spinal cord. Lamina i, ii, and x of the spinal
cord receive noxious sensory input from somatic and visceral organs. The grey matter of the
spinal cord is divided according to the cellular structure of the neurons present in each lamina.
Aδ and C- fibers terminate in lamina i, ii, and x as depicted.
Central terminals of primary afferent neurons, interneurons and dendritic processes of
projection neurons are all subject to descending modulation from cortical and subcortical
structures. In addition to the direct synaptic interactions between neurons in the dorsal horn,
volume transmission also occurs. In volume transmission, neurotransmitters diffuse from the
site of release to act on distant receptors resulting in a widespread and persistent modulation
of neuronal activity. This mechanism of neurotransmitter release is similar to that of intrathecal
drug application which allows activation of receptors and neurons that are not part of a
synapse (Millan 2002; Todd 2010) .
While multiple brain structures have been implicated in descending modulation, the
specific neurotransmitters and mechanisms of pain modulation are unknown. The most
extensively studied neurotransmitters involved in descending modulation are serotonin,
11

norepinephrine (NE) and gamma amino butyric acid (GABA). The net effect of each
neurotransmitter on pain signaling depends on the type and location of receptor being
activated or inhibited. For instance, NE is predominantly involved in descending inhibition via
activation of alpha-2 adrenergic receptors (2ARs) on central terminals of primary afferent
neurons. At the same time, it could also enhance facilitation by inhibiting GABA release from
interneurons (2ARs -mediated) or by increasing glutamate release from excitatory
interneurons (α1AR- mediated) (Millan 2002; Llorca-Torralba, Borges et al. 2016).
1.1.3 Serotonergic signaling in pain transmission
Serotonin activates intrinsic and extrinsic primary afferent neurons in the GI tract to
regulate gut absorption, secretion, motility and sensory functions. The gut is the most abundant
source of 5-HT in humans and other animal species including rodents. 5-HT is synthesized by
specialized neuroendocrine cells called enterochromaffin cells (ECs). Serotonin is synthesized
from the amino acid tryptophan in EC cells and serotonergic neurons. Tryptophan hydroxylase
(TPH) is the rate limiting enzyme in 5-HT synthesis. TPH isoform 1 is expressed in EC cells while
isoform 2 is expressed in 5-HT neurons (Gershon 2004; Spohn and Mawe 2017) . The release of
5-HT from ECs can be accomplished via mechanical or chemical stimuli of ECs as shown in
Figure 1.7. Serotonin released from EC cells activates different types of 5-HT receptors to
modulate its actions (Gershon 2004). To date seven classes of 5-HT receptor types have been
identified some of which have subtypes as shown in Figure 1.6.

12

Figure 1.6 5-HT receptors types. Seven different 5-HT receptors have been identified. 5-HT1,
5-HT2 and 5-HT5 receptors have different subtypes. 5-HT1, 5-HT2, 5-HT3, 5-HT4 and 5-HT7
have been identified in the GI tract.
5-HT1 and 5-HT5 receptors are Gi- coupled receptors, 5-HT2 receptors are Gq-coupled, 5HT4, 5-HT6, and 5-HT7 receptors are Gs-coupled, and the 5-HT3 receptor is a pentameric
cation channel (Jorgensen 2007). The activity of 5-HT is terminated via reuptake by the
serotonin transporter (SERT) which is expressed on enterocytes and serotonergic neurons in
the ENS (figure 1.7). Serotonin can also be cleared by high capacity organic cation transporter
(OCT) and dopamine transporters in the gut. Once inside the cell, 5-HT is metabolized into 5hydroxyindoleacetic acid (5-HIAA) by monoamine oxidase (MOA) in enterocytes and
serotonergic neurons. (Gershon 2004; Gershon and Tack 2007).

13

Figure 1.7 5-HT homeostasis in the gut. 5-HT is released from enterochromaffin (EC-cells)
upon chemical or mechanical stimulation. The activity of 5-HT is terminated via reuptake by
the serotonin transporter (SERT) expressed on epithelial cell and serotonergic neurons in the
gastrointestinal tract. 5-HT3R indicates 5-HT3 receptor.
The net effects of 5-HT signaling in the GI tract depend on the type and location of the
receptors being activated. Intrinsic primary afferent neurons express 5-HT1p, 5-HT3 and 5-HT4
receptors. Since the publication of Gershon, 2004, 5-HT1p receptors have been identified as 5HT7 receptors (Kim and Khan 2014). 5-HT7 receptors are expressed on submucosal intrinsic
primary afferent neurons (IPANs) and mediate peristaltic stimulation and secretory reflexes.
Similarly, 5-HT4 receptors are also expressed in IPANs and once activated enhance the release
of acetylcholine and CGRP from excitatory interneurons in the myenteric plexus to cause
smooth muscle contraction and increased gut motility (Gershon 2004). 5-HT3 receptors are

14

expressed on IPAN and myenteric motor neurons. Their activation leads to initiation of fast
excitatory neurotransmission leading to increased gut motility. In addition, 5-HT3 receptors
are also expressed on extrinsic primary afferents neurons and once activated lead to neuronal
depolarization and signal transmission to the brainstem(via the vagus nerve) and spinal cord
(Gershon 2004; Sengupta 2009).
Centrally, 5-HT is synthesized by neurons localized to the raphe nuclei (RN). Rostral RN
nuclei send serotonergic output to various structures in the brain, while the caudal nuclei send
serotonergic projections to the spinal cord (Hornung 2003) . Many 5-HT receptors have been
identified in the brain and spinal cord and can either enhance or dampen pain perception.
Experiments done in rodents suggest that 5-HT1A receptors are predominantly antinociceptive
since their activation leads to reduction in glutamatergic signaling and reduction in pain
behaviors (Jeong, Mitchell et al. 2012; Choi, Cho et al. 2013). Recent work done using GAD
65/GAD 67 knock-in mice show expression of 5-HT1A receptors on GABAergic neurons as well.
5-HT2 receptors have also been implicated in central pain modulation. Pharmacological studies
suggest that 5-HT2A receptors are pronociceptive, while 5-HT2C receptors are anti-nociceptive
(Rahman, Bannister et al. 2011). Similarly, 5-HT3 receptors appear to have a dual role in pain
modulation and will be discussed in great detail in chapter 2.
1.2 Irritable Bowel Syndrome
Irritable bowel syndrome (IBS) is a chronic functional gastrointestinal disorder (FGID)
characterized by sensory and motility dysfunction. The sensory dysfunction results in abdominal
pain while the motility disorder manifests as diarrhea and/or constipation. The diagnosis of IBS

15

is based on the Rome criteria which were updated in 2016. The most updated Rome IV criteria
placed a greater emphasis on abdominal pain as a main diagnostic symptom that must occur at
least once a week versus 3 times per month per Rome III criteria (Simren, Palsson et al. 2017).
IBS is not a life threating disorder but it negatively impacts the quality of life of affected
individuals. In fact, IBS patients have a quality of life scores worse than patients suffering from
depression, diabetes mellitus and end stage renal disease (Gralnek, Hays et al. 2000; Buono,
Carson et al. 2017) . In addition, IBS imposes a large economic burden resulting from doctors’
visits and work absenteeism (Hoekman, Rutten et al. 2015; Buono, Carson et al. 2017; Buono,
Mathur et al. 2017).
Treatment of IBS depends on the associated bowel dysfunction and is often inadequate
due to its’ multifactorial pathogenesis. Patients with IBS-diarrhea (IBS-D) are treated with drugs
that slow gut transit and motility to decrease diarrhea. IBC-constipation (IBS-C) patients are
treated with pro-kinetic drugs and/or stool bulking agents to alleviate the constipation. IBSalternating or mixed are treated with a combination of drugs to address the symptoms as they
present. Of the available IBS-C treatments only linaclotide, which is a CG-receptor agonist,
relieves the abdominal pain. The exact mechanism by which linaclotide relives pain is unknown
but it may simple be due to treating the constipation. The major side effect of linaclotide is
diarrhea resulting in lower patient compliance. Eluxadoline is a peripherally restricted δ-opioid
receptor antagonist and μ-opioid receptor agonist that has been shown to treat diarrhea and
reduce visceral sensitivity in IBS-D patients. While eluxadoline is effective in treating IBS-D
symptoms it has a black box waring due to increased risk pancreatitis in patients with gall
bladder diseases. Other conventional pain medications such as NSAIDS, acetaminophen, aspirin
16

and narcotics do not adequately treat the pain associated with IBS due to complication that
result from long term use. Many clinical studies have been conducted to assess the efficacy of
benzodiazepine, tricyclic antidepressants (TCAs), selective serotonin reuptake inhibitors (SSRI)
and serotonin norepinephrine reuptake inhibitors (SNRI )for treating abdominal pain in IBS
patients. It appears that only TCAs are efficacious at reducing pain when used in IBS patients
with comorbid psychological disorders. Other pain medications such as clonidine, gabapentin,
and pregabalin have been shown to reduce overall pain in IBS. Unfortunately, all three lead to
drowsiness and sedation making them unsuitable for long term pain management in IBS. Drugs
that target the serotonergic system have also been used to treat IBS. Alosetron, a 5-HT3
receptor antagonist is FDA approved for the treatment of severe IBS-D in women. Alosetron can
only be prescribed under risk management protocol due to serious side effect of severe
constipation and ischemic colitis. Teagserod, a 5-HT4 receptor agonist, was also used to treat
IBS-C. However, it was pulled of the market due to increased risk of ischemic heart disease.
(Chen, Ilham et al. 2017) The adverse events of the effective drugs and lack of efficacy of the
other treatments discussed underscores the importance of developing medications to
effectively treat the visceral pain associated with IBS.
1.3 Visceral Hypersensitivity
The cause of pain in some IBS patients is thought to be due to neuronal dysfunction which
leads to visceral hypersensitivity. Visceral hypersensitivity is defined as enhanced perception of
pain to noxious stimuli or pain perception to non-noxious stimuli. IN 1973, the first account of
increased pain perception to rectal distension was made. Since then multiple clinical and basic
science studies have shown that IBS patients and IBS animal models have lower pain threshold
17

than healthy controls. It has been reported that 50-80% of IBS patients exhibit VHS which does
not correlate with IBS symptom severity (Drossman, Camilleri et al. 2002). A more recent study
done in Japan confirmed that visceral hypersensitivity is observed in IBS patients and
significantly correlated with IBS symptom severity (Kanazawa, Hongo et al. 2011). This group
also found that rectal distention in IBS patients caused increased blood flow in specific brain
structures implicated in pain processing such as ACC, prefrontal cortex, insula and thalamus. In
pediatric patients pain threshold to rectal distension was also lower in IBS patients compared to
healthy controls (CAMILLERI, COULIE et al. 2001; Camilleri, Coulie et al. 2001; Halac, Noble et al.
2010). A study performed by Boubin et al. showed that VHS should be used as a diagnostic
marker to distinguish between different causes of abdominal pain since patients with IBS,
unlike patients suffering from other GI disorders exhibited lower pain threshold to rectal
distension (Bouin, Plourde et al. 2002).
1.4 Serotonin signaling dysfunction in IBS
In the early 2000s multiple case-control studies showed that certain polymorphisms in
the SLC6A4 gene which codes for serotonin transporter (SERT) could be associated with IBS
(Camilleri 2004; Jin, Cao et al. 2016). Of the identified polymorphisms the 5-HTT gene linked
polymorphic region (5-HTTLRP) upstream of the transcription start site is the most extensively
studied. This polymorphism is 41 base-pair deletion or insertion in the promotor region of the
SERT gene (park, choi et al. 2006; Jin, Cao et al. 2016). The short (s) allele leads to a reduction in
SERT expression compared to the long (l) allele (park, choi et al. 2006). In 2004, Patel et al.
showed that the s-allele is associated with Turkish IBS patients with a strong association of l/s
genotype in IBS-D patients. Other studies performed in Korea and India also showed a strong
18

association between s-allele and IBS-D patients (Yeo 2004; park, choi et al. 2006). The largest
study was performed in the United States and showed no association between SERT
polymorphism and IBS. Experts in the field speculated that this might be due to ethnic
heterogeneity of the subjects(Camilleri 2004). In addition, other SERT gene polymorphisms
have been identified and could also alter SERT expression regardless of 5-HTTLRP
polymorphism(Jin, Cao et al. 2016). Of specific importance is the rs25531 polymorphism which
is a functional single nucleotide polymorphism found in intron 2 of the SERT gene. The G allele
increases the odds of developing IBS by 3-fold in carriers and is capable of reducing SERT
expression even in patients with l/l allele (Kohen, Jarrett et al. 2009). Therefore the SERT gene
deserves further investigation to understand its role in IBS pathogenesis.
In addition to the polymorphisms in the SERT gene, other alterations in serotonergic
signaling in IBS patients have been identified. For instance, IBS patients exhibit higher mucosal
and plasma 5-HT levels, decreased SERT expression, and increased TPH1 expression (Foley,
Garsed et al. 2011). Additional studies showed a reduction in 5-HT uptake by platelets from IBS
patients compared to healthy controls suggesting aberrant SERT function (Cremon, Carini et al.
2011).
Another clinical observation that corroborates serotonergic dysfunction in IBS is the
effectiveness of the 5-HT3 receptor antagonist, alosetron, in relieving global IBS symptoms
(Mangel and Northcutt 1999; Houghton, Foster et al. 2000; Viramontes, Camilleri et al. 2001;
Zheng, Yu et al. 2017). Alosetron can cause severe constipation and ischemic colitis in IBS-C
patients and as a result was withdrawn from the market in 2000. However, the FDA

19

reintroduced it back to the marker under restricted prescription following patients’ request to
treat severe IBS-D in women (Zheng, Yu et al. 2017). On the other hand, the 5-HT3 receptor
antagonists approved in Europe are used to treat nausea but not IBS symptoms. In the far east,
ramosetron and azasetron are approved for the treatment of IBS-diarrhea (Thompson and
Lummis 2007). Tegaserod, a 5-HT4 receptor agonist, is efficacious in treating IBS-C but was
pulled of the market due to cardiovascular risks in 2007 (Layer, Keller et al. 2007).
1.5 Sex differences in IBS
IBS is considered a women’s health issue because it is twice as prevalent in females as in
males. Until recently this female predominance was thought to occur in western countries only
(Meleine and Matricon 2014; Mulak, Tache et al. 2014). However, a study done in China
showed that IBS is indeed more prevalent in female patients, especially those in more affluent
regions (Chang, Lu et al. 2010; Bhattarai, Muniz Pedrogo et al. 2017). The female predominance
in IBS is associated with the severity of constipation relative to that of diarrhea (Herman,
Pokkunuri et al. 2010). In addition to the female predominance in IBS, women IBS patients are
more likely to have IBS-C, comorbid anxiety, depression, and somatic pain. Furthermore,
alosetron has been shown to be more efficacious in female patients in comparison to male IBSD patients (Mulak, Tache et al. 2014).
The incidence of IBS in women decreases with age to equal that of males after the 6th
decade of life. Studies comparing IBS symptom severity across the menstrual cycle, pregnancy,
and menopause are equivocal. For instance, while the incidence of IBS decreases with age,
symptoms severity increases in menopause. Another study showed that post-menopausal

20

women experience IBS symptoms more frequently compared to men. However, the difference
was lost when the data was corrected for age (Cain, Jarrett et al. 2009). It has been established
that the high levels of ovarian hormones during pregnancy result in an analgesic state. It has
also been shown that during the third trimester of pregnancy gastrointestinal transit is slowed
(Chiloiro, Darconza et al. 2001). However, no studies were performed to specifically determine
the effect of pregnancy on IBS. In female IBS patients, abdominal is exacerbated during
menses when ovarian hormone levels reach their nadir (Houghton, Lea et al. 2002). Despite
these observations, the exact role of ovarian hormones in modulating abdominal pain remains
unclear. The role of estrogen in mediating visceral hypersensitivity is discussed in chapter 3.
1.6 Serotonin transporter knockout (SERT KO) rat, sex specific animal model of VHS
Clinical studies emphasized the importance of serotonergic signaling in the pathogenesis
of IBS inspiring the investigation of serotonergic dysfunction in animal models. For instance,
infusion of 5-HTP caused an increase in pain response to colorectal distension (CRD) in rats
following an inflammatory insult (Choi, Sung et al. 2008). Similarly, SERT KO mice have been
used to study gastrointestinal dysfunction. SERT KO mice exhibit GI motility dysfunction which
manifests as alternating periods of diarrhea and constipation (Chen, Li et al. 2001). However,
assessment of VHS in mice is difficult due to small size. Therefore, studies assessing visceral
sensation in these animals were not made.
Homberg et al developed a global SERT KO rat via N-ethyl-N-nitrourea (ENU) mutagenesis
which resulted in a single point mutation leading to premature stop codon in the part of the
gene that codes for the second extracellular loop of SERT (Figure 1.8) (Homberg, Olivier et al.

21

2007). While SERT KO rats exhibits decreased whole tissue levels of 5-HT in the brain, they have
a 9-fold increase in extracellular 5-HT concentrations in the hippocampus which was the only
brain region investigated. This is thought to occur due to the reduction in reuptake of 5-HT in
the absence of increased expression or activity of TPH. These rats also exhibit anxiety and
depression-like behaviors in a sex-independent manner which recapitulates the comorbidities
observed in some IBS patients (Olivier, Van Der Hart et al. 2008). Galligan et al. showed that
SERT KO rats is as an animal model to study sex-specific visceral hypersensitivity since the
female but not male rats exhibit increased visceromotor response (VMR) to colorectal
distension (CRD) (Galligan, Patel et al. 2013). A recent article written by Meleine and Matricon
highlighted the need for an integrated animal model that can recapitulate the multifactorial
and complex nature of the pathogenesis of IBS (Meleine and Matricon 2014). SERT KO rat
serves as such a model since it will allow us to understand how interactions between ovarian
hormones in presence of a dysfunction in serotonergic signaling contribute to the development
of visceral hypersensitivity.

Figure 1.8 Location of stop codon in SERT protein. In SERT KO rat, a stop codon in the part of
the gene that codes for the second extracellular loop (red arrow) results in a nonfunctional
SERT protein.
22

1.7 Hypothesis and specific aims
The overall aim of this study was to understand the paradoxical increase in visceral
sensitivity observed in SERT KO female rats following the administration of some 5-HT3
receptor antagonists and how estrogen could be modulating these effects. We hypothesized
that the location of 5-HT3 receptors and their interaction with estrogen receptors mediated this
response. We tested this hypothesis in 2 specific aims.
Specific Aim 1: To determine the location of 5-HT3 receptors mediating the enhanced pain
perception by testing VMR to CRD following subcutaneous, intrathecal and
intracerebroventricular administration of 5-HT3 receptor antagonists and to determine the
mechanism by which inhibition of 5-HT3 receptors increased VMR to CRD.
Specific Aim 2: To determine the mechanism by which estrogen could modulate the increase in
VMR to CRD in SERT KO rats by measuring VMR following 1) ovariectomy and 2) inhibition of
genomic and non-genomic estrogen receptors in intact SERT KO and WT female rats.

23

BIBLIOGRAPHY

24

BIBLIOGRAPHY

Bhattarai Y, Muniz Pedrogo DA, Kashyap PC (2017) Irritable bowel syndrome: a gut microbiotarelated disorder? American Journal of Physiology-Gastrointestinal and Liver Physiology
312:G52-G62.
Blackshaw LA, Brookes SJ, Grundy D, Schemann M (2007) Sensory transmission in the
gastrointestinal tract. Neurogastroenterology Motility 19:1-19.
Bouin M, Plourde V, Boivin M, Riberdy M, Lupien F, Laganière M, Verrier P, Poitras P (2002)
Rectal distention testing in patients with irritable bowel syndrome: Sensitivity,
specificity, and predictive values of pain sensory thresholds. Gastroenterology
122:1771-1777.
Brookes SJ, Spencer NJ, Costa M, Zagorodnyuk VP (2013) Extrinsic primary afferent signalling in
the gut. Nature Reviews of Gastroenterology and Hepatolology 10:286-296.
Buono JL, Carson RT, Flores NM (2017a) Health-related quality of life, work productivity, and
indirect costs among patients with irritable bowel syndrome with diarrhea. Health
Quality Life Outcomes 15:017-0611.
Buono JL, Mathur K, Averitt AJ, Andrae DA (2017b) Economic Burden of Irritable Bowel
Syndrome with Diarrhea: Retrospective Analysis of a U.S. Commercially Insured
Population. Journal of Managed Care and Specialty Pharmcy 23:453-460.
Camilleri M (2004) Is there a SERT-ain association with IBS? Gut 53:1396-1399.
Camilleri M, Coulie B, Tack JF (2001a) Visceral hypersensitivity facts sepculations and challenges
camelliri. Gut 48:7.
Camilleri M, Coulie B, Tack JF (2001b) Visceral hypersensitivity: facts, speculations, and
challenges. Gut 48:125-131.
Chang FY, Lu CL, Chen TS (2010) The current prevalence of irritable bowel syndrome in Asia. J
Neurogastroenterology and Motility 16:389-400.
Chen JJ, Li Z, Pan H, Murphy DL, Tamir H, Koepsell H, Gershon MD (2001) Maintenance of
serotonin in the intestinal mucosa and ganglia of mice that lack the high-affinity
serotonin transporter: Abnormal intestinal motility and the expression of cation
transporters. Journal of Neuroscience 21:6348-6361.
Chen L, Ilham SJ, Feng B (2017) Pharmacological Approach for Managing Pain in Irritable Bowel
Syndrome: A Review Article. Anesthesiology and Pain Medicine 7.
25

Choi IS, Cho JH, Jang IS (2013) 5-Hydroxytryptamine 1A receptors inhibit glutamate release in
rat medullary dorsal horn neurons. Neuroreport 24:399-403.
Choi YD, Sung TS, Kim HJ, La JH, Kim TW, Yang IS (2008) Increased 5-hydroxytryptamine
mediates post-inflammatory visceral hypersensitivity via the 5-hydroxytryptamine 3
receptor in rats. Digestive Disease and Sciences 53:2909-2916.
Cremon C, Carini G, Wang B, Vasina V, Cogliandro RF, De Giorgio R, Stanghellini V, Grundy D,
Tonini M, De Ponti F, Corinaldesi R, Barbara G (2011) Intestinal Serotonin Release,
Sensory Neuron Activation, and Abdominal Pain in Irritable Bowel Syndrome. The
American Journal of Gastroenterology 106:1290-1298.
Drossman DA, Camilleri M, Mayer EA, Whitehead WE (2002) AGA technical review on irritable
bowel syndrome. Gastroenterology 123:2108-2131.
Foley S, Garsed K, Singh G, Duroudier NP, Swan C, Hall IP, Zaitoun A, Bennett A, Marsden C,
Holmes G, Walls A, Spiller RC (2011) Impaired uptake of serotonin by platelets from
patients with irritable bowel syndrome correlates with duodenal immune activation.
Gastroenterology 140:1434-1443 e1431.
Furness JB, Rivera LR, Cho HJ, Bravo DM, Callaghan B The gut as a sensory organ: Nature
Reviews Gastroenterology and Hepatolology 2013 Dec;10(12):729-40. doi:
10.1038/nrgastro.2013.180. Epub 2013 Sep 24.
Galligan JJ, Patel BA, Schneider SP, Wang H, Zhao H, Novotny M, Bian X, Kabeer R, Fried D,
Swain GM (2013) Visceral hypersensitivity in female but not in male serotonin
transporter knockout rats. Neurogastroenterololgy and Motililty 25:e373-381.
Gebhart GF (1999) Peripheral contributions to visceral hyperalgesia. Canadian Journal of
Gastroenterology 13:37A-41A.
Gebhart GF, Bielefeldt K (2016) Physiology of Visceral Pain. Comprehensive Physiology 6:16091633.
Gershon MD (2004) Review article: serotonin receptors and transporters -- roles in normal and
abnormal gastrointestinal motility. Alimentary Pharmacology and Therapeutics 7:3-14.
Gershon MD, Tack J (2007) The serotonin signaling system: from basic understanding to drug
development for functional GI disorders. Gastroenterology 132:397-414.
Gralnek IM, Hays RD, Kilbourne A, Naliboff B, Mayer EA (2000) The impact of irritable bowel
syndrome on health-related quality of life. Gastroenterology 119:654-660.

26

Gunawardene AR, Corfe BM, Staton CA (2011) Classification and functions of enteroendocrine
cells of the lower gastrointestinal tract. International Journal of Experimental Pathology
92:219-231.
Halac U, Noble A, Faure C (2010) Rectal sensory threshold for pain is a diagnostic marker of
irritable bowel syndrome and functional abdominal pain in children. Journal of
Pediatrics 156:60-65 e61.
Herman J, Pokkunuri V, Braham L, Pimentel M (2010) Gender distribution in irritable bowel
syndrome is proportional to the severity of constipation relative to diarrhea. Gender
Medicine 7:240-246.
Hoekman DR, Rutten JM, Vlieger AM, Benninga MA, Dijkgraaf MG (2015) Annual Costs of Care
for Pediatric Irritable Bowel Syndrome, Functional Abdominal Pain, and Functional
Abdominal Pain Syndrome. Journal of Pediatrics 167:1103-1108.
Homberg JR, Olivier JD, Smits BM, Mul JD, Mudde J, Verheul M, Nieuwenhuizen OF, Cools AR,
Ronken E, Cremers T, Schoffelmeer AN, Ellenbroek BA, Cuppen E (2007) Characterization
of the serotonin transporter knockout rat: a selective change in the functioning of the
serotonergic system. Neuroscience 146:1662-1676.
Hornung JP (2003) The human raphe nuclei and the serotonergic system. Journal of Chemical
Neuroanatomy 26:331-343.
Houghton LA, Foster JM, Whorwell PJ (2000) Alosetron, a 5-HT3 receptor antagonist, delays
colonic transit in patients with irritable bowel syndrome and healthy volunteers.
Alimentary Pharmacology and Therapeutics 14:775-782.
Iyengar S, Ossipov MH, Johnson KW (2017) The role of calcitonin gene-related peptide in
peripheral and central pain mechanisms including migraine. Pain 158:543-559.
Jeong HJ, Mitchell VA, Vaughan CW (2012) Role of 5-HT(1) receptor subtypes in the modulation
of pain and synaptic transmission in rat spinal superficial dorsal horn. British Journal of
Pharmacology 165:1956-1965.
Jin D-C, Cao H-L, Xu M-Q, Wang S-N, Wang Y-M, Yan F, Wang B-M (2016) Regulation of the
serotonin transporter in the pathogenesis of irritable bowel syndrome. World Journal of
Gastroenterology 22:8137.
Jorgensen HS (2007) Studies on the neuroendocrine role of serotonin. Danish Medical Bulletin
54:266-288.
Kanazawa M, Hongo M, Fukudo S (2011) Visceral hypersensitivity in irritable bowel syndrome.
Journal of Gastroenterology and Hepatology 26:119-121.
27

Kirkup AJ, Brunsden AM, Grundy D (2001) Receptors and transmission in the brain-gut axis:
potential for novel therapies. I. Receptors on visceral afferents. American Journal of
Physiology-Gastrointestinal and Liver Physiology 280:G787-794.
Kohen R, Jarrett ME, Cain KC, Jun S-E, Navaja GP, Symonds S, Heitkemper MM (2009) The
Serotonin Transporter Polymorphism rs25531 Is Associated with Irritable Bowel
Syndrome. Digestive Diseases and Sciences 54:2663-2670.
Layer P, Keller J, Loeffler H, Kreiss A (2007) Tegaserod in the treatment of irritable bowel
syndrome (IBS) with constipation as the prime symptom. Therapeutic Clinical Risk
Management 3:107-118.
Lee KN, Lee OY (2016) The Role of Mast Cells in Irritable Bowel Syndrome. Gastroenterology
Research and Practice 2031480:28.
Llorca-Torralba M, Borges G, Neto F, Mico JA, Berrocoso E (2016) Noradrenergic Locus
Coeruleus pathways in pain modulation. Neuroscience 338:93-113.
Mangel AW, Northcutt AR (1999) Review article: the safety and efficacy of alosetron, a 5-HT3
receptor antagonist, in female irritable bowel syndrome patients. Alimentary
Pharmacology and Therapeutics 2:77-82.
Meleine M, Matricon J (2014) Gender-related differences in irritable bowel syndrome: potential
mechanisms of sex hormones. World Journal of Gastroenterology 20:6725-6743.
Millan MJ (2002) Descending control of pain. Progress Neurobiology 66:355-474.
Mulak A, Tache Y, Larauche M (2014) Sex hormones in the modulation of irritable bowel
syndrome. World Journal of Gastroenterology 20:2433-2448.
Olivier JD, Van Der Hart MG, Van Swelm RP, Dederen PJ, Homberg JR, Cremers T, Deen PM,
Cuppen E, Cools AR, Ellenbroek BA (2008) A study in male and female 5-HT transporter
knockout rats: an animal model for anxiety and depression disorders. Neuroscience
152:573-584.
Palecek J, Paleckova V, Willis WD (2002) The roles of pathways in the spinal cord lateral and
dorsal funiculi in signaling nociceptive somatic and visceral stimuli in rats. Pain 96:297307.
Park J. M, Choi M-G, Park J-A, Oh JH, Cho YK, Lee IS, Kim SW, Choi KY, Chung I-S (2006)
Serotonin transporter gene polymorphism and irritable bowel syndrome.
Neurogastroenterology and Motility 18:995-1000.

28

Rahman W, Bannister K, Bee LA, Dickenson AH (2011) A pronociceptive role for the 5-HT2
receptor on spinal nociceptive transmission: an in vivo electrophysiological study in the
rat. Brain Research 25:29-36.
Sengupta JN (2009) Visceral Pain: The Neurophysiological Mechanism. 194:31-74.
Simren M, Palsson OS, Whitehead WE (2017) Update on Rome IV Criteria for Colorectal
Disorders: Implications for Clinical Practice. Current Gastroenterology Reports 19:0170554.
Spohn SN, Mawe GM (2017) Non-conventional features of peripheral serotonin signalling - the
gut and beyond. Nature Reviews of Gastroenterology and Hepatology 14:412-420.
Su X, Gebhart GF (1998) Mechanosensitive pelvic nerve afferent fibers innervating the colon of
the rat are polymodal in character. Journal of Neurophysiology 80:2632-2644.
Todd AJ (2010) Neuronal circuitry for pain processing in the dorsal horn. Nature Reviews of
Neuroscience 11:823-836.
Viramontes BE, Camilleri M, McKinzie S, Pardi DS, Burton D, Thomforde GM (2001) Genderrelated differences in slowing colonic transit by a 5-HT3 antagonist in subjects with
diarrhea-predominant irritable bowel syndrome. American Journal of Gastroenterology
96:2671-2676.
Xu D, Wu X, Grabauskas G, Owyang C (2013) Butyrate-induced colonic hypersensitivity is
mediated by mitogen-activated protein kinase activation in rat dorsal root ganglia. Gut
62:1466-1474.
Yeo A (2004) Association between a functional polymorphism in the serotonin transporter gene
and diarrhoea predominant irritable bowel syndrome in women. Gut 53:1452-1458.
Zagorodnyuk VP, Kyloh M, Nicholas S, Peiris H, Brookes SJ, Chen BN, Spencer NJ (2011) Loss of
visceral pain following colorectal distension in an endothelin-3 deficient mouse model of
Hirschsprung's disease. Journal of Physiology 589:1691-1706.
Zheng Y, Yu T, Tang Y, Xiong W, Shen X, Jiang L, Lin L (2017) Efficacy and safety of 5hydroxytryptamine 3 receptor antagonists in irritable bowel syndrome: A systematic
review and meta-analysis of randomized controlled trials. PLoS One 12.

29

Chapter 2
Spinal 5-HT3 receptor inhibition increases visceral hypersensitivity in SERT KO female rats.
2.1 Abstract
The irritable bowel syndrome (IBS) is a functional gastrointestinal motor and visceral
sensation disorder that is more common in women compared to men. 5-Hydroxytryptamine (5HT, serotonin) signaling is disrupted in some IBS patients possibly due to polymorphic variations
in the gene encoding the serotonin transporter (SERT) which result in increased extracellular 5HT availability. Female SERT knockout (KO) rats exhibit visceral hypersensitivity to colonic
distention that mimics colonic hypersensitivity known to occur in female IBS patients.
Alosetron, a 5-HT3 receptor antagonist, is used to treat severe IBS-D in female patients. The
exact mechanism by which alosetron reduced abdominal pain is not well understood since it is
capable of crossing the blood brain barrier (BBB). Thus, it can act on peripheral and central 5HT3 receptors to reduce pain. We measured the visceromotor response (VMR) to colorectal
distension (CRD) in SERT KO and wild type (WT) rats following treatment with several 5-HT3
receptor antagonists and agonist. Specifically, we tested subcutaneous (s.c.), intrathecal (i.t.),
and intracerebroventricular (icv) administration of various BBB-permeable 5-HT3 receptor
antagonists and the partially BBB-permeable 5-HT3 receptor antagonist ramosetron (s.c.). We
also tested the effects of intrathecal administration of the 5-HT3 receptor agonist SR 57227 in
SERT KO and WT rats. Depletion of spinal serotonin via intrathecal administration of the
neurotoxin 5, 7-dihydroxytryptamine (5,7-DHT) was performed to determine the role of
descending serotonergic input in modulating pain transmission in the dorsal horn of the spinal
cord. Immunohistochemistry (IHC) studies were performed to determine the pattern of 5-HT3 A

30

receptor subunit and -aminobutyric acid (GABA) expression in the spinal cord of SERT KO and
WT rats. The 5-HT3 receptor antagonists alosetron and granisetron caused an increase in VMR
to CRD when given peripherally in SERT KO female rats. Alosetron also caused an increase in
VMR to CRD in WT male rats. Intrathecal alosetron increased VMR to CRD in SERT KO female
rats only, while alosetron (25 nmol, i.c.v) had no effect on VMR to CRD in all rats. In SERT KO
male rats intrathecal administration of SR 52772 increased visceral sensitivity. Depletion of
spinal 5-HT by 5, 7-DHT prevented the increase in VMR to CRD in SERT KO female and male rats
treated with intrathecal alosetron and SR 52772, respectively. IHC studies showed increased 5HT A subunit and GABA colocalization in the dorsal horn of the spinal cord in SERT KO female
rats compared to SERT KO males rats and WT rats. In conclusion, the increase in visceral
hypersensitivity in SERT KO female rats treated with 5-HT3 receptor antagonists is mediated by
blockade of spinal 5-HT3 receptors that are likely expressed on inhibitory GABAergic
interneurons. While in male SERT KO rats activation of 5-HT3 receptors caused an increase in
visceral sensitivity since 5-HT3 receptors are likely predominantly expressed on central
terminals of primary afferent neurons.
2.2 Introduction
Irritable bowel syndrome (IBS) is a functional gastrointestinal disorder characterized by
altered bowel habits and abdominal pain (Chey, Kurlander et al. 2015). The abdominal pain is
believed to occur due to visceral hypersensitivity secondary to neuronal dysfunction. 5-HT
signaling at 5-HT3 receptors may contribute to visceral pain observed in IBS patients. 5-HT3
receptors are expressed in the central and peripheral nervous system. (Jensen, Davies et al.
2008; Lummis 2012). They are found on ENS neurons and the peripheral and central terminals
31

of spinal and vagal extrinsic primary afferent neurons that innervate the gut (Figure
2.1)(Glatzle, Sternini et al. 2002). 5-HT3 receptors may also be expressed by colonic enterocytes
in humans as suggested by one study. (Kapeller, Houghton et al. 2008). Thus, the 5-HT3
receptors can contribute to gut motility and pain transmission from the gut (Morales,
Battenberg et al. 1998; Browning 2015).
In the CNS 5-HT3 receptors are expressed with the highest density in the nucleus tractus
solitarious and area postrema of the brainstem where the vomit reflex is mediated. In fact,
antagonists targeting 5-HT3 receptors are a mainstay for the treatment of post-surgical,
chemotherapy and radiation induced nausea (Barnes, Hales et al. 2009; Kovac 2016; Simino,
Marra et al. 2016).

32

Figure 2.1 5-HT3 receptor expression along the pain pathway. 5-HT3 receptors are expressed
in the peripheral and central nervous system. Extrinsic spinal and vagal primary afferents
express 5-HT3 receptors on their peripheral and central terminals.
5-HT3 receptors are ligand-gated cation channels composed of 5 subunits. They belong
to the Cys-loop receptor family which includes ionotropic GABAergic receptor and nicotinic
acetylcholine receptors (Jensen, Davies et al. 2008). At the time of their discovery it was
believed that pentameric 5-HT3 receptors where comprised of one type of subunit. However,
the electrophysiological properties of 5-HT3 receptors composed of only -subunits expressed
in cell lines differ from the properties of 5-HT3 receptors expressed in the nervous system of
laboratory animals suggesting the existence of other subunits. Butler et al. noticed differences

33

in 5-HT ED50 values in electrophysiological studies performed across species and different
tissues within a species(Butler, Hill et al. 1988; Wolf 2000). Since the 5-HT3A receptor subunit
was cloned, several other subunits and splice variants have been discovered confirming the
existence of multiple types of 5-HT3 receptors. In humans, 5 different subunits (A-E) have been
identified some of which have splice variants. The A-C subunits are expressed centrally and
peripherally, while the D and E subunits have been only identified in the gastrointestinal tract
(Barnes, Hales et al. 2009). It is important to note that for a 5-HT3 receptor to be functional it
must express at least one A-subunit. Polymorphisms in the A and E subunits have been
associated with increased IBS-diarrhea risk (Cheung and Wu 2014; Gu, Zhang et al. 2015). The
functional polymorphisms in the 5-HT3A and 5-HT3E gene result in increased 5-HT3 receptor
density in the cell membrane of transfected HEK293 cells suggesting that receptor upregulation
might occur in patients with these polymorphisms (Kapeller, Houghton et al. 2008).
In rodents only A and B subunits have been identified. While it is agreed that the 5-HT3A
receptor subunit is expressed both centrally and peripherally, the expression of 5-HTB subunit
in the central nervous system remains debatable due to discrepancies between
electrophysiological and immunohistochemistry studies (Morales and Wang 2002; Jensen,
Davies et al. 2008). Furthermore, in rodents the 5-HT3A subunit has two splice variants, a short
allele (5-HT3b or 5-HT3s) and long allele (5-HT3a or 5-HT3l) (Miquel, Emerit et al. 1995). The
short allele is more ubiquitous with 90% expression versus the long allele and is similar to the 5HT3A subunit in humans. For a 5-HT3 receptor to be functional, it must have an at least one Asubunit both in humans and other animals. Furthermore, the single channel conductance of 5HT3 receptors composed of 5 A-subunits is less than 1 picosecond. 5-HT3 receptors composed
34

of A and B-subunits exhibit a slower single channel conductance (Lummis 2012). . The
permeability of 5-HT3 receptors to cations is also dependent on their subunit composition. 5HT3A receptors are permeable to divalent and monovalent cations, while 5-HT3AB receptor are
selective for monovalent cations (Barnes, Hales et al. 2009; Thompson, Verheij et al. 2013).
These properties support the importance of distinguishing the different types of 5-HT3
receptors.
Alosetron is FDA approved for the treatment of severe IBS-diarrhea in female patients
only. Alosetron is not approved for the treatment of IBS-diarrhea in males because it failed to
improve symptoms. The reasons behind this observation remains unclear (Rahimi, Nikfar et al.
2008). Furthermore, the mechanism by which alosetron exerts its antinociceptive effect in
females is unknown. Positron emission topography (PET) scans showed that alosetron
treatment reduced cerebral blood flow in the amygdala and other limbic structures involved in
pain processing during rectal distension. These data suggest that central 5-HT3 receptor
antagonism caused pain relief in IBS patients. Furthermore, the authors also deduced from
their data that peripheral inhibition of 5-HT3 receptors by alosetron is unlikely to produce
analgesia since the regions of the brain receiving input from visceral afferent such as the
thalamus had either unchanged or reduced blood flow during distension (Mayer, Berman et al.
2002).
The role of 5-HT3 receptors in mediating pain transmission is even more controversial in
animal studies. Over the past 20 years many studies have been performed, while most support
a pronociceptive role of 5-HT3 receptors, some indicate that 5-HT signaling at 5-HT3 receptors

35

is antinociceptive. What is agreed upon is the importance of 5-HT3 receptors in mediating pain
transmission. 5-HT3A subunit knockout mice exhibit reduced pain response following
intraperitoneal 5-HT injection indicating that 5-HT3 receptors on primary afferent neurons are
required to mediate pain transmission. Furthermore, treatment of the 5-HT3A KO mice with
intrathecal 5-HT resulted in a damped scratching behavior compared to WT mice. This further
supports a pronociceptive role of spinal 5-HT3 receptors (Zeitz, Guy et al. 2002). Mice treated
with dextran sulfate sodium solution to induce colitis, exhibit increased pain behavior and
increased 5-HT3 receptor expression in dorsal root ganglia. (Matsumoto, Lo et al. 2012).
Similarly, rats treated with intracolonic acetic acid also develop colitis and exhibit 5-HT3
mediated increases in visceral sensitivity (Choi, Sung et al. 2008; Greenwood-Van Meerveld,
Mohammadi et al. 2014). Sengupta et al, 2009 showed that rats treated with an acidic saline
(pH4) intramuscular injection into gastrocnemius muscle develop somatic and visceral
hypersensitivity. Treatment with chronic intravenous or intrathecal alosetron reduces the
somatic pain but didn’t affect the visceral pain experienced in response to colorectal distension.
The authors concluded that while the inhibition of 5-HT3 receptors didn’t reduce the pain
response compared to controls it prevented the sensitization observed in acid treated rats
(Miranda, Peles et al. 2006). In a study where mast cell degranulation was induced by BrX-537A
to cause visceral hypersensitivity, 5-HT3 receptor inhibition didn’t affect the pain response.
However, 5-HT1A receptor inhibition reduced the pain response associated with mast cell
degranulation. (Coelho, Fioramonti et al. 1998). Infusion of 5-HT precursor 5hydroxytryptophan (5-HTP) caused bladder hyperalgesia in female rats and this response was
attenuated by intrathecal but not subcutaneous ondansetron suggesting a pronociceptive role

36

of 5-HT (Hall, DeWitte et al. 2015). While, 5-HT3 receptors on the vagus nerve play an
antinociceptive role in intact rats, spinal 5-HT3 receptors play a nociceptive role in rats
subjected to water avoidance stress (Bradesi, Lao et al. 2007). Coelho et al and Bradesi et al
concluded that upregulation of a spino-bulbo-spinal loop leads to increased activity of primary
afferent neurons to enhance pain perception via signaling at 5-HT3 receptors as shown in
Figure 2.2.

Figure 2.2 Spino-bulbo-spinal loop in the dorsal horn of the spinal cord. Descending
serotonergic input can cause pain facilitation via 5-HT3 receptors activation on central terminal
of primary afferent neurons. This will lead to increased excitatory neurotransmitter release and
enhanced nociceptive signaling via projecting neurons.
Saria et al showed that activation of spinal 5-HT3 receptors caused the release of
substance P, neurokinin A and calcitonin-gene related peptide, all of which are excitatory
37

neurotransmitters that cause increased pain transmission to corroborate the spino-bulbo-spinal
loop theory (Saria, Javorsky et al. 1990). Other 5-HT receptors are expressed in the spinal cord
and have been shown to modulate pain. For instance, 5-HT1A receptor agonists cause a
reduction in pain in response to hind paws formalin injection, while 5-HT3 receptors mediate
nociception (Oyama, Ueda et al. 1996). Depletion of 5-HT in the spinal cord by the neurotoxin 5,
7-dihydroxytryptamine reversed the antinociceptive effects of 5-HT3 receptor antagonists
suggesting that endogenous 5-HT activates 5-HT3 receptors to enhance pain transmission.
Replacing 5-HT via intrathecal injection in the 5, 7-DHT treated rats at a high and low dose
resulted in a variable response. Rats treated with a high dose of 5-HT intrathecally developed
hyperalgesia via 5-HT3 receptor activation. On the other hand, low dose 5-HT resulted in
antinociception by acting of 5-HT1A receptors (Oyama, Ueda et al. 1996; Sawynok and Reid
1996).
Other studies suggested that 5-HT signaling at spinal 5-HT3 receptors is antinociceptive.
Mice treated with intrathecal 5-HT or 5-HT3 receptor agonist (2-methy-5-HT) exhibit increased
latency of tail flick and decreased pain behaviors in response to intrathecal administration of Nmethyl-D-aspartate (NMDA) or substance P. This increase in latency and pain behaviors were
reversed following treatment with 5-HT3 receptor antagonists (Alhaider, Lei et al. 1991). In rats,
hot plate latency was increased following treatment with intrathecal 5-HT or 2-methy-5-HT and
was reversed with 5-HT3 receptor inhibition (Glaum, Proudfit et al. 1990). In both studies it was
concluded that 5-HT3 receptor mediated analgesia was produced by release of GABA from
inhibitory interneurons since the GABAA receptor antagonist, ICS-205-930 blocked the analgesic
effects of 5-HT3 receptor agonist.
38

It is clear from clinical and basic research discussed that the role of 5-HT signaling at 5HT3 receptors in pain modulation is not well understood. The discrepancies arise from animal
model, pain assay and route of administration of various 5-HT3 receptor antagonists and
agonist. For instance, animal models used to study IBS rely on inflammatory insults such as
intracolonic instillation of acetic acid or dextran sulfate to induce visceral pain. This is a good
animal model to study post-infectious IBS. However, post-infectious IBS is seen in only 10% of
IBS patients (Beatty, Bhargava et al. 2014). Therefore, relying on inflammation in animal models
to study IBS pathophysiology is not optimal. Stress-induced visceral hypersensitivity animal
models are also used extensively as 50 % of IBS patients have a comorbid anxiety disorder
(Grzesiak, Beszlej et al. 2014). Despite the increased prevalence of IBS in female patients most
basic research conducted relies on male animals. SERT KO rats circumvent these issues since
inflammatory insult, treatment with 5-HTP or stress stimuli are not required to induce visceral
hypersensitivity. Galligan et al. 2013 showed that SERT KO rat serves as a sex-specific animal
model of visceral hypersensitivity. Therefore the SERT KO rat serves as a great resource to help
us understand the mechanism by which a dysfunction in serotonergic system, which is observed
in some IBS patients, can lead to sex-specific visceral hypersensitivity. The studies performed
herein will test the hypothesis that that inhibition of 5-HT3 receptor will decrease VMR to CRD
in SERT KO female rats. This will allow us to gain insights on the specific role of 5-HT3 receptors
in mediating visceral hypersensitivity with underlying disruption in serotonergic signaling.

39

2.3 Methods
2.3.1 Animals
All animal use protocols were approved by the Institution Animal Use and Care
Committee at Michigan State University. The serotonin transporter (SERT) knockout (KO) and
wild type (WT) control rats were purchased under license from Genoway, Inc
(http://genoway.com/). The WT rats used in this study and the background of SERT KO rats are
the Wistar strain. Following surgery all animals were singly housed to prevent post-surgical
complications. Age matched 3-4 months old rats were used for all experiments described in this
chapter.
2.3.2 Surgery
Electromyographic (EMG) electrode implantation. Rats were deeply anaesthetized with
4% isoflurane gas and placed on a heating pad (~30oC). A midscapular incision was made
followed by a ventral midline incision. A hemostat was used to gently separate the skin from
the muscles. Teflon coated silver wires (A-M systems, Sequim, WA) were stitched into the
external oblique muscles above the inguinal ligaments and the incision was closed with wound
clips. The electrodes were tunneled subcutaneously, secured to the back musculature and
eternalized in the midscapular region for future access. Sutures were used to close the
midscapular incision. Rats were allowed to recover for 3 days before assessing visceromotor
response to colorectal distension. During recovery the rats were acclimatized to a plexiglass rat
restrainer for an hour a day over two consecutive days. Carprofen (5mg/kg, s.c.) and
piperacillin/tazobactam (120mg/kg, i.m.) were administered at the time of surgery to control
pain and reduce risk of infection respectively.
40

ICV cannula implantation. Rats were deeply anaesthetized with 4% isoflurane gas and
positioned in a stereotactic apparatus on a heating pad (~30oC). An incision was made to
expose the skull and a hole was drilled to place the cannula in the lateral ventricle. Jeweler’s
screws and dental cement were used to anchor the cannula in place. Carprofen (5mg/kg,
subcutaneous) and piperacillin/tazobactam (120mg/kg, intramuscular) were administered at
the time of surgery to control pain and reduce risk of infection respectively.
2.3.3 Visceromotor response (VMR) to colorectal distension (CRD) and volume pressure loop
data acquisition.
Under light Isoflurane anesthesia, a flexible latex balloon (4.5 cm, females; 7 cm, males)
was inserted intra-anally into the colon. The distal end of the balloon was located in the rectum
1 cm away from the anal sphincter and secured to the base of the tail using surgical tape to
prevent excretion of the balloon during experiments. After recovery from anesthesia, the
animals were placed in the plexiglass restrainer and allowed to acclimate for 30 minutes before
the initiation of the colorectal distension procedure. The balloon was connected to an animal
barostat (G&J electronics, Toronto, Ontario, Canada) and the electrode leads were connected
to amplifier (7P1, Grass-Instruments-Astro-Med Inc., West Warwick, RI, USA). The barostat and
amplifier were connected to an A/D converter amplifier (Digidata 1223A, Axon InstrumentsMolecular Devices, Sunnyvale, CA, USA) and signals digitized at 1KHz and recorded using
Axoscope 10 software (Axon-instruments-Molecular Devices).
The VMR to CRD was assessed before and after treatment with the drug being studied.
Each CRD procedure consisted of a series of 5 phasic distensions at pressures of 10, 20, 40, 60,

41

80 mmHg (10-s duration; 5-min interval between distension). The VMR to CRD was measured
by recording electromyographic (EMG) activity of the external oblique muscles 10 s before, 10 s
during, and 10 s after each distension episode. The EMG activity was analyzed using Clampfit
10 (Axon-instruments-Molecular Devices). The signal was rectified and the area under the curve
calculated and normalized to baseline which is measured 10-s before each distension episode.
The data is reported as normalized visceromotor response (VMR).
Volume pressure loop data were collected by measuring the pressure of the colon and
rectum in response to increased volume. The volume inside of the balloon was increased by 0.1
mL increments to reach a maximum of 1 mL. The pressure inside the colon recorded
throughout the balloon inflation period. This protocol was performed before and after drug
treatments before the start of VMR recoding. Using the barostat, the volume pressure loop
data was collected by recording the volume needed to increase the intracolonic pressure by 1.0
mmHg up to 10 mmHg.
2.3.4 Intrathecal injection
Rats were lightly anesthetized with 4% isoflurane, the hair along the dorsal aspect of the
lower spine was clipped. The L5-L6 intervertebral space was exaggerated by placing a 50 mL
centrifuge tube under the lower limbs. A 27 G ½ inch needle connected to a 50 μL glass syringe
was gently inserted into the vertebral canal. Tail flick confirmed entry of the needle into the
vertebral canal. Drugs were dissolved in 5-10 μL solvent and administered using a 50 μL glass
syringe to minimize back flow of CSF upon removal of the needle following injection.

42

2.3.5 Cerebrospinal fluid (CSF) fluid collection
Rats were deeply anaesthetized with 4% isoflurane gas and position in a stereotactic
apparatus to exaggerate the atlanto-occiptal (AO) joint. A midline incision was made at the base
of the skull and retractor was used to expose the AO joint. A pulled capillary was used to
puncture the ligaments and access the CSF in the cisterna magna. CSF samples were collected in
Eppendorf tubes and stored at – 80 degrees Celsius until time of analysis.
2.3.6 High-performance liquid chromatography (HPLC)
For ramosetron detection a Phenomenex Luna 5u C-18 250x4.6mm column was used.
A Photo Diode Array detector was employed and ramosetron was read at 213 nm. The mobile
phase was 70% 0.05M sodium phosphate (pH 5.2) and 30% acetonitrile with flow rate at 1.0
ml/min. 5-HT detection was performed using a Thermo Scientific ODS Hypersil 3 u 150 x 3mm
column with detection at 200 mV. The mobile phase used was 90mM Dihydrogen Sodium
Phosphate, 50 mM Citrate, 50 uM EDTA, and 1.7mM sodium octyl sulfate with 10% acetonitrile
with flow rate at 0.6 ml/min. Norepinephrine was detected using a Thermo Scientific HR-80 3u
80X4.6mm column with Cat-A-Phase II mobile phase with flow rate at 1.1 ml/min at detection
at -300mV.
2.3.7 Immunohistochemistry
The lumbosacral spinal cord and DRG were collected and fixed in 4 % paraformaldehyde
(PFA) overnight. Following fixation the tissue was cryopreserved in 30% (w/vol) sucrose
solution. Optimal cutting temperature (OCT) matrix compound was used to embed the tissue
for sectioning using a cryostat. Ten-20 μm sections were collected on glass slides and stored at -

43

80 oC. Thawed sections were washed with 0.01 M PB-T (PB with 0.4 % Triton X-100) 3 times for
a total of 30 minutes. To prevent non-specific binding, sections were incubated in 5% animal
serum (GS) for 1 hr at room temperature. Primary antibody incubations were performed
overnight at 4 oC. Following incubation with primary antibody, the sections were washed 3
times with PB for 10 minutes each and incubated with secondary antibody at room
temperature for 1 hr. Secondary antibody was washed off and sections were rinsed with PB 3
times for 10 minutes each. Prolong gold anti-fade reagent was applied to sections and cover
slips were placed to preserve slides for future imaging.
All primary antibodies were diluted in 1% animal serum and all secondary antibodies were
diluted in 0.01 M PB.

Table 2.1 Primary and secondary antibodies used for IHC
Antibody type
Vendor
Catalogue #
Primary:
5-HT3 BIOSS
Bs-12051R
receptor antibody
Primary:
GABA Abcam
Ab17413
antibody
Secondary: Alexa 488 Life
A11073
technologies
Secondary: Alexa 594 Life
A21207
technologies

Lot #
AE090712

Dilution
1:10

GR3175268-1

1:300

1841755

1:500

1668652

1:500

2.3.8 Statistics
Electromyographic recordings from visceromotor response to colorectal distension were
analyzed using two way-repeated measures ANOVA and Bonferonni’s post hoc test to
determine the effects of drugs on VMR at each distending pressure. We used 6-8 animals per
group to achieve a power of 0.8 unless specified otherwise for a given experiment. Data will be

44

presented as mean ± SEM. P-values less than 0.05 were considered statistically significant. HPLC
data were analyzed using Student’s-t test. P < 0.05 was considered statistically significant.
2.3.9 Time-line of studies performed

1

2

3

4

5

6

7

Day 1:
Days 3 and 4:
Day 5:
Electrode
Acclimate
Experiment
implant for
EMG study

8

9

10

Day 8:
Experiment

11
Day 11:
Experiment
and sacrifice
rats for tissue
collection

Figure 2.3 Time-line for studies of subcutaneous drug administration. Alosetron (0.1 mg/kg),
granisetron (0.1 mg/kg), and sterile saline vehicle were administered in random order. Rats
were equipped with electrodes on day one and allowed to recover on day 2. On day 3 and 4 the
rats were acclimated to the restrainer for one hour each day. On day 5, 8 and 11 VMR to CRD
was assessed before and after treatment with the drug or vehicle. Rats were euthanized at day
11 following the final experiment.

45

1

2

3

4

5

6

7

8

Days 3 and 4:
Day 5:
Day 1:
Acclimate
Experiment
Electrode
implant for
EMG study

9

10

11

12

13

14

Day 14:
Day 11:
Experiment Experiment
and sacrifice
rats for tissue
collection

Day 8:
Experiment

Figure 2.4 Ramosetron and alosetron dose response and intrathecal study time-lines. Three
different doses of ramosetron (0.01, 0.1 and 1 mg/kg) and sterile saline vehicle were
administered to each rat in random order. Rats were equipped with electrodes on day one and
allowed to recover on day 2. On day 3 and 4 the rats were acclimated to the restrainer for one
hour each day. On day 5, 8, 11 and 14 VMR to CRD was assessed before and after treatment
with the drug or vehicle. Rats were euthanized at day 14 following the final experiment. A
similar procedure was performed for another group of rats to determine the alosetron dose
response curve. Alosetron (0.01, 0.1 and 1 mg/kg) or sterile saline vehicle were given to each
rat. For intrathecal studies alosetron, granisetron and ondansetron were administered in
random order at a dose of 25 nmol in 10 μl of sterile saline.

1

2 3

Day 1:
Electrode
implant for
EMG study
and ICV
cannula

4

5 6

7

8

9

10 11 12

Days 7 and 8: Day 9:
Acclimate Experiment

13

14 15

Day 12:
Experiment

Day 15:
Experiment
and sacrifice
rats for tissue
collection

Figure 2.5 Intracerebroventricular study timeline. On day 1, rats were equipped with
electrodes for electromyographic recording and cannula in the right lateral cerebral ventricle.
Rats were allowed to recover for 6 days post-surgery. On days 7 and 8 rats were acclimated to
the restrainer for an hour each day. On days 9, 12 and 15 rats were treated with alosetron 25
nmol/10μl), alosetron (2.5 nmol/ 10 μl) or 10 μL of sterile saline vehicle in random order.

46

1

2

3

4

5

6

7

8

9

10

11

12

13

14

15

16

Day 16:
Experiment
and sacrifice
rats for tissue
collection
Figure 2.6 5,7-DHT experiment time-line. On day 1 rats were equipped with electrodes. On day
3 rats were treated with desipramine (25 mg/kg) via an intraperitoneal injection (i.p). Forty five
minutes after desipramine treatment rats were treated with 100 μg of 5, 7-DHT dissolved in 10
μL of sterile saline or sterile saline vehicle (10 μL). Rats were allowed to recover for 7 days
following 5, 7-DHT treatment. On days 8 and 9 rats were acclimated to the restrainer and on
days 10, 13 and 16 rats were treated with alosetron (25 nmol, i.t.), SR57227 (100 pmol, i.t.) or
sterile saline vehicle in random order. Visceromotor response to colorectal distension was
recorded before and after treatment.
Day 1:
Electrode
implant

Day 3: DMI
and 5,7 DHT
injections

Day 13:
Days 8 and 9: Day 10:
Acclimate Experiment Experiment

2.4 Results
To test the hypothesis that inhibition of 5-HT3 receptors decreases VMR to CRD we
administered various 5-HT3 receptor antagonists via subcutaneous injection. Alosetron and
granisetron increased VMR to CRD in SERT KO but not WT female rats as shown in Figure 2.7.
Alosetron increased VMR to CRD in WT male rats only and granisetron did not enhance pain
perception in WT and SERT KO male rats (Figure 2.8). Volume-pressure loop analysis which
measures the compliance of the GI tract indicates that inhibition of 5-HT3 receptors doesn’t
increase the VMR by reducing the relaxation of the gut walls in response to increased volume
as shown in Figure 2. 9.

47

Figure 2.7 Subcutaneous administration of 5-HT3 receptor inhibitor increased VMR to CRD in
SERT KO but not WT female rats. VMR recorded at the indicated pressure before (control) and
after treatment with 5-HT3R antagonists granisetron (0.1 mg/kg, s.c.), alosetron (0.1 mg/kg,
s.c.) or saline. Saline didn’t affect VMR to CRD in WT (top left) and SERT KO female rats (top
right). Granisetron didn’t affect VMR to CRD in WT female rats (bottom left) but increased VMR
to CRD in SERT KO female rats (middle right). Alosetron didn’t affect VMR to CRD in WT female
rats (middle left) but increased VMR to CRD in SERT KO female rats (bottom right). *, ** Indicate
significantly different from control (* P<0.05,** P<0.01). Data are mean ± SEM.

48

Figure 2.8 Subcutaneous administration of the 5-HT3 receptor inhibitor, alosetron, increased
VMR to CRD in WT but not SERT knockout (KO) male rats. VMR recorded at the indicated
pressure before (control) and after treatment with 5-HT3R antagonists granisetron (0.1 mg/kg,
s.c.), alosetron (0.1 mg/kg, s.c.) or saline. Saline didn’t affect VMR to CRD in WT (top left ) and
SERT KO male rats (top right). Granisetron didn’t affect VMR to CRD in WT (middle left) and
SERT KO male rats (middle right). Alosetron increased VMR to CRD in WT (bottom left) but not
SERT KO male rats (bottom right). **Indicates statistical significance from control (P<0.01). Data
are mean ± SEM.

49

30

8

KO, female
Pressure (mmHg)

Pressure (mmHg)

40

Control

20
10

Alosetron, s.c

0
0.0

0.5

1.0

KO, males

6

Control
4
2

Alosetron. s.c.
0
0.0

1.5

0.5

0.5

Pressure (mmHg)

Volume (mL)

3.0

WT, female

0.4

Alosetron, s.c.

0.3
0.2

Control
0.1
0.0
0

5

10

1.0

1.5

Volume (mL)

Volume (mL)

2.5
Control

2.0

pressure (mmHg)

Alosetron, s.c.

1.5
1.0
0.0

15

WT, males

0.5

1.0

1.5

Volume (mL)

Figure 2.9 Volume-Pressure loop analysis. Inhibition of 5-HT3R does not affect the compliance
of the colon and distal rectum in WT and SERT KO female and male rats. Data are mean ± SEM.
Alosetron (0.01 mg/kg, s.c) and (1mg/kg, s.c.) didn’t not affect VMR to CRD.
However, an intermediate dose of 0.1 mg/kg alsoetron caused an increase in VMR to CRD as
shown in Figure 2.10.

50

SERTKO Alosetron 0.01mg/kg, s.c.

SERTKO Alosetron 0.1mg/kg, s.c.
20

10

Normalized VMR

Normalized VMR

15

Alosetron

5

Control

0

Alosetron

15

*

10

Control

5
0

0

20

40

60

80

100

0

20

Pressure(mmHg)

40

60

80

100

Pressure(mmHg)

SERTKO Alosetron 1mg/kg, s.c.

Normalized VMR

30

20

Alosetron

Control

10

0
0

20

40

60

80

100

Pressure(mmHg)

Figure 2.10 Alosetron dose-response in SERT KO female rats. VMR recorded at the indicated
pressure before (control) and after treatment with alosetron at 0.01 mg/kg, 0.1 mg/kg, and 1.0
mg/kg, s.c. *Indicate significantly different from control (* P<0.05), Data are mean ± SEM.
It is evident from clinical and basic research that despite their similar affinities to 5-HT3
receptors, 5-HT3 receptor antagonists have variable effectiveness in treating pain and nausea.
For instance, ondansetron and granisetron reduce chemotherapy induced nausea, but
alosetron has no effect on nausea. Instead nausea is a side effect of alosetron (Cremonini,
Nicandro et al. 2012). Therefore, we decided to treat SERT KO females with ondansetron to
determine if we will observe a similar response to granisetron and alosetron. Ondansetron did
not increase VMR to CRD in SERT KO female rats (data not shown).

51

Alosetron and granisetron cross the BBB. Therefore, the increase in pain might be
mediated by central inhibition of 5-HT3 receptors. To test this hypothesis we administered
ramosetron, a partially BBB permeable 5-HT3 receptor antagonist at 3 doses. We hypothesized
that at the lower dose of 0.1 mg/kg, the drug will not have sufficient central nervous system
penetration while the higher dose will be able to accumulate in the CNS. To confirm our
hypothesis we measured ramosetron concentration in cerebrospinal fluid (CSF) using HPLC.
Ramosetron (1 mg/kg, s.c) was detected in CSF and caused an increase in VMR to CRD. The
lower dose of ramosetron (0.1 mg/kg, s.c.) was not detected in CSF and didn’t affect the pain
response in SERT KO rats as shown in Figure 2.11.

52

A

B

15

0.1 mg/kg, s.c.
Control

10

5

Ramosetron

Normalized VMR

0

0.01 mg/kg, s.c.
Control

10

Ramosetron

5

0
0

20

40

60

80

100

0

20

Pressure (mmHg)

60

80

100

Pressure (mmHg)

D

*
Control

0
0

20

40

60

80

10

5

0

100
0.
1

m

g/
kg

Pressure (mmHg)

m

5

*

(N
=4
)

Ramosetron

10

15

g/
kg

1 mg/kg, s.c.

(N
=6
)

15

CSF Ramosetron concentration
(ng/ml)

C
Normalized VMR

40

1.
0

Normalized VMR

15

Figure 2.11 Dose-response curve of s.c. ramosetron in SERT KO female rats. VMR recorded at
the indicated pressure before (control) and after treatment with ramosetron at 0.01 mg/kg (A),
0.1 mg/kg (B), and 1.0 mg/kg (C), s.c. HPLC analysis of CSF indicates that at 1.0 mg/kg, s.c. dose,
Ramosetron was detected (D). *Indicate significantly different from control (* P<0.05), Data are
mean ± SEM.
A more direct way of testing the effects of central inhibition of 5-HT3 receptors is to
directly administer the drugs into the CNS. This was accomplished by i.t. and i.c.v. injections.
Alosetron (25 nmol), granisetron (25 nmol), ondansetron (25 nmol) and saline vehicle were
administered i.t. to SERT KO and WT rats. Alosetron increased VMR to CRD in SERT KO female
rats only (Figure 2.12). Intrathecal administration of alosetron, granisetron and ondansetron did
not affect VMR to CRD in WT female rats, and male rats regardless of genotype (Figure 2.13).

53

C

10

E

15

KO female
Normalized VMR

Ondansetron

5

Control
0

10

Granisetron

5

Control

20

40

60

80

100

0

20

Pressure (mmHg)

Normalized VMR

Normalized VMR

WT, female

10

Ondansetron
5

Alosetron
5

0

20

40

60

Pressure (mmHg)

80

Control

0

100

0

20

F

15

WT, female
Granisetron

10

40

5

100

80

100

15

WT, female

10

Alosetron

5

Control

0
80

60

Pressure (mmHg)

Control

Control

0

60

***
***

Pressure (mmHg)

D

15

40

***

KO female

10

0
0

B

15

KO female

Normalized VMR

Normalized VMR

15

Normalized VMR

A

0
0

20

40

60

Pressure (mmHg)

80

100

0

20

40

60

80

100

Pressure (mmHg)

Figure 2.12 Intrathecal administration of alosetron increased VMR to CRD in serotonin
transporter (SERT) knockout (KO) but not WT female rats. VMR recorded at the indicated
pressure before (control) and after treatment with 5-HT3R antagonists granisetron (25 nmol,
i.t.), alosetron (25 nmol, i.t.) and ondansetron (25 nmol, i.t.). Ondansetron didn’t affect VMR to
CRD in WT (N=6) (A) and SERT KO female rats (B). Granisetron didn’t affect VMR to CRD in WT
(N=5) (C) and SERT KO female rats (D). Alosetron didn’t affect VMR to CRD in WT female rats
(N=5) (E) but increased VMR to CRD in SERT KO female rats. N=8 for SERT KO rats. *** indicates
significantly different than control (P<0.001) (F). Data are mean ± SEM.

54

20

15

Control
10
5

Alosetron

Granisetron

15
10
5

Control

0
20

40

60

80

100

20

Pressure(mmHg)
20

20

Normalized VMR

15

Alosetron

5

40

60

80

60

Pressure(mmHg)

0

20

20

15

Granisetron

10

80

40

60

100

80

5

WT, male

15

Ondansetron

10
5

Control
N=4

0
0

20

40

60

Pressure(mmHg)

100

Pressure(mmHg)

0
40

Control

Control

0
20

5

100

WT, male

Control
0

10

Pressure(mmHg)

WT, male

10

Ondansetron

0
0

Normalized VMR

0

KO, male

15

N=5

0

Normalized VMR

20

KO, male
Normalized VMR

KO, male
Normalized VMR

Normalized VMR

20

80

100

0

20

40

60

80

100

Pressure(mmHg)

Figure 2.13 Inhibition of spinal 5-HT3 receptors did not affect VMR to CRD in SERT KO or WT
male rats. VMR recorded at the indicated pressure before (control) and after treatment with 5HT3R antagonists granisetron (25 nmol, i.t.), alosetron (25 nmol, i.t..), .) or saline. Data are
mean ± SEM.
Drugs administered i.t. circulate throughout the central nervous system via CSF.
Therefore, the increase in VMR to CRD observed with i.t. alosetron could be mediated in the
brain or brainstem. To test this possibility we administered alosetron via i.c.v. cannula
implanted into the right lateral ventricle of the brain in SERT KO and WT rats. Confirmation of
proper placement of the ICV cannula was determined at the end of the last experiments by
injecting methylene blue and grossly inspecting the ventricles. ICV administration of alosetron
did not increase in VMR to CRD in all rats (Figure 2.14).

55

A

B
20

15

Alosetron

10
5

Control

WT, female

15

Control

10
5

0

Alosetron

Pressure (mmHg)

80

60

Pressure (mmHg)

C

D

20

20

KO, male

15

Normalized VMR

Normalized VMR

40

20

0

10
0

80

60

40

20

0

0

10
0

KO, female
Normalized VMR

Normalized VMR

20

Alosetron

10
5

WT male

15
10

Alosetron

5

Control

Control
0

0
0

20

40

60

80

100

0

Pressure (mmHg)

20

40

60

80

100

Pressure (mmHg)

Figure 2.14 Intracerebroventricular administration of alosetron didn’t affect VMR to CRD in
SERT KO and WT rats. VMR recorded at the indicated pressure before (control) and after
treatment with 5-HT3R antagonists alosetron (25nmol, i.t.) or saline. Data are mean ± SEM. KO
female N=7, WT female N=5, KO male N=6, WT male N=5.
Since the inhibition of 5-HT3 receptors caused an increase in VMR to CRD in SERT KO
female rats we wanted to test if activation of 5-HT3 receptors using an agonist could decrease
the pain response. The 5-HT3 receptor agonist SR 57227 caused an increase in VMR to CRD in
SERT KO males but not SERT KO female rats and WT rats (Figure 2.15).

56

A

B

15

KO female
Normalized VMR

10

SR 57227
5

Control

WT, female

10

SR 57227

5

Control

0

0
0

20

40

60

80

100

0

20

40

Pressure (mmHg)

80

100

D

25

25

KO, male
SR 57227

15

**

*

Normalized VMR

20

10
5

WT, male

20
15

Control

10
5

Control

SR 57227

Pressure (mmHg)

60

40

20

0

10
0

80

60

40

20

0

0

0

10
0

C
Normalized VMR

60

Pressure (mmHg)

80

Normalized VMR

15

Pressure (mmHg)

Figure 2.15 Activation of 5-HT3 receptor increased VMR to CRD in male SERT KO rat. The 5HT3 receptor agonist SR 57227 (100 pm/10μl) increased visceral sensitivity in SERT KO male
rats. VMR recorded at the indicated pressure before (control) and after treatment with 5-HT3R
agonist SR 57227 (100 pmol, i.t.). ). *, ** Indicate significantly different from control (* P<0.05),
(** P<0.01). Data are mean ± SEM.
Blocking the action of 5-HT from descending serotonergic neurons at 5-HT3 receptors
on inhibitory interneurons might be the mechanism by which alosetron increases VMR to CRD
in SERT KO female rats. To test this hypothesis the effects of intrathecal alosetron on VMR to
CRD were assessed after reduction of descending 5-HT neurons using the neurotoxin 5,7dihydroxytrptamine (5,7-DHT). Intrathecal treatment with 5,7 DHT reduced serotonin levels in
the lumbosacral spinal cord (figure 2.16). The reduction of 5-HT was more prominent in the WT
animals. HPLC analysis of lumbosacral spinal cord homogenate showed that 5-HT was reduced

57

by 60 % in WT female rats, 40% in WT male rats, 32 % in SERT KO male rats and 25% in SERT KO
female rats. Norepinephrine levels were not affected by 5,7 DHT treatment as shown in figure
2.17. IHC analysis of spinal cord samples from 5,7-DHT treated and untreated rats exhibited a
reduction in 5-HT staining in the treated rats versus untreated rats regardless of genotype.
Functional studies showed that the 25% reduction in 5-HT was sufficient to prevent the
increase in VMR to CRD in SERT KO female rats (figure 2.18). 5,7-DHT did not affect the
response to 5-HT3 receptor inhibition in WT female rats and male rats as shown in Figure 2.19.

58

Figure 2.16 5,7 DHT (100 μg, i.t.) treatment decreased 5-HT levels in the lumbar and sacral
spinal cord of KO and WT rats. IHC studies showed a reduction in 5-HT intensity in SERT KO
female rat treated with 5,7 DHT (A). Quantification of intensity of 5-HT signal from IHC
experiments shows a reduction in intensity in 5,7 DHT treated rats (B). HPLC analysis of lumbar
and sacral spinal cord indicates a reduction in 5-HT levels following 5,7 DHT treatment (C).

59

Figure 2.17 Norepinephrine levels are not affected by 5, 7-DHT. HPLC analysis was used to
determine the amount of norepinephrine in lumbosacral spinal cord. Desipramine treatment
prevented the uptake of 5, 7-DHT by noradrenergic neurons in WT and SERT KO rats. Data are
mean ± SEM

60

10

SR 57227

5

Control
0

Alosetron
10

5

Control

40

60

80

100

0

20

Pressure (mmHg)
15

WT, female

10

Control
5

60

80

40

60

Pressure (mmHg)

Alosetron
0

20

15

10

80

100

40

60

80

100

Pressure (mmHg)

Control

5

WT, female

10

Saline

5

Control

Alosetron
0

20

Control
5

100

WT, female

SR 57227
0
0

10

Pressure (mmHg)

Normalized VMR

Normalized VMR

40

Normalized VMR

20

KO, female

0

0
0

15

15

KO, female
Normalized VMR

15

KO, female
Normalized VMR

Normalized VMR

15

0
0

20

40

60

Pressure (mmHg)

80

100

0

20

40

60

80

100

Pressure (mmHg)

Figure 2.18 Treatment with 5,7-DHT prevented the increased VMR to CRD in SERT KO female
rats following alosetron treatment. SERT KO and WT female rats showed no change in VMR to
CRD following treatment with alosetron (25 nmol. i.t.), SR 57227 (100 pmol, i.t.) and sterile
saline vehicle (10 μL). Data are mean ± SEM

61

10

SR 57227
5

Control

Saline

SR 57227

5

Control
0

Pressure (mmHg)

10
0

80

60

KO, male
Control

10

Alosetron
5

Pressure (mmHg)

10
0

80

60

40

20

10
0

80

60

40

0

20

10
0

80

60

0

40

0

10
0

60

80

Normalized VMR

10

15

0

Saline

Normalized VMR

5

20

Control

Pressure (mmHg)

KO, male

0

Normalized VMR

Control

10

0

Alosetron
5

Pressure (mmHg)
15

KO, male

40

20

0

10
0

80

60

40

20

Pressure (mmHg)
15

10

0

0

0

0

WT, male

40

Control
5

15

20

10

WT, male
Normalized VMR

15

WT, male
Normalized VMR

Normalized VMR

15

Pressure (mmHg)

Figure 2.19 Treatment with 5,7-DHT prevented the increase in the VMR to CRD in SERT KO
male rats following SR 57227 treatment. SERT KO and WT female rats showed no change in
VMR to CRD following treatment with alosetron (25 nmol. i.t.), SR 57227 (100 pmol, i.t.) and
sterile saline vehicle (10 μL). Data are mean ± SEM

To determine the pattern of 5-HT3 receptor expression we used IHC. We also stained
spinal cord sections with GABA antibody to determine if 5-HT3 receptors are expressed on
GABAergic inhibitory interneurons (Figure 2.20). 5-HT3A receptor subunit and GABA are
colocalized to in all rats. A higher degree of colocalization is observed in SERT KO female rats as
shown in Figure 2.20 and Figure 2.21.

62

Figure 2.20 5-HT3A subunit and GABA expression in the superficial dorsal horn of the spinal cord in SERT KO rats. Alexa 594 (red)
was used to visualize 5-HT3A subunits expression. Alexa 488 (green) was used to visualize GABA expression. The top panel shows 5HT3A (A), GABA (B) and superimposed 5-HT3A and GABA images (C) in SERT KO female rats. The bottom panel shows 5-HT3A (D),
GABA (E) and superimposed 5-HT3A and GABA images (F) in SERT KO male rats. White arrows indicate regions of colocalization.

63

Figure 2.21 5-HT3A subunit and GABA expression in the superficial dorsal horn of the spinal cord in WT rats. Alexa 594 (red) was
used to visualize 5-HT3A subunits expression. Alexa 488 (green) was used to visualize GABA expression. The top panel shows 5-HT3A
(A), GABA (B) and superimposed 5-HT3A and GABA images (C) in WT female rats. The bottom panel shows 5-HT3A (D), GABA (E) and
superimposed 5-HT3A and GABA images (F) in WT male rats. White arrows indicate regions of colocalization.

64

2.5 Discussion
Peripheral administration of the 5-HT3 receptor antagonists alosetron and granisetron
at 0.1 mg/kg dose via subcutaneous injection resulted in an increase in VMR to CRD in SERT KO
female rats only (table 2.2). Alosetron but not granisetron caused an increase in VMR to CRD in
WT male rats. Volume-pressure loop analysis shown in figure 2.9 indicates that the increase in
VMR to CRD in response to alosetron in SERT KO female rats and WT male rats is not due to
reduced compliance of the colon and rectum. Compliance is a measure of wall tension in
response to change in pressure. (Gregersen and Kassab 1996). A reduction in compliance would
suggest that inhibition of 5-HT3 receptors within the gastrointestinal tract prevents the normal
relaxation reflex of the colon walls in response to increased pressure leading to increased pain.
A similar experiment to ours was performed by Bradesi et al, 2007 where they determined the
effects of alosetron (0.3 mg/kg, s.c.) on VMR to CRD. Their data was identical to our data which
showed an increase in VMR to CRD following subcutaneous administration of alosetron. They
showed that capsaicin induced vagal ablation prevented the increase in VMR to CRD observed
following treatment with alosetron. They concluded that inhibition of 5-HT3 receptors on
peripheral terminals of the vagus nerve decreased descending inhibition at the level of the
spinal cord which increased the VMR to CRD. They ruled out the possibility that alosetron is
acting centrally by showing that intrathecal administration of alosetron didn’t affect VMR to
CRD. This is in line with our intrathecal studies. In WT male rats, inhibition of spinal 5-HT3
receptors with alosetron, granisetron and ondansetron didn’t affect the VMR to CRD ruling out
direct central effects. Similarly, intrathecal 5-HT3 receptor inhibition did not affect the pain
response in WT female rats and SERT KO male rats. In SERT KO female rats, intrathecal

65

alosetron caused an increase in VMR to CRD. However, granisetron and ondansetron didn’t
affect the pain response.
5-HT3 receptor antagonists administered intrathecally can circulate to supraspinal
structures via CSF to exert their effects at the level of the brain. To determine if the effects of
alosetron are mediated in the brain we administered alosetron directly into the lateral ventricle
of the brain via an icv cannula. Alosetron (2.5 nmol and 25 nmol) did not affect VMR to CRD in
all rats when administered directly into the brain.
In SERT KO female rats we also tested the effects of the 5-HT3 receptor inhibitor,
ramosetron. Unlike the other 5-HT3 receptor antagonist tested, ramosetron is a partially BBB
permeable. While it does cross the BBB it is transported out of CNS via P-glycoprotein efflux
pumps (Yamamoto, Murakami et al. 2002). We tested the VMR to CRD in SERT KO female rats
after administration of 3 doses of ramosetron (0.01, 0.1 and 1 mg/kg, s.c.). The low dose of 0.1
mg/kg was not detected in CSF and did not affect VMR to CRD. The 1.0 mg/kg dose was
detected in CSF samples and increased visceral sensitivity confirming that central but not
peripheral inhibition of 5-HT3 receptors in SERT KO female rats leads to the increase in
discomfort.

66

Table 2.2 Summary of results from 5-HT3 receptor antagonist studies
SERT KO females
Increased VMR
Increased VMR

Alosetron, s.c.
Granisetron, s.c
Ondansetron, s.c
Alosetron, it
Increased VMR
Granisetron, it
Ondansetron, it
Alosetron, ICV
Ramosetron, s.c.
Low dose
Ramosetron, s.c
Increased VMR
High dose

SERT KO males

WT females

WT males
Increased VMR

np

np

np

np

np

np

np

np

np

Blank cells indicate no change; np indicates not performed
Our results indicate that alosetron causes an increase in visceral sensitivity in SERT KO
female rats by inhibiting lumbosacral spinal 5-HT3 receptors. However, while granisetron
caused an increase in visceral sensitivity with peripheral administration, it failed to increase
VMR following intrathecal and ICV application. Furthermore, alosetron but not granisetron
caused an increase in VMR to CRD in male rats. These observations are in line with multiple
animal studies which showed different 5-HT3 receptor antagonists exhibit variable responses.
Rats treated with intracolonic acetic acid show reduction in visceral hypersensitivity with s.c
granisetron but not ondansetron even when administered at a dose 10-fold higher than
granisetron (Langlois, Pascaud et al. 1996). In another study where visceral sensitivity was
induced via systemic injection of 5-hydroxytrptophan, granisetron but not ondansetron was
able to relief visceral sensitivity (Banner and Sanger 1995). In both studies it was speculated
that the variable response to 5-HT3 receptor antagonists is most likely due to the different
potencies for different 5-HT3 receptors. Perhaps granisetron has higher affinity for 5-HT3
receptors composed of 2-A and 3-B subunits, while ondansetron has higher potency for 5-HT3

67

receptor composed of A subunits only. This hypothesis has yet to be tested. It is worth
mentioning that studies characterizing 5-HT3 receptor antagonists are performed in 5-HT3
receptors composed of the A-subunit only (Banner and Sanger 1995). Clinically, variable
responses to 5-HT3 receptor antagonists have also been observed as summarized below.
In a study with 5 participants ondansetron increased pain threshold to rectal electric
stimulation but decreased maximum tolerated volume in rectal distension studies (Goldberg,
Kamm et al. 1996). A recent study assessing clinical endpoints in 120 IBS patients showed that
while ondansetron decreased stool frequency, urgency, and bloating it failed to improve pain
scores (Garsed, Chernova et al. 2014). However, granisetron was able to reduce pain response
in 12 IBS patients by assessing responses to rectal distension. Aside from the fact that this
study had only 12 participants, pain as a clinical endpoints was not measured in this study
which weakens the claim of the authors that granisetron reduces pain in IBS patients (Chen,
Ilham et al. 2017). Alosetron has been extensively studied in IBS patients with sufficient number
of participants and is the only FDA-approved 5-HT3 receptor antagonist shown to reduce
abdominal pain in female IBS-diarrhea patients (Rahimi, Nikfar et al. 2008; Cremonini, Nicandro
et al. 2012). The efficacy of alosetron in treating IBS in male patients has been questioned. A
recent study with over 600 IBS-D male patients showed that while alosetron improved stool
consistency, it failed to reduce the number of days with increased stool frequency, bloating and
pain (Grover and Camilleri ; Chang, Ameen et al. 2005). The difference in efficacy of alosetron
between males and females could be due to sex differences in drug metabolism (CYP2C19),
SERT gene polymorphism, and limbic system activation (women exhibit higher activity of limbic
system during rectal distension) (Berman, Munakata et al. 2000; Naliboff, Berman et al. 2003;
68

Koch, Corrigan et al. 2004). Furthermore, alosetron is more potent in female IBS-D patients
expressing the l/l allele of the gene encoding SERT (Acosta and Camilleri 2015). It has been
postulated that this due to increased 5-HT clearance in l/l carriers. Our studies support the
observation that the effect of alosetron depends on polymorphisms in the SERT gene since in
SERT KO rats, alosetron caused an increase in visceral sensitivity in female rats.
In SERT KO female rats alosetron exhibited a bell-shaped dose response curve as shown
in Figure 2.10. This was observed in more than one study where the potency of alosetron was
studied (Langlois, Pascaud et al. 1996; Clayton, Sargent et al. 1999). We can explain this
antagonist response by assuming that at high dose steric hindrance at the binding site of the
drug or action at other types of receptors could result in reduced potency (Wolf 2000). Indeed,
alosetron has been shown to bind albeit with low affinity to H3 histamine receptors and 5-HT1C
receptors (Clayton, Sargent et al. 1999).
Data from my i.t. studies indicate that the mechanism by which alosetron causes an
increase in visceral sensitivity in SERT KO female rats is different than that in WT male rats.
Descending serotonergic input to the dorsal horn of the spinal cord has been extensively
studied and plays a pivotal role in pain modulation in the presence of inflammation, nerve
injury or serotonergic dysfunction (Yoshimura 2006). SERT KO mice exhibit mechanical allodynia
but not thermal hyperalgesia following sciatic nerve injury. This is thought to be due to specific
changes in Aδ fibers (Vogel, Mossner et al. 2003). When assessing visceral pain produced by
intraperitoneal injection of acetic acid, SERT KO mice showed a similar response to WT mice.
(Hall, Schwarzbaum et al. 2011). Since sex-specific response to visceral pain is observed in SERT

69

KO rats, it is worth noting that the sex of the mice used in this study was not reported. A
potential mechanism by which 5-HT3 receptor inhibition could mediate increased pain
perception could be due to blocking the effects of 5-HT released from raphe neurons onto 5HT3 receptors expressed on spinal dorsal horn inhibitory interneurons as shown in Figure 2.22.
To test this hypothesis, we measured the effect of alosetron on visceral sensitivity in rats
treated with the neurotoxin 5,7-DHT. Treatment with 5,7-DH reduced spinal 5-HT levels
suggesting decreased descending 5-HT innervation to the lumbosacral spinal cord. Forty five
minutes before administration of 5,7 DHT, rats were treated with norepinephrine transporter
blocker, desipramine to prevent damage to nerve terminals of descending noradrenergic
neurons. Reduction in spinal 5-HT prevented the increase in visceral sensitivity observed with
intrathecal alosetron treatment suggesting that endogenous 5-HT signaling at 5-HT3 receptors
in SERT KO female rats is antinociceptive. A similar observation was reported in somatic pain
animal models where formalin was injected into the hind paw to induce somatic pain. Oyama,
Ueda et al. 1996 showed that reduction of spinal 5-HT reduced 5-HT3 receptor mediated
analgesia. In their study 5,7-DHT caused a 15% reduction in spinal 5-HT which is comparable to
the 5-HT reduction we saw in SERT KO female rats. In another study, HPLC analysis of
microdialysis samples from rat spinal cord treated with intrathecal 5-HT3 receptor agonist
showed an increased in GABA release compared to control rats (Kawamata, Omote et al. 2003).
Therefore, a potential mechanism by which 5-HT3 receptors mediate nociception is via
activating inhibitory GABAergic interneurons (Alhaider, Lei et al. 1991; Song, Meyerson et al.
2011). To test this possibility we performed immunohistochemistry analysis to determine coexpression of 5-HT3A subunits and GABA in dorsal horn of lumbar spinal cord. IHC experiments

70

showed that SERT KO female rats exhibit increased colocalization of 5-HT3A and GABA in the
superficial dorsal horn of the spinal cord compared to SERT male rats and WT rats.
Since inhibition of 5-HT3 receptors led to an increase in the VMR in SERT KO female rats,
we wanted to test if activation of 5-HT3 receptors will produce analgesia. We treated rats with
the selective 5-HT3 receptor agonist, SR 57227, via intrathecal injection. 5-HT3 receptor
activation didn’t affect the VMR in SERT KO female rats or WT rats. However, SR 57227 caused
an increase in VMR to CRD in SERT KO male rats. These data are summarized in table 2.3. This
may be observed due to expression of 5-HT3 receptors on central terminals of primary afferent
neurons in SERT KO male rats as shown in Figure 2.2. Depletion of 5-HT from descending
serotonergic neurons prevented the increase in visceral sensitivity in SERT KO male rats.
Table 2.3 Summary of 5-HT3 receptor antagonist and agonist studies
Alosetron, s.c.
Granisetron, s.c
Ondansetron, s.c
Alosetron, it

SERT KO females
Increased
sensitivity
Increased
sensitivity

SERT KO males

WT females

WT males
Increased
sensitivity

np

np

np

np

np

np

np

np

np

Increased
sensitivity

Granisetron, it
Ondansetron, it
Alosetron, ICV
Ramosetron, s.c.
Low dose
Ramosetron, s.c Increased
High dose
sensitivity
SR 57277, it

Increased
sensitivity

Alosetron, it
In 5,7-DHT
treated rats

Blank cells indicate no change; np indicates not performed

71

In conclusion, our data suggest that sex-difference in 5-HT3 receptor mediated pain
transmission is observed in SERT KO rats. In SERT KO female rats inhibition of spinal 5-HT3
receptors led to an increase in visceral sensitivity. On the other hand, spinal 5-HT3 receptor
activation caused an increase in pain transmission in SERT KO males rats. This may be due to
differential expression of 5-HT3 receptors in a sex-dependent manner. Functional and IHC data
suggest that 5-HT3 receptors are predominantly expressed on inhibitory GABAergic neurons in
SERT KO female rats and on central terminals of primary afferent neurons in SERT KO male rats.

Figure 2.22 Differential 5-HT3 receptor expression in female and male SERT KO rats. 5-HT
from descending serotonergic raphe nuclei acting on 5-HT3 receptors expressed on GABAergic
interneurons causes the release of GABA to reduce pain transmission.

72

BIBLIOGRAPHY

73

BIBLIOGRAPHY

Acosta, A. and M. Camilleri (2015). "Pharmacogenetics in irritable bowel syndrome." Expert
Opinion on Drug Metabolisim and Toxicology 11(8): 1187-1191.
Alhaider, A. A., S. Z. Lei, et al. (1991). "Spinal 5-HT3 receptor-mediated antinociception: possible
release of GABA." Journal of Neuroscience 11(7): 1881-1888.
Banner, S. E. and G. J. Sanger (1995). "Differences between 5-HT3 receptor antagonists in
modulation of visceral hypersensitivity." British Journal of Pharmacology 114(2): 558562.
Barnes, N. M., T. G. Hales, et al. (2009). "The 5-HT3 receptor--the relationship between
structure and function." Neuropharmacology 56(1): 273-284.
Beatty, J. K., A. Bhargava, et al. (2014). "Post-infectious irritable bowel syndrome: mechanistic
insights into chronic disturbances following enteric infection." World Journal of
Gastroenterology 20(14): 3976-3985.
Berman, S., J. Munakata, et al. (2000). "Gender differences in regional brain response to visceral
pressure in IBS patients." European Journal of Pain 4(2): 157-172.
Bradesi, S., L. Lao, et al. (2007). "Dual role of 5-HT3 receptors in a rat model of delayed stressinduced visceral hyperalgesia." Pain 130(1-2): 56-65.
Browning, K. N. (2015). "Role of central vagal 5-HT3 receptors in gastrointestinal physiology and
pathophysiology." Frontiers of Neuroscience 9(413).
Butler, A., J. M. Hill, et al. (1988). "Pharmacological properties of GR38032F, a novel antagonist
at 5-HT3 receptors." British Journal of Pharmacology 94(2): 397-412.
Chang, L., V. Z. Ameen, et al. (2005). "A dose-ranging, phase II study of the efficacy and safety of
alosetron in men with diarrhea-predominant IBS." American Journal of
Gastroenterology 100(1): 115-123.
Chen, L., S. J. Ilham, et al. (2017). "Pharmacological Approach for Managing Pain in Irritable
Bowel Syndrome: A Review Article." Anesthesiology and Pain Medicine 7(2).
Cheung, C. K. and J. C. Wu (2014). "Genetic polymorphism in pathogenesis of irritable bowel
syndrome." World J Gastroenterology 20(47): 17693-17698.

74

Chey, W. D., J. Kurlander, et al. (2015). "Irritable bowel syndrome: a clinical review." Journal of
Ameican Medical Association 313(9): 949-958.
Choi, Y. D., T. S. Sung, et al. (2008). "Increased 5-hydroxytryptamine mediates postinflammatory visceral hypersensitivity via the 5-hydroxytryptamine 3 receptor in rats."
Digestive Disease and Sciences 53(11): 2909-2916.
Clayton, N. M., R. Sargent, et al. (1999). "The pharmacological properties of the novel selective
5-HT3 receptor antagonist, alosetron, and its effects on normal and perturbed small
intestinal transit in the fasted rat." Neurogastroenterology and Motility 11(3): 207-217.
Coelho, A. M., J. Fioramonti, et al. (1998). "Mast cell degranulation induces delayed rectal
allodynia in rats: role of histamine and 5-HT." Digestive Disease and Sciences 43(4): 727737.
Cremonini, F., J. P. Nicandro, et al. (2012). "Randomised clinical trial: alosetron improves quality
of life and reduces restriction of daily activities in women with severe diarrhoeapredominant IBS." Alimentary Pharmacology Ther 36(5): 437-448.
Garsed, K., J. Chernova, et al. (2014). "A randomised trial of ondansetron for the treatment of
irritable bowel syndrome with diarrhoea." Gut 63(10): 1617-1625.
Glatzle, J., C. Sternini, et al. (2002). "Expression of 5-HT3 receptors in the rat gastrointestinal
tract." Gastroenterology 123(1): 217-226.
Glaum, S. R., H. K. Proudfit, et al. (1990). "5-HT3 receptors modulate spinal nociceptive
reflexes." Brain Research 510(1): 12-16.
Goldberg, P. A., M. A. Kamm, et al. (1996). "Modification of visceral sensitivity and pain in
irritable bowel syndrome by 5-HT3 antagonism (ondansetron)." Digestion 57(6): 478483.
Greenwood-Van Meerveld, B., E. Mohammadi, et al. (2014). "Synergistic effect of 5hydroxytryptamine 3 and neurokinin 1 receptor antagonism in rodent models of somatic
and visceral pain." Journal of Pharmacology and Experimental Therapeutics 351(1): 146152.
Gregersen, H. and G. Kassab (1996). "Biomechanics of the gastrointestinal tract."
Neurogastroenterology and Motility 8(4): 277-297.
Grover, M. and M. Camilleri Ramosetron in irritable bowel syndrome with diarrhea: new hope
or the same old story?, Clinical Gastroenterology and Hepatology. 2014 Jun;12(6):960-2.
doi: 10.1016/j.cgh.2013.12.025. Epub 2014 Jan 3.

75

Grzesiak, M., J. A. Beszlej, et al. (2014). "The lifetime prevalence of anxiety disorders among
patients with irritable bowel syndrome." Advanced Clinical Experimental Medicine
23(6): 987-992.
Gu, Q. Y., J. Zhang, et al. (2015). "Association of genetic polymorphisms in HTR3A and HTR3E
with diarrhea predominant irritable bowel syndrome." International Journal of Clinical
and Experimental Medicine 8(3): 4581-4585.
Hall, F. S., J. M. Schwarzbaum, et al. (2011). "A greater role for the norepinephrine transporter
than the serotonin transporter in murine nociception." Neuroscience 175: 315-327.
Hall, J. D., C. DeWitte, et al. (2015). "Serotonin enhances urinary bladder nociceptive processing
via a 5-HT3 receptor mechanism." Neuroscience Letters 604: 97-102.
Jensen, A. A., P. A. Davies, et al. (2008). "3B but which 3B and that's just one of the questions:
the heterogeneity of human 5-HT3 receptors." Trends in Pharmacological Sciences
29(9): 437-444.
Kapeller, J., L. A. Houghton, et al. (2008). "First evidence for an association of a functional
variant in the microRNA-510 target site of the serotonin receptor-type 3E gene with
diarrhea predominant irritable bowel syndrome." Human Molecular Genetics 17(19):
2967-2977.
Kawamata, T., K. Omote, et al. (2003). "The activation of 5-HT(3) receptors evokes GABA release
in the spinal cord." Brain Research 978(1-2): 250-255.
Koch, K. M., B. W. Corrigan, et al. (2004). "Alosetron repeat dose pharmacokinetics, effects on
enzyme activities, and influence of demographic factors." Alimentary Pharmacology and
Therapeutics 20(2): 223-230.
Kovac, A. L. (2016). "Comparative Pharmacology and Guide to the Use of the Serotonin 5-HT3
Receptor Antagonists for Postoperative Nausea and Vomiting." Drugs 76(18): 17191735.
Langlois, A., X. Pascaud, et al. (1996). "Response heterogeneity of 5-HT3 receptor antagonists in
a rat visceral hypersensitivity model." European Journal of Pharmacology 318(1): 141144.
Lummis, S. C. (2012). "5-HT(3) receptors." Journal of Biological Chemistry 287(48): 4023940245.
Matsumoto, K., M. W. Lo, et al. (2012). "Experimental colitis alters expression of 5-HT receptors
and transient receptor potential vanilloid 1 leading to visceral hypersensitivity in mice."

76

Laboratory Investigation 92(5): 769-782.
Mayer, E. A., S. Berman, et al. (2002). "The effect of the 5-HT3 receptor antagonist, alosetron,
on brain responses to visceral stimulation in irritable bowel syndrome patients."
Alimentary Pharmacology and Therapeutics 16(7): 1357-1366.
Miquel, M. C., M. B. Emerit, et al. (1995). "Developmental changes in the differential expression
of two serotonin 5-HT3 receptor splice variants in the rat." Journal of Neurochemistry
65(2): 475-483.
Miranda, A., S. Peles, et al. (2006). "Effects of the 5-HT3 receptor antagonist, alosetron, in a rat
model of somatic and visceral hyperalgesia." Pain 126(1-3): 54-63.
Morales, M., E. Battenberg, et al. (1998). "Distribution of neurons expressing immunoreactivity
for the 5HT3 receptor subtype in the rat brain and spinal cord." Journal of Comparative
Neurology 402(3): 385-401.
Morales, M. and S. D. Wang (2002). "Differential composition of 5-hydroxytryptamine3
receptors synthesized in the rat CNS and peripheral nervous system." Journal of
Neuroscience 22(15): 6732-6741.
Naliboff, B. D., S. Berman, et al. (2003). "Sex-related differences in IBS patients: central
processing of visceral stimuli." Gastroenterology 124(7): 1738-1747.
Oyama, T., M. Ueda, et al. (1996). "Dual effect of serotonin on formalin-induced nociception in
the rat spinal cord." Neuroscience Research 25(2): 129-135.
Rahimi, R., S. Nikfar, et al. (2008). "Efficacy and tolerability of alosetron for the treatment of
irritable bowel syndrome in women and men: a meta-analysis of eight randomized,
placebo-controlled, 12-week trials." Clinical Therapeutics 30(5): 884-901.
Saria, A., F. Javorsky, et al. (1990). "5-HT3 receptor antagonists inhibit sensory neuropeptide
release from the rat spinal cord." Neuroreport 1(2): 104-106.
Sawynok, J. and A. Reid (1996). "Interactions of descending serotonergic systems with other
neurotransmitters in the modulation of nociception." Behavioral Brain Research 73(1-2):
63-68.
Simino, G. P., L. P. Marra, et al. (2016). "Efficacy, safety and effectiveness of ondansetron
compared to other serotonin-3 receptor antagonists (5-HT3RAs) used to control
chemotherapy-induced nausea and vomiting: systematic review and meta-analysis."
Expert Review of Clinical Pharmacology 9(9): 1183-1194.

77

Song, Z., B. A. Meyerson, et al. (2011). "Spinal 5-HT receptors that contribute to the painrelieving effects of spinal cord stimulation in a rat model of neuropathy." Pain 152(7):
1666-1673.
Thompson, A. J., M. H. Verheij, et al. (2013). "Structure-activity relationships of quinoxalinebased 5-HT3A and 5-HT3AB receptor-selective ligands." ChemMedChem 8(6): 946-955.
Vogel, C., R. Mossner, et al. (2003). "Absence of thermal hyperalgesia in serotonin transporterdeficient mice." Journal of Neuroscience 23(2): 708-715.
Wolf, H. (2000). "Preclinical and clinical pharmacology of the 5-HT3 receptor antagonists."
Scandinavian Journal Rheumatology Suppl 113: 37-45.
Yamamoto, C., H. Murakami, et al. (2002). "Contribution of P-glycoprotein to efflux of
ramosetron, a 5-HT3 receptor antagonist, across the blood-brain barrier." Journal of
Pharmacy and Pharmacology 54(8): 1055-1063.
Yoshimura, M. (2006). "Plastic changes of the descending serotonergic system in the spinal
dorsal horn." Current Anaesthesia & Critical Care 17(1): 3-8.
Zeitz, K. P., N. Guy, et al. (2002). "The 5-HT3 subtype of serotonin receptor contributes to
nociceptive processing via a novel subset of myelinated and unmyelinated nociceptors."
Journal of Neuroscience 22(3): 1010-1019.

78

Chapter 3
Classical estrogen receptors play an antinociceptive role in SERT KO female rats
3.1 Abstract
Serotonin transporter knockout (SERT KO) female rats exhibit an increase in visceromotor
response (VMR) to colorectal distension (CRD) compared to wild type (WT) female rats. This
recapitulates clinical studies that show female IBS-D patients carrying the s/s polymorphism in
the SERT gene experience more severe abdominal pain. Studies performed in this chapter
investigated the effects of fluctuating ovarian hormone levels during the estrous cycle on VMR
to CRD. Ovariectomy and estrogen receptor (ER) antagonist studies were also performed in
SERT KO and WT female rats. VMR to CRD was enhanced in the proestrus phase of the estrous
cycle in SERT KO but not WT female rats. Ovariectomy increased discomfort to CRD in SERT KO
female rats in a time dependent manner. VMR to CRD increased on day 7 post-ovariectomy and
lasted for up to 3 weeks. Intrathecal administration of ICI 182 780 (ER-α and ER-β) antagonist
increased VMR to CRD in SERT KO female rats. The G-protein coupled ER (GPR30) antagonist,
G15, did not affect VMR to CRD in SERT KO and WT female rats. These studies suggest that in
SERT KO female rats classical ERs (ERα and ERβ) play an antinociceptive role in the presence of
serotonergic dysfunction.

79

3.2 Introduction
Irritable bowel syndrome (IBS) is considered a women’s health issue since it is more
prevalent in females compares to males. Questionnaire based and epidemiological studies
show a 2:1 and 4:1 female IBS predominance, respectively (Meleine and Matricon 2014; Mulak,
Tache et al. 2014; Harris, Umar et al. 2016). The motility disorder in IBS also exhibits a sex
difference. Female IBS patients are more likely to suffer from constipation while males have
more diarrhea (Adeyemo, Spiegel et al. 2010). Furthermore, women experience more
abdominal pain and as a result have lower IBS quality of life scores compared to males. (Cain,
Headstrom et al. 2006; Choghakhori, Abbasnezhad et al. 2017). In addition to the abdominal
pain, female IBS patients often present with comorbid chronic pain disorders such as
fibromyalgia and chronic pelvic pain (Meleine and Matricon 2014).
Most female IBS patients are of childbearing age. In fact the prevalence of IBS begins to
decrease by the 4th decade of life and equals that of males by the 7th decade (Heitkemper and
Chang 2009). Studies were performed to determine the influence of menstrual cycle, oral
contraceptive use, pregnancy and menopause on IBS symptoms. Unfortunately, no conclusions
were reached from these studies due to variation in study design. In pre-menses and menses
when ovarian hormone levels reach their nadir, IBS symptoms worsen (Heitkemper and Chang
2009; Mulak, Tache et al. 2014; Harris, Umar et al. 2016). Balloon rectal distension studies
showed that in IBS patients but not healthy controls rectal sensitivity is enhanced during
menses. In addition to the enhanced rectal sensitivity these patients also reported increased
abdominal pain and bloating during menses (Houghton, Lea et al. 2002).

80

Ovarian hormones can influence pain by affecting gut motility, permeability and the
stress response. It has been shown that patients with IBS have increased gut permeability
which correlates with symptom severity and that this is mediated via an estrogen dependent
mechanism (Harris, Umar et al. 2016). Unlike male IBS patients, female IBS patients exhibit
decreased activity of brain regions that mediate the hypothalamic-pituitary-axis response and
increased activation of the limbic system in response to colorectal distension (Naliboff, Berman
et al. 2003). These data suggest that there are sex differences in central processing of
abdominal pain. Ovarian hormone effects on motility are more complex. Studies suggest that
estrogen slows gut motility by upregulating nitric oxide signaling in the GI tract of pregnant
women and progesterone increases SERT expression to decrease extracellular 5-HT to slow gut
motility (Meleine and Matricon 2014).
While experiments done in rodents corroborate the clinical observation that females
exhibit higher pain response compared to males, the mechanisms by which this occurs remain
unclear. Like humans, rodents also exhibit fluctuations in their response to CRD across different
phases of the estrous cycle. An artificial stone implanted in the ureter of rats resulted in
increased pain behaviors in the metestrus and diesterus stages of the estrous cycle. Similarly,
noxious stimulation of the uterus increased escape behaviors during the metestrus and diestrus
stages of the estrous cycle (Giamberardino, Affaitati et al. 1997; Bradshaw, Temple et al. 1999).
On the other hand, visceral hypersensitivity to colorectal distension in rats is the highest during
proestrus phase (Ji, Tang et al. 2008) or proestrus and diestrus (Gustafsson and Greenwood-Van
Meerveld 2011; Galligan, Patel et al. 2013; Moloney, Sajjad et al. 2016).

81

There is strong evidence from the studies discussed herein that estrogen plays an
important role in pain modulation. However, the mechanisms by which estrogen modulates
nociceptive signal transmission are poorly understood. Estrogen signaling is mediated via
steroidal estrogen receptors (ER) and membrane-bound G-protein coupled estrogen receptors
(mER). The steroidal estrogen receptors ERα and ERβ mediate classical estrogen signaling which
affects protein expression. On the other hand, mERs mediate rapid estrogen receptor signaling
and include GPR30, ERα, ERβ, ER-αΔ4, ER-X and mER-Gαq (Wierman 2007; Sinchak and Wagner
2012). In addition to rapid signaling via activation of various protein kinases, m-ERs also affect
gene expression via downstream cyclic adenosine monophosphate (cAMP) and response
element protein (pCREP)(Wierman 2007).
Similar to the strong evidence linking estrogen to pain modulation, it is clear that an
interaction between estrogen and 5-HT also plays a role in mediating pain. Most of the clinical
studies investigating the link between the two signaling systems aim at understanding their role
in headaches. Data from a study investigating the pathogenesis of migraines suggests that
estrogen directly affects serotonergic signaling in the brain by upregulating tryptophan
hydroxylase 1 (TPH1) expression and down regulating SERT expression (Gupta, McCarson et al.
2011). Other studies suggest a direct effect of estrogen on 5-HT1 and 5-HT2 receptors in
mediating depression, osteoporosis and blood clotting disorders (Rybaczyk, Bashaw et al.
2005). Few experiments focus on signaling mechanisms that link 5-HT and ovarian hormones in
the pathogenesis of abdominal pain. Male and female IBS-diarrhea patients have higher fasting
and postprandial platelet-depleted plasma 5-HT levels compared to healthy controls.
Furthermore female IBS-diarrhea patients have higher 5-HT plasma levels during the luteal
82

phase when progesterone and estrogen levels are high compared to female IBS-diarrhea
patients during menses where ovarian hormone levels are low (Houghton, Brown et al. 2009).
This study suggests that 5-HT metabolism differs across phases of the menstrual cycle. Together
the data from this study and the clinical observation that 5-HT3 receptor inhibition reduces
abdominal pain in females strongly suggests that an interaction between ovarian hormones and
5-HT signaling contribute to the pathogenesis of IBS.
Galligan et al, 2013 showed that the SERT KO female rat exhibits increased visceral
hypersensitivity compared to WT female rats. SERT KO and WT male rats have similar VMR to
CRD. Furthermore, the increase is visceral sensitivity in SERT KO female rats is estrous cycle
depend since VMR to CRD is enhances in the proestrus and estrus phase. In this chapter I will
test the hypothesis that ovarian hormones cause the increase in visceral hypersensitivity in
SERT KO rat by exerting their effects on spinal estrogen receptors specifically.
3.3 Methods
3.3.1 Animals
All animal use protocols were approved by the Institution Animal Use and Care
Committee at Michigan State University. The serotonin transporter (SERT) knockout (KO) and
wild type (WT) control rats were purchased under license from Genoway, Inc
(http://genoway.com/). The WT rats used in this study and the background of SERT KO rats are
the Wistar strain. Following surgery all animals were singly housed to prevent post-surgical
complications. Age matched 3-4 months old rats were used for all experiments described in this
chapter.

83

3.3.2 Surgery
Electromyographic (EMG) electrode implantation. Rats were deeply anaesthetized with
4% isoflurane gas and placed on a heating pad (~30oC). A midscapular incision was made
followed by a ventral midline incision. A hemostat was used to gently separate the skin from
the muscles. Teflon coated silver wires (A-M systems, Sequim, WA) were stitched into the
external oblique muscles above the inguinal ligaments and the incision was closed with wound
clips. The electrodes were tunneled subcutaneously, secured to the back musculature and
eternalized in the midscapular region for future access. Sutures were used to close the
midscapular incision. Rats were allowed to recover for 3 days before assessing visceromotor
response to colorectal distension. During recovery the rats were acclimatized to a plexiglass rat
restrainer for an hour a day over two consecutive days. Carprofen (5mg/kg, s.c.) and
piperacillin/tazobactam (120mg/kg, i.m.) were administered at the time of surgery to control
pain and reduce risk of infection respectively.
Ovariectomy. Ovariectomies or sham operation of rats (n=48, 24 WT and 24 SERT KO
rats) were performed under isoflurane anesthesia (4%) and aseptic conditions. Surgical sites
were scrubbed with chlorhexidine and alcohol wipes. Bilateral flank incisions 5 mm in length
were made through the skin and then muscle wall. The ovaries were pulled through the
incisions, and the ovarian artery and vein were tied off tightly with suture silk before cutting off
the ovaries. The muscle wall incision was closed with suture silk and the skin incision was
closed with wound clips. Uterine weight was recorded at the time of euthanasia to confirm
ovariectomy.

84

3.3.3 Visceromotor response (VMR) to colorectal distension (CRD)
Under light Isoflurane anesthesia, a flexible latex balloon (4.5 cm, females; 7 cm, males)
was inserted intra-anally into the colon and secured to the base of the tail using surgical tape.
After recovery from anesthesia, the animals were placed in the plexiglass restrainer and
allowed to acclimate for 30 minutes before the initiation of the colorectal distension procedure.
The balloon was connected to an animal barostat (G&J electronics, Toronto, Ontario, Canada)
and the electrode leads were connected to amplifier (7P1, Grass-Instruments-Astro-Med Inc.,
West Warwick, RI, USA). The barostat and amplifier were connected to an A/D converter
amplifier (Digidata 1223A, Axon Instruments-Molecular Devices, Sunnyvale, CA, USA) and
signals digitized at 1KHz and recorded using Axoscope 10 software (Axon-instrumentsMolecular Devices).
The VMR to CRD was assessed before and after treatment with the drug being studied.
Each CRD procedure consisted of a series of 5 phasic distensions at pressures of 10, 20, 40, 60,
80 mmHg (10-s duration; 5-min interval between distension). The VMR to CRD was measured
by recording electromyographic (EMG) activity of the external oblique muscles 10 s before, 10 s
during, and 10 s after each distension episode. The EMG activity was analyzed using Clampfit
10 (Axon-instruments-Molecular Devices). The signal was rectified and the area under the curve
calculated and normalized to baseline which is measured 10-s before each distension episode.
The data is reported as normalized VMR.

85

3.3.4 Intrathecal injection
Rats were lightly anesthetized with 4% isoflurane, the hair along the dorsal aspect of the
lower spine was clipped. The L5-L6 intervertebral space was exaggerated by placing a 50 mL
centrifuge tube under the lower limbs. A 27 G ½ inch needle connected to a 50 μL glass syringe
was gently inserted into the vertebral canal. Tail flick confirmed entry of the needle into the
vertebral canal. Drugs were dissolved in 5-10 μL solvent and administered using a 50 μL glass
syringe to minimize back flow of CSF upon removal of the needle following injection.
3.3.5 Vaginal lavage and cytology
The vaginal canal was flushed with a few drops of sterile saline 2-3 times. The resulting
solution was placed on a glass slide and visualized at 20X magnification to determine the phase
of the estrous cycle.
3.3.6 Statistics
Electromyographic recordings of the visceromotor response to colorectal distension
were analyzed using two way-repeated measures ANOVA and Bonferonni’s post hoc test to
determine the effects of drugs on VMR at each distending pressure. We used 6-8 animals per
group to achieve a power of 0.8 unless specified otherwise for a given experiment. Data will be
presented as mean ± SEM. P-values less than 0.05 were considered statistically significant.

86

3.3.7 Time-line of studies performed

1

2

3

4

5

6

7

8

9

10 11 12

Day 1:
Days 3 and 4: Day 5: VMR to
OvariectomyAcclimate
CRD
and Electrode
experiment
implant for
EMG study

13 14 15

Day 7: VMR to
CRD
experiment

16 17 18 19 20 21

Day 14: VMR to
CRD
experiment

Day 21: VMR to
CRD
experiment

Figure 3.1 Timeline of ovariectomy studies. On day 1 rats underwent ovariectomy or sham
ovariectomy and electrode implant. Following a 2 day recovery, rats were acclimated to the
restrainer on days 3 and 4 for one hour each day. On days 5, 7, 14 and 21 VMR to CRD
experiments were performed and the rats were euthanized at the end of the last experiment.

1

2

Day 1:
Electrode
implant for
EMG study

3

4

5

6

Days 6 and 7:
Day 5:
Experiment Second and

Days 3 and 4:
Acclimate

First injection

third
Injections

7

Day 8: Fourth
injection,
Experiment
and sacrifice

Figure 3.2 Estrogen receptor antagonist experiment timeline. Electrode implants were
performed on day 1. On days 3 and 4 rats were acclimated to the restrainer. On day 5 VMR to
CRD was performed before and 30 minutes after the first injection of ER antagonist (ICI 182
780, 100 nM/10 μL, i.t) or G15 10ng/10 μL, i.t.) or vehicle ( 20% (2-Hydroxypropyl)-βcyclodextrin ). Injections were repeated on days 6 and 7. On day 8 animals received final
injection and VMR to CRD experiments were performed. At the end of the experiment the
animals were euthanized for tissue collection.

87

8

3.4 Results
Female SERT KO rats exhibit an enhanced VMR to CRD compared to WT female rats. On
the other hand, SERT KO and WT male rats do not demonstrate a difference in their response to
CRD (Figure 3.3). The discomfort in response to CRD is dependent on the estrus cycle as shown
in Figure 3.4. SERT KO female rats experience increased VMR to CRD compared to WT female
rats in the proestrus phase of estrous cycle.

B

A

25

10

***
SERT KO

***

6

**
4
2

WT

Male
Normalized VMR

Normalized VMR

Female
8

0

20

SERT KO

15
10

WT
5
0

0

20

40

60

80

0

Pressure (mmHg)

20

40

60

80

100

Pressure(mmHg)

Figure 3.3 SERT KO rats exhibit a sex dependent increase in VMR to CRD. Female SERT KO rats
exhibit an increase in VMR to CRD compared to WT female rats (n=7) (A). There is no difference
in visceral sensitivity to CRD between SERT KO and WT male rats (n=7) (B). **, *** indicates
significantly different than control (** P<0.01), (***P<0.001) (F). Data are mean ± SEM.

88

A

B
Estrus

15

Normalized VMR

Normalized VMR

15

SERT KO

10

5

Diestrus/metestrus
SERT KO

10

5

WT

WT
0

0
0

20

40

60

80

100

0

20

Pressure (mmHg)

40

60

80

100

Pressure (mmHg)

C
Normalized VMR

15

Proestrus
SERT KO

****

****

10

**
5

WT
0
0

20

40

60

80

100

Pressure (mmHg)

Figure 3.4 The increase VMR to CRD is estrous cycle dependent. In the estrus and
diestrus/metestrus phase of the estrous cycle VMR to CRD did not differ between SERT KO and
WT female rats(n=9) (A,B). In the proestrus phase SERT KO female rats exhibit increased VMR
to CRS compared to WT female rats (n-=8) (C). **, *** indicates significantly different than
control (** P<0.01), (***P<0.001) (F). Data are mean ± SEM.
Ovariectomy further enhanced VMR to CRD in SERT KO female rats but not WT female
rats. The increase in VMR to CRD occurred 7 days post-ovariectomy and persisted for up to 3
weeks as shown in Figure 3.5. On the other hand, ovariectomy did not affect VMR to CRD in WT
female rats (Figure 3.6). Sham WT female rats were unable to be maintained for 3 weeks since
animals damaged their electrodes or aggravated the back incision where the electrodes were
exteriorized.

89

Day 5 post-OVX

A

25

OVX (n=5)
SHAM (n=5)

20

Normalized VMR

Normalized VMR

25

Day 7 post-OVX

B

15
10
5
0

OVX (n=5)
SHAM (n=5)

20

**

15
10
5
0

0

20

40

60

80

100

0

20

Pressure (mmHg)

Day 14 post-OVX
25

OVX n=5
SHAM=4

20

D
Normalized VMR

Normalized VMR

C

**

15

*

*

10

40

60

80

100

80

100

Pressure (mmHg)

5
0

Day 21 post-OVX
25

OVX=5
20 SHAM=4
15
10
5
0

0

20

40

60

80

100

0

Pressure (mmHg)

20

40

60

Pressure (mmHg)

Figure 3.5 Ovariectomy increased VMR to CRD in SERT KO female rats in a time dependent
manner. Ovariectomy caused an increase in VMR to CRD in SERT KO female rats compared to
sham SERT KO female rats on days 7 and 14 post-ovariectomy (B –C). VMR to CRD was not
different between ovariectomized and sham rats on days 5 and 21 (A, D). *, ** indicates
significantly different than control (* P<0.05), (**P<0.01) (F). Data are mean ± SEM. OVX:
ovariectomy.

90

Day 5 post-OVX
20

B

OVX (n=5)
SHAM (n=3)

Normalized VMR

Normalized VMR

A

15
10
5

Day 7 post-OVX
20

OVX (n=5)
SHAM (n=3)

15
10

0

5
0

0

20

40

60

80

100

0

20

Pressure (mmHg)

Normalized VMR

C

40

60

80

100

Pressure (mmHg)

Days 5, 7,14, 21 post-OVX
20

Day 5
Day 7
DAY14 (n=3)
DAY 21(n=3)

15
10
5
0
0

20

40

60

80

100

Pressure (mmHg)

Figure 3.6 Ovariectomy did not affect VMR to CRD in WT female rats. VMR to CRD did not
differ between ovariectomized and sham operated WT female rats on days 5 and 7 potsovariectomy (A,B). VMR to CRD did not differ among ovariectomized rats on day 5, 7, 14 and
21. Data are mean ± SEM. OVX: ovariectomy.
Intrathecal administration of the steroidal estrogen receptor antagonist ICI 182 780 (100
nM/10μL) caused an increase in VMR to CRD in SERT KO female rats one hour after the first
injection. The increase in VMR to CRD was maintained following the fourth injection as shown
in Figure 3.7. On the other hand, the mER receptor antagonist G15 did not affect VMR to CRD
in SERT KO and WT female rats.

91

A

B

C
10

8
6
4
2

8
6
4
2
0

0
0

20

40

60

80

20

40

60

80

4
2
0

Pressure (mmHg)

20

80

100

40

60

80

100

Pressure (mmHg)
10

WT, G 15

8
6
4
2

KO, ICI 182 780

**

8

*

6
4
2
0

0
60

0

Normalized VMR

Normalized VMR

6

40

2

F
10

8

20

4

100

E
KO, vehicle

0

6

Pressure (mmHg)

D
10

WT, ICI 182 780

8

0
0

100

Pressure (mmHg)

Normalized VMR

10

WT, G 15
Normalized VMR

WT, vehicle
Normalized VMR

Normalized VMR

10

Control
First injection
Fourth injection

0

20

40

60

Pressure (mmHg)

80

100

0

20

40

60

80

100

Pressure (mmHg)

Figure 3.7 The effect of intrathecal administration of ER receptor antagonists. The vehicle and
GPR30 antagonist G15 (10 ng/ 10μL, i.t.) did not affect VMR to CRD in SERT KO and WT female
rats (A, B, E, D). The steroidal estrogen receptor antagonist ICI 182780 (100 nM/ 10 μL, i.t.) did
not affect VMR to CRD in WT female rats (E) but increased VMR to CRD in SERT KO female rats
following the first injection and fourth injection (F). *, ** indicates significantly different than
control (* P<0.05), (**P<0.01) (F). Data are mean ± SEM.
3.5 Discussion
The SERT KO female rat exhibits increased visceral sensitivity to CRD compared to WT
female rats. This recapitulates the clinical observation that female IBS patients with s/s allele of
the SERT gene experience severe abdominal pain compared to IBS patients expressing s/l and l/l
allele (park, choi et al. 2006). Fluctuations in ovarian hormone levels occurring during the
estrous cycle affect the level of discomfort experienced during CRD. In intact Sprague-Dawley
rats VMR to CRD was highest during the proestrus phase of the estrous cycle (Ji, Tang et al.
2008). Similarly, Moloney et al, 2016 showed that the Sprague-Dawley female rat exhibited
increased pain behaviors during CRD in the proestrus and estrus phase of estrus cycle.
92

However, this response is lost in rats subjected to maternal separation stress as they exhibit
increased pain behaviors in all phases of the estrus cycle (Moloney, Sajjad et al. 2016). Galligan
et al, 2013 showed that the VMR to CRD is enhanced in the proestrus and estrus phase of the
estrous cycle in SERT KO female rats. However, in my hands I was able to show an increased in
pain response only in the proestrus phase of the estrous cycle as shown in Figure 3.4. Most
studies equate the proestrus cycle with high estrogen levels and the rapid change in estrogen
levels is often overlooked (Goldman, Murr et al. 2007). Estrogen levels begin to decline at in
the early afternoon in the proestrus stage. Therefore, the fluctuation in estrogen levels might
contribute to the increase in VMR and pain behaviors in response to CRD (Figure 3.8). Studies
discussed herein, similar to my studies, were performed between 9am-2pm. Therefore the
declining levels of estrogen in early afternoon might contribute to the increase in discomfort.
Clinically, abdominal pain in IBS patients is exaggerated in pre-menses and menses when
estrogen levels drop to reach their nadir (Houghton, Lea et al. 2002; Heitkemper and Chang
2009). Experts in the field suggest that the fluctuations in ovarian hormones levels leads to the
increase in pain observed in peri-menses and menses.
The ovaries produce both estrogen and progesterone. While many studies investigated
the role of estrogen which will be discussed later, the role of progesterone in mediating visceral
pain has not been adequately investigated. Thus, its role in mediating pain is equivocal. In one
study progesterone administration into the amygdala of ovariectomized rats caused an
increases in abdominal contraction in response to VMR (Myers, Schulkin et al. 2011). On the
other hand, progesterone reduced estrogen induced visceral hypersensitivity in ovariectomized
rats treated with intracolonic mustard oil to induce inflammation (Ji, Tang et al. 2005). In
93

another study progesterone administration to ovariectomized rats did not affect VMR to CRD
indicating that progesterone signaling in not important in pain modulation (Lu, Hsieh et al.
2009).

Figure 3.8 Fluctuation of ovarian hormones during the estrus cycle. Estrogen levels peak
during the morning of the estrus cycle and begin to drop during the afternoon. While
progesterone peaks in the early afternoon and rapidly drops before the evening hours.
Modified from (Goldman, Murr et al. 2007).
As stated earlier the role of estrogen in mediating abdominal pain has been extensively
studied. Ovariectomy is often used to study the effects of ovarian hormones in modulating
pathogenesis of various diseases. Most ovariectomy experiments done in rats to study visceral
pain mechanisms suggest that ovarian hormones are pronociceptive. For instance, ovariectomy
decreased VMR to CRD in unsensitized and sensitized rats treated with either intracolonic

94

mustard-oil , maternal separation or subcutaneous 5-HTP injections (Ji, Murphy et al. 2003; Ji,
Tang et al. 2005; Lu, Hsieh et al. 2009; Gustafsson and Greenwood-Van Meerveld 2011) .
However, few studies suggest that estrogen signaling can be antinociceptive. A study
performed in mice showed that ovariectomy increased pain behavior induced by intracolonic
mustard-oil administration and was reversed by estrogen but not progesterone administration
(Sanoja and Cervero 2010). Similarly, a study done in rats showed that ovariectomy decreased
pain response to repeated 20 mmHg colorectal balloon distension and that this response was
reversed with estrogen replacement. Similarly, electrophysiological recording from dorsal horn
neurons showed that ovariectomy reduced firing of neurons in the dorsal horn that was
reversed with estrogen replacement suggesting that estrogen modulates pain transmission at
the level of the spinal cord (Ji, Murphy et al. 2003). In SERT KO rats ovariectomy enhanced VMR
to CRD in a time dependent manner as shown in figure 3.5. Visceral hypersensitivity was
increased between days 7-21 post-ovariectomy. This suggests that in presence of serotonergic
dysfunction, ovarian hormones play an antinociceptive role. However the mechanism by which
this occurs is unclear.
My 5-HT3 receptor antagonist studies discussed in Chapter 2 suggest that inhibition of
spinal 5-HT3 receptors contributes to the enhanced VMR to CRD observed in SERT KO female
rats. Similarly, studies focused on understanding the role of estrogen in mediating visceral pain
also suggest that spinal estrogen receptors play an important role in pain transmission. In the
rat lumbosacral dorsal root ganglia (DRG) neurons ER-α receptors are found in 17% of L6-S1
DRG neurons, ER-β receptors are found in 23% of L6-S1 DRG neurons and 5% of these neuron
express both receptors (Papka and Storey-Workley 2002). Furthermore, it has been shown that
95

ER-α receptors are predominantly expressed in the superficial layers (lamina i and ii) of dorsal
horn of the spinal cord. ER-β receptors are expressed in lamina iii and iv but not lamina i and ii
were most primary afferent terminate (Papka, Storey-Workley et al. 2001; Papka and StoreyWorkley 2002) (Vanderhorst, Gustafsson et al. 2005). Ji et al 2011 showed that treatment of
ovariectomized rats with ER-α receptor agonist, propyl pyrazole triol (PPT), caused an increased
in VMR to CRD. While treatment with steroidal ERs antagonist ICI 182 780 decreased VMR to
CRD observed in ovariectomized rats treated with estrogen. Furthermore, they performed invivo electrophysiological recordings from dorsal root and dorsal horn neurons responsive to
CRD and showed that activation of ER-α receptors increased neuronal firing of Abrupt neurons
whose response to CRD is terminated after 4 seconds (Ji, Tang et al. 2011). Estrogen signaling at
ER-β receptors is believed to be antinociceptive in animal models of visceral hypersensitivity.
The ER-β receptor agonists diarylpropionitrile (DPN) and WAY-200070 reduced VMR to CRD in
intact and ovariectomized rats when administered subcutaneously. DPN was also able to
reduce VMR to CRD when administered intrathecally suggesting that spinal ER-β receptors are
antinociceptive (Cao, Ji et al. 2012). The antinociceptive effects of ER-β agonist took 4 hours to
occur, therefore the authors concluded that in their animal model genomic ER-β signaling is
responsible for the reduction in VMR. Lu et al , 2009 showed that estrogen signaling at spinal
GPR30 membrane bound estrogen receptor is pronociceptive in 5-HTP-induced visceral
hypersensitivity animal model (Lu, Hsieh et al. 2009).
Downstream mechanisms that mediates estrogen signaling in visceral hypersensitivity
include activation of neurokinin 1 (NK1) receptor and upregulation of cAMP signaling in the
dorsal horn of the spinal cord (Bradesi, Eutamene et al. 2003; Lu, Hsieh et al. 2007). (Bradesi,
96

Eutamene et al. 2003). It has been shown that ERα receptor activation increased the activity of
NMDA receptors via phosphorylation on NR1 subunit and up regulation of NMDA receptor
expression in the lumbosacral dorsal horn to enhance VMR to CRD (Tang, Ji et al. 2008).
Furthermore, ER-α and ER-β KO mice exhibit an increase in P2X3 and TRPV1 receptors in
lumbosacral DRG neurons indicating that estrogen might play an antinociceptive role since both
receptors are found on primary afferent neurons and mediate enhanced pain transmission (Cho
and Chaban 2012).
Intrathecal administration of the steroidal estrogen receptor antagonist ICI 182780
caused an increase in VMR to CRD following the first and fourth injection. Inhibition of GPR30
via intrathecal administration of the GPR30 antagonist G15 did not affect VMR to CRD as shown
in figure 3.7. These data suggest that inhibition of either ER-α or ER-β increases discomfort in
SERT KO female rats similar to the increase in discomfort observed in ovariectomy studies.
Since VMR to CRD is measured one hour following the first intrathecal injection non-genomic
estrogen signaling at ER receptors is likely to be responsible for the increase in VMR to CRD.
This response was maintained on the fourth day following treatment with the antagonist for 4
days.
Estrogen signaling can be mediated via a genomic response where cytoplasmic estrogen
receptor dimerization followed by translocation to the nucleus is required to affect gene
expression. This response required multiple hours to occur (McEwen and Alves 1999; Marino,
Galluzzo et al. 2006). Estrogen signaling at ERα and ERβ receptors can also occur via rapid nongenomic signaling. In fact, the ER-α agonist PPT exerts it effects 15 minutes following drug

97

administration. The exact mechanism by which activation leads to increase in pain appears to
be mediated via MAPK activation in this case. (Amandusson, Hallbeck et al. 1999) (Ji, Tang et al.
2011).
In conclusion, SERT KO female rats exhibit an increase in VMR to CRD compared to WT
female rats. This increase in discomfort occurs in the proestrus phase of the estrus cycle.
Ovariectomy increased VMR to CRD in SERT KO female rats which developed 7 days postovariectomy and lasted for up to 3 weeks. Similarly, inhibition of spinal ER-α and ER-β estrogen
receptors with ICI 1822 780 enhanced discomfort in SERT KO female rats. However, the
inhibition of GPR30 did not affect VMR to CRD. This suggests that the spinal ER-α and/or ER-β
signaling in the spinal cord is antinociceptive. ER-α is expressed on inhibitory enkephalin
interneurons in the spinal cord (Amandusson, Hermanson et al. 1996; Amandusson, Hallbeck et
al. 1999). Therefore, might mediate antinociception by activating inhibitory opioid neurons
located in the dorsal horn of the spinal cord. Another study showed that activation of ER-β
receptors increased the expression of TPH2 in dorsal raphe nuclei suggesting that estrogen
signaling at ER-β receptors can increase inhibitory descending serotonergic input to the spinal
cord. Furthermore, ER-δ and ER-β receptors have been colocalized to inhibitory GABAergic
interneurons (Blurton-Jones and Tuszynski 2002). Thus, estrogen can exert its antinociceptive
effect via GABAergic interneurons located in the spinal cord.

98

BIBLIOGRAPHY

99

BIBLIOGRAPHY

Adeyemo, M. A., B. M. Spiegel, et al. (2010). "Meta-analysis: do irritable bowel syndrome
symptoms vary between men and women?" Alimentary Pharmacology and Therapeutics
32(6): 738-755.
Amandusson, A., M. Hallbeck, et al. (1999). "Estrogen-induced alterations of spinal cord
enkephalin gene expression." Pain 83(2): 243-248.
Amandusson, A., O. Hermanson, et al. (1996). "Colocalization of oestrogen receptor
immunoreactivity and preproenkephalin mRNA expression to neurons in the superficial
laminae of the spinal and medullary dorsal horn of rats." European Journal of
Neuroscience 8(11): 2440-2445.
Blurton-Jones, M. and M. H. Tuszynski (2002). "Estrogen receptor-beta colocalizes extensively
with parvalbumin-labeled inhibitory neurons in the cortex, amygdala, basal forebrain,
and hippocampal formation of intact and ovariectomized adult rats." Journal of
Comparative Neurology 452(3): 276-287.
Bradesi, S., H. Eutamene, et al. (2003). "Stress-induced visceral hypersensitivity in female rats is
estrogen-dependent and involves tachykinin NK1 receptors." Pain 102(3): 227-234.
Bradshaw, H. B., J. L. Temple, et al. (1999). "Estrous variations in behavioral responses to
vaginal and uterine distention in the rat." Pain 82(2): 187-197.
Cain, K. C., P. Headstrom, et al. (2006). "Abdominal pain impacts quality of life in women with
irritable bowel syndrome." American Journal of Gastroenterology 101(1): 124-132.
Cao, D. Y., Y. Ji, et al. (2012). "Estrogen receptor beta activation is antinociceptive in a model of
visceral pain in the rat." J Pain 13(7): 685-694.
Cho, T. and V. V. Chaban (2012). "Expression of P2X3 and TRPV1 receptors in primary sensory
neurons from estrogen receptors-alpha and estrogen receptor-beta knockout mice."
Neuroreport 23(9): 530-534.
Choghakhori, R., A. Abbasnezhad, et al. (2017). "Sex-Related Differences in Clinical Symptoms,
Quality of Life, and Biochemical Factors in Irritable Bowel Syndrome." Digestive Disease
and Science 62(6): 1550-1560.
Galligan, J. J., B. A. Patel, et al. (2013). "Visceral hypersensitivity in female but not in male
serotonin transporter knockout rats." Neurogastroenterology and Motility 25(6): e373381.

100

Giamberardino, M. A., G. Affaitati, et al. (1997). "Changes in visceral pain reactivity as a function
of estrous cycle in female rats with artificial ureteral calculosis." Brain Research 774(12): 234-238.
Goldman, J. M., A. S. Murr, et al. (2007). "The rodent estrous cycle: characterization of vaginal
cytology and its utility in toxicological studies." Birth Defects Research B Developmental
Reproduction and Toxicology 80(2): 84-97.
Grzesiak, M., J. A. Beszlej, et al. (2014). "The lifetime prevalence of anxiety disorders among
patients with irritable bowel syndrome." Advances in Clinical and Experimental
Medicine 23(6): 987-992.
Gupta, S., K. E. McCarson, et al. (2011). "Mechanisms of pain modulation by sex hormones in
migraine." Headache 51(6): 905-922.
Gustafsson, J. K. and B. Greenwood-Van Meerveld (2011). "Amygdala activation by
corticosterone alters visceral and somatic pain in cycling female rats." American Journal
of Physiology-Gastrointestinal and Liver Physiology 300(6): 31.
Harris, L. A., S. B. Umar, et al. (2016). "Irritable Bowel Syndrome and Female Patients."
Gastroenterology Clinics of North America 45(2): 179-204.
Heitkemper, M. M. and L. Chang (2009). "Do fluctuations in ovarian hormones affect
gastrointestinal symptoms in women with irritable bowel syndrome?" Gender Medicine
2: 152-167.
Heitkemper, M. M. and L. Chang (2009). "Do fluctuations in ovarian hormones affect
gastrointestinal symptoms in women with irritable bowel syndrome?" Gender Medicine
6 Suppl 2: 152-167.
Houghton, L. A., H. Brown, et al. (2009). "5-hydroxytryptamine signalling in irritable bowel
syndrome with diarrhoea: effects of gender and menstrual status." Alimentary
Pharmacology and Therapeutics 30(9): 919-929.
Houghton, L. A., R. Lea, et al. (2002). "The menstrual cycle affects rectal sensitivity in patients
with irritable bowel syndrome but not healthy volunteers." Gut 50(4): 471-474.
Ji, Y., A. Z. Murphy, et al. (2003). "Estrogen modulates the visceromotor reflex and responses of
spinal dorsal horn neurons to colorectal stimulation in the rat." Jornal of Neuroscience
23(9): 3908-3915.
Ji, Y., B. Tang, et al. (2005). "Modulatory effects of estrogen and progesterone on colorectal
hyperalgesia in the rat." Pain 117(3): 433-442.

101

Ji, Y., B. Tang, et al. (2008). "The visceromotor response to colorectal distention fluctuates with
the estrous cycle in rats." Neuroscience 154(4): 1562-1567.
Ji, Y., B. Tang, et al. (2011). "Spinal estrogen receptor alpha mediates estradiol-induced
pronociception in a visceral pain model in the rat." Pain 152(5): 1182-1191.
Lu, C. L., J. C. Hsieh, et al. (2009). "Estrogen rapidly modulates 5-hydroxytrytophan-induced
visceral hypersensitivity via GPR30 in rats." Gastroenterology 137(3): 1040-1050.
Lu, C. L., J. C. Hsieh, et al. (2007). "Estrogen rapidly modulates mustard oil-induced visceral
hypersensitivity in conscious female rats: A role of CREB phosphorylation in spinal dorsal
horn neurons." American Journal of Physiology Gastrointestinal Liver Physiology 292(1):
14.
Marino, M., P. Galluzzo, et al. (2006). "Estrogen signaling multiple pathways to impact gene
transcription." Current Genomics 7(8): 497-508.
McEwen, B. S. and S. E. Alves (1999). "Estrogen actions in the central nervous system."
Endocrine Reviews 20(3): 279-307.
Meleine, M. and J. Matricon (2014). "Gender-related differences in irritable bowel syndrome:
potential mechanisms of sex hormones." World Journal of Gastroenterology 20(22):
6725-6743.
Moloney, R. D., J. Sajjad, et al. (2016). "Estrous cycle influences excitatory amino acid transport
and visceral pain sensitivity in the rat: effects of early-life stress." Biology of Sex
Differences 7(33): 016-0086.
Mulak, A., Y. Tache, et al. (2014). "Sex hormones in the modulation of irritable bowel
syndrome." World Journal of Gastroenterology 20(10): 2433-2448.
Myers, B., J. Schulkin, et al. (2011). "Sex steroids localized to the amygdala increase pain
responses to visceral stimulation in rats." Journal of Pain 12(4): 486-494.
Naliboff, B. D., S. Berman, et al. (2003). "Sex-related differences in IBS patients: central
processing of visceral stimuli." Gastroenterology 124(7): 1738-1747.
Papka, R. E. and M. Storey-Workley (2002). "Estrogen receptor-alpha and -beta coexist in a
subpopulation of sensory neurons of female rat dorsal root ganglia." Neuroscience
Letters 319(2): 71-74.
Park, J. M., Choi, et al. (2006). "Serotonin transporter gene polymorphism and irritable bowel
syndrome." Neurogastroenterology and Motility 18(11): 995-1000.

102

Rybaczyk, L. A., M. J. Bashaw, et al. (2005). "An overlooked connection: serotonergic mediation
of estrogen-related physiology and pathology." BMC Womens Health 5: 12.
Sanoja, R. and F. Cervero (2010). "Estrogen-dependent changes in visceral afferent sensitivity."
Autonomic Neuroscience 153(1-2): 84-89.
Sinchak, K. and E. J. Wagner (2012). "Estradiol signaling in the regulation of reproduction and
energy balance." Frontiers in Neuroendocrinology 33(4): 342-363.
Tang, B., Y. Ji, et al. (2008). "Estrogen alters spinal NMDA receptor activity via a PKA signaling
pathway in a visceral pain model in the rat." Pain 137(3): 540-549.
Vanderhorst, V. G., J. A. Gustafsson, et al. (2005). "Estrogen receptor-alpha and -beta
immunoreactive neurons in the brainstem and spinal cord of male and female mice:
relationships to monoaminergic, cholinergic, and spinal projection systems." Journal of
Comparative Neurology 488(2): 152-179.
Wierman, M. E. (2007). "Sex steroid effects at target tissues: mechanisms of action." Advances
in Physiology Educaction 31(1): 26-33.

103

Chapter 4
General discussion
4.1 Ovarian hormones, serotonin and visceral hypersensitivity
Clinical studies suggest that the abdominal pain observed in some IBS patients may be
due to serotonergic dysfunction. However, the mechanism by which this occurs is not
understood. SERT gene polymorphisms have been identified in a subset of IBS patients
(Camilleri 2004; Acosta and Camilleri 2015). Female IBS-patients carrying the s/s allele exhibit
decreased SERT function and increased abdominal pain severity (Kumar, Ranjan et al. 2012).
Furthermore, variations in plasma 5-HT levels across the menstrual cycle have been identified
(Houghton, Atkinson et al. 2003). In the luteal phase of the menstrual cycle when estrogens and
progesterone levels are high, post-prandial plasma 5-HT levels were higher in female IBSdiarrhea patients compared to healthy controls (Houghton, Atkinson et al. 2003). A follow up
study showed that this increase in post-prandial 5-HT does not occur during menses when
abdominal pain is reported to be the worst (Heitkemper and Chang 2009). Furthermore, the 5HT3 receptor antagonist alosetron is effective in reducing IBS-D symptoms in female but not
male patients. While some speculate that this could be due to reduced alosetron clearance in
women resulting in 30-50 percent increased concertation of alosetron following a given dose
(Koch, Palmer et al. 2002). This could also be due to differences in serotonergic signaling
between males and females since alosetron results in increased 5-HT synthesis in specific brain
regions in males only (Nakai, Diksic et al. 2005).
In rodents it has been shown that estrogen upregulates TPH2, SERT and 5-HT2A
receptor expression in the brain (Blurton-Jones and Tuszynski 2002; Sumner, Grant et al. 2007).
104

In a partial restraint-stress induced rat animal model of visceral hypersensitivity, 17β-estradiol
and progesterone replacement in ovariectomized rats caused down regulation in 5-HT3
receptor expression in the colon. This suggests that estrogen plays an antinociceptive role since
5-HT3 receptor are expressed on primary afferent neuron terminals and lead to neuronal
activation (Li, Yu et al. 2004).
These clinical and basic research observations demonstrate the importance of studying
the interaction between ovarian hormones and serotonergic signaling in mediating nociceptive
signaling. The SERT KO rat serve as an excellent animal model to investigate the interaction
between ovarian hormones and 5-HT signaling. Galligan et al, 2013 showed that the SERT KO
female rat exhibits increased discomfort in response to CRD. Furthermore, the increase in
discomfort is estrous cycle dependent. SERT KO rats exhibit an increased visceral sensitivity to
colorectal distension (CRD) in the proestrus (high estrogen) and estrus (low estrus) stage of the
estrus cycle. While colonic mucosal levels of 5-HT are the same in SERT KO and WT rats,
amperometric studies showed that extracellular 5-HT levels are higher in SERT KO female and
male rats compared WT rats. Whole-cell patch-clamp analysis of colon projecting DRG neurons
showed that SERT KO female DRG neurons exhibit increased rate of action potential firing
compared to WT female and male rats (Galligan, Patel et al. 2013). These data suggest that in
presence of serotonergic dysfunction, ovarian hormones might modulate increased visceral
sensitivity by affecting neuronal signaling at the peripheral or central terminals of primary
afferent neurons.

105

As discussed in chapter 2, 5-HT3 receptors play an important role in mediating sensory
information from the gastrointestinal tract to the central nervous system. We showed that in
the SERT KO female rat spinal 5-HT3 receptors play an antinociceptive role. On the other hand,
in the SERT KO male rat spinal 5-HT3 receptor activation led to enhanced VMR to CRD. These
functional differences may be due to differential 5-HT3 receptor expression in the dorsal horn
of the spinal cord. In female SERT KO female rats 5-HT3 receptors appear to be predominantly
expressed on inhibitory interneurons in the dorsal horn of the spinal cord. While in SERT KO
male rats 5-HT3 receptors are likely to be expressed predominantly on the central terminals of
primary afferent neurons where their activation results in an increase in excitatory
neurotransmitters release (Figure 4.1).
It has been shown that the competitive GABAa receptor antagonist SR95531 was able
to reverse receptor desensitization in cell culture (Xu, Roberts et al. 2016). Therefore we could
also hypothesize that in female SERT KO rats, alosetron which is a competitive receptor
antagonist could reverse 5-HT3 receptor desensitization leading to increased discharge of
excitatory neurotransmitters from central terminals of primary afferent neurons (Machu 2011).
Furthermore, the SERT KO female rat exhibits increased VMR to CRD in the proestrus
phase of the estrous cycle where estrogen levels reach their peak in the late morning and drop
to their minimum by the end of the day (Goldman, Murr et al. 2007). This indicates that
perhaps the fluctuations in ovarian hormones rather than the absolute concentration at a given
time contributes to the development of visceral hypersensitivity. This is also observed clinically
where female IBS patients report increased abdominal pain during around menses where

106

ovarian hormone levels begin to drop. Ovariectomy increased VMR to CRD between days 7 and
21 post ovariectomy. Inhibition of the steroidal estrogen receptors ERα and ERβ in the spinal
cord caused an increase in VMR to CRD one hour following intrathecal administration of the ICI
182 780. This increase in discomfort was also observed after intrathecal administration of the
drugs for 4 days. This data suggest that the increase in VMR to CRD could be mediated by nongenomic and genomic signaling of the ERα and ERβ. In fact, ER α and ER β have been reported
to mediate non-genomic signaling. Specifically, it has been shown that membrane ER-α
receptors forms a complex with metabotropic glutamate receptors to mediate its non-genomic
signaling (Sinchak and Wagner 2012). Estrogen mediated analgesia could also occur via
GABAergic neurotransmission. For instance, ER-α receptors have been colocalized with
GABAergic inhibitory interneurons (Lu, Willcockson et al. 2005). It has also been shown that in
proestrus phase membrane estrogen receptors (ER-α and ER-β) cause heterodimerization of μopioid and κ-opioid receptors to mediate the antinociceptive effects of spinal dynorphins. In the
diestrus/estrus phase spinal dynorphins mediate nociception via κ-opioid receptor monomer
signaling (Gintzler and Liu 2012). Therefore, we can hypothesize that blockade of membrane
ER-α and/or ER-β can result in hyperalgesia due to a reduction in opioid receptor
heterodimerization.

107

Figure 4.1 Inhibitory and excitatory 5-HT signaling in the dorsal horn of the spinal cord.
Various 5-HT receptors are expressed on inhibitory interneuron, primary afferent and second
order neurons. For instance decreased pain transmission occurs when descending 5-HT
activates 5-HT3 receptors on inhibitory interneurons and 5-HT1A receptors on primary and
secondary afferents. On the other hand increased nociceptive signaling occurs when 5-HT1A
receptors are activated on inhibitory interneurons and 5-HT3 receptors are activated on
primary and secondary afferent neurons.
4.2 Technical considerations
In this section I will discuss some of the techniques used to perform my experiments and
discuss the strength and limitation of some of these techniques.
4.2.1 Visceromotor response (VMR) to colorectal distension (CRD)
CRD is a reliable method to study acute visceral pain since it is minimally invasive and
unlike electrical or chemical stimulation is reproducible. Noxious response to CRD can be
measured by monitoring changes in blood pressure, heart rate and aversive behavior. VMR is
also used to measure noxious effects of CRD. VMR is an abrupt contraction of the abdominal

108

and hind-limb muscles in response to CRD. It is equivalent to the abdominal muscle rigidity that
occurs in humans experiencing abdominal pain. The VMR to CRD is diminished following injury
to the spinal cord at either the thoracic or cervical level but is not affected by midcollicular
lesion which interrupts the communication between the brainstem and the brain. This suggests
that the VMR is mediated at the level of the brainstem. CRD results in both a non-noxious
response caused by low pressure distensions of 20 mmHg or lower and noxious response
occurring above 30 mmHg (Ness and Gebhart 1988). Morphine and clonidine both of which are
known for their analgesic effects diminished the VMR to CRD as shown by Ness, et al 1988. We
were also able to show that morphine when given subcutaneously and intrathecally diminished
the VMR to CRD (Figure 4.2). Similarly, clonidine was also able to completely inhibit VMR to
CRD as shown in Figure 4.3. This suggests that increased intraabdominal pressure at or above
40 mmHg results in noxious stimulus that can be employed to study visceral hypersensitivity.
It is worth noting that while the VMR to CRD is reproducible and reliable, differences in
noxious response have been observed in the rat depending on the strain of the rat. For
instance, Sprague-Dawley rats do not exhibit noxious response to CRD at intracolonic pressure
lower than 60 mmHg and show minimal number of pain behaviors at higher pressure. On the
other hand Wistar Kyoto rats exhibit pain response at 40 mmHg and increased number of pain
behaviors (Hyland, O'Mahony et al. 2015). The control rats used in our studies were Wistar
strain which is also the background of the SERT KO rats.

109

4.2.2 5,7-DHT mechanism of action.
The neurotoxin 5 7 DHT results in destruction of 5-HT nerve terminals and axons at the
site of injection. At 200 μg dose, 5,7 DHT reduced 5-HT levels by up to 50% in the rat brain
when administered via an intracerebroventricular cannula (Choi, Jonak et al. 2004).
Pretreatment with the norepinephrine transport (NET) blocker desipramine prevented the
destruction of norepinephrine neurons. We were also able to reproduce this observation as
shown in chapter 2 confirming that uptake of 5,7-DHT via NET is required to cause toxicity to
noradrenergic neurons. However, the use of selective 5-HT transporter inhibitors (SSRI) such as
fluoxetine and other 5-HT blocker (chlorimipramine, alaproclate)) were unable to prevent 5-HT
neuronal toxicity by 5,7-DHT suggesting that uptake via SERT is not required for toxicity.
Furthermore, the blockade of 5-HT auto-receptors using metergoline was also unable to block
5,7 DHT toxicity (Choi, Jonak et al. 2004). Pretreatment with reserpine failed to protect 5-HT
neurons from 5,7 DHT induced neurotoxicity suggesting that synaptic vesicles are not the site of
neurotoxicity. In fact, studies performed with labeled 5,7-DHT indicate that the toxin
accumulates in the mitochondria of 5-HT neurons (Baumgarten 1994 ).

110

Figure 4.2 Effects of morphine on VMR to CRD. Morphine (3mg/kg, s.c.) suppresses VMR to CRD in SERT KO female rats (n=3) (A) WT
female rats (saline, n=4; morphine, n=7) (B) SERT KO male rats (n =4) (C) and WT male rats (saline, n=4; morphine, n=7) (D). .
Morphine (10µg, i.t.) suppresses VMR to CRD in SERT KO female rats (=3) (E) WT female rats (n=4) (F) SERT KO male rats (n =4) (G)
and WT male rats (n=2) (H). Two-Way ANOVA was used to detect statistical significance. *,**,*** Indicate significantly different
from control (*P< 0.05),(** P<0.01) and (***P<0.001). Data are mean ± SEM.

111

20

WT male, s.c.
**

15

Control

Normalized VMR

Normalized VMR

20

**

10
5

Clonidine

0
0

20

40

60

80

WT male, i.t.
Control

15
10

Clonidine

5
0

100

0

Pressure (mmHg)

20

40

60

80

100

Pressure (mmHg)

Figure 4.3 Effect of clonidine on VMR to CRD in WT male rats. Clonidine (α2 adrenergic
agonist) reduced VMR to CRD in WT male rats when administered subcutaneously at done of
100μl/kg (A) and intrathecally at dose of 8 μg/10 μL (B). Two-Way ANOVA was used to detect
statistical significance (n=2). ** Indicate significantly different from control (* P<0.05), (**
P<0.01). Data are mean ± SEM.

5,7-DHT produced a robust decrease in spinal 5-HT levels in both SERT KO and WT rats.
Our studies, like the studies described above suggest the SERT is not required for 5,7 DHT
neurotoxicity One mechanism which has not been investigated and could account for uptake of
5,7-DHT by serotonergic neurons when SERT is inhibited or absent is uptake via organic cation
transporters (OCTs) . OCTs are capable of transporting a wide variety of cations including 5-HT.
In SERT KO mice, OCT3 expression is upregulated in the hippocampus but not other brain
regions. However, OCT1 which is predominantly expressed in the liver and kidney was not
upregulated in SERT KO mice (Schmitt, Mossner et al. 2003). Unpublished work performed in
our lab showed that OCT3 expression in the colon is not upregulated in SERT KO rats compared
to WT rats. However, this may not be reflective of OCT3 expression in the central nervous

112

system. Therefore, we cannot rule out the possibility of 5,7-DHT uptake via OCTs expressed in
the spinal cord.
4.2.3 Ovariectomy limitations
Ovariectomy increased VMR to CRD between 7 and 21 post-ovariectomy. The reversal of
the increase in VMR to CRD 3 weeks after ovariectomy suggests that the increase in pain was
due to physiological adaptations in response to the rapid reduction in ovarian hormones.
Indeed, it has been shown that estrogen levels reach their nadir 1-2 weeks post-ovariectomy in
rodents. Therefore, the endpoints measured during the first month post-ovariectomy may be
an reflection of physiological adaptations and should be considered carefully (Diaz Brinton
2012).
Furthermore, the assumption that the levels of steroids measured in the plasma are
reflective of steroid concentration in organs after ovariectomy has also been challenged. In a
study where neuroactive steroid levels were measured in plasma, peripheral nervous system
and central nervous system after gonadectomy in rats showed that the levels of neuroactive
hormones were significantly different in plasma compared to levels in sciatic nerve, cerebral
cortex, brainstem and spinal cord (Diaz Brinton 2012). In fact, aromatase is expressed in lamina
I and V in the spinal cord in neurons that stain positive for inhibitory interneuron markers. This
suggests that estrogen can be synthesized locally in the spinal cord to modulate pain
transmission (Tran, Kuhn et al. 2017). These studies suggest that neuroactive steroidal levels
are controlled locally and therefore, interpretation of ovariectomy studies should be performed
with these caveats in mind.

113

4.3 Clinical significance and future directions
Our studies showed that inhibition of spinal 5-HT3 receptors caused an increase in VMR
to CRD in SERT KO female rats which is in line with the clinical observation that patients with
reduced SERT activity (s/s allele carriers) respond poorly to alosetron (Acosta and Camilleri
2015). On the other hand, activation of spinal 5-HT3 receptors in male SERT KO rats caused an
increase in discomfort to CRD. However, inhibition of 5-HT3 receptors didn’t affect VMR to CRD
in SERT KO male rats. This recapitulates the clinical observation that alosetron does not
alleviate abdominal pain in male IBS-D patients (Koch, Palmer et al. 2002).
Furthermore, we showed that ovariectomy and inhibition of classocal ERs also increased
discomfort. While clinical studs assessing oral contraceptives for treatment of IBS are sparse.
One study showed that progestin only birth control is associated with increased IBS diagnosis.
This suggests that disruption of ovarian hormone balance may contribute to the development
of IBS (Bird, Liu et al. 2012). We observed that SERT KO female rats exhibit normal estrous cycle
and are capable of reproducing without any complications or reduced capacity. This suggests
that the dysfunction in serotonergic signaling does not alter reproductive function in SERT KO
rats. Yet, ovarian hormones appear to influence response to noxious stimulation of the colon.
The sex difference in VMR to CRD response in SERT KO rats and the response to 5-HT3
receptor inhibition suggest that SERT KO rats serves as a valuable resource to understand the
connection between ovarian hormones and 5-HT in mediating visceral hypersensitivity. Future
work should focus on determining how inhibition of ERs leads to increase visceral sensitivity.

114

Employing various estrogen receptor antagonist and agonist to intact rats is a better approach
than ovariectomy given the ovariectomy limitations discussed above.
Furthermore, Investigation the role of other 5-HT receptors in mediating visceral
hypersensitivity in SERT KO rats will further enhance our understanding of the role of 5-HT
signaling in mediating pain. Specifically, 5-HT1A, 5-HT2 and 5-HT7 receptors have been shown
to mediate pain transmission at the level of the spinal cord (figure 4.1) (Millan 2002). Such
work will allow us to combine different 5-HT receptor mediators to effectively treat pain
instead of focusing on once receptor in a clinical setting.
As discussed in chapter 2, reducing 5-HT levels in the spinal cord from descending 5-HT
neurons prevented the increase in VMR to CRD observed following alosetron treatment in SERT
KO female rats and SR 57227 in SERT KO male rats. This could be observed due to unmasking
the antinociceptive effects of descending noradrenergic signaling at α2-adregenrgic receptors.
In fact, co-administration of clonidine (α2 agonist) and alosetron prevented the increase in VMR
to CRD in SERT KO female rats (data not shown). Therefore, we can employ SERT KO rats to
further investigate the interactions between descending noradrenergic and serotonergic pain
modulation in mediating visceral hypersensitivity. Most studies performed to determine
descending pain modulation employ somatic pain assays such as hot plate, tail flick and sciatic
nerve ligation. Very few experiments focus on understanding descending modulation of visceral
pain. Therefore, performing such experiments will fill a large gap in the field.

115

BIBLIOGRAPHY

116

BIBLIOGRAPHY

Acosta, A. and M. Camilleri (2015). "Pharmacogenetics in irritable bowel syndrome." Expert
Opinion on Drug Metabolism & Toxicology 11(8): 1187-1191.
Adeyemo, M. A., B. M. Spiegel, et al. (2010). "Meta-analysis: do irritable bowel syndrome
symptoms vary between men and women?" Alimentary Pharmacology & Therapeutics
32(6): 738-755.
Alhaider, A. A., S. Z. Lei, et al. (1991). "Spinal 5-HT3 receptor-mediated antinociception: possible
release of GABA." Journal of Neuroscience 11(7): 1881-1888.
Amandusson, A., M. Hallbeck, et al. (1999). "Estrogen-induced alterations of spinal cord
enkephalin gene expression." Pain 83(2): 243-248.
Amandusson, A., O. Hermanson, et al. (1996). "Colocalization of oestrogen receptor
immunoreactivity and preproenkephalin mRNA expression to neurons in the superficial
laminae of the spinal and medullary dorsal horn of rats." European Journal of
Neuroscience 8(11): 2440-2445.
Banner, S. E. and G. J. Sanger (1995). "Differences between 5-HT3 receptor antagonists in
modulation of visceral hypersensitivity." British Journal of Pharmacology 114(2): 558562.
Barnes, N. M., T. G. Hales, et al. (2009). "The 5-HT3 receptor--the relationship between
structure and function." Neuropharmacology 56(1): 273-284.
Baumgarten, H. G., Zimmerman B (1994 ). Neurotoxic phenylalkylamines and indolealkylamines.
Selective Neurotoxicity, Springer-Verlag: 225-229.
Beatty, J. K., A. Bhargava, et al. (2014). "Post-infectious irritable bowel syndrome: mechanistic
insights into chronic disturbances following enteric infection." World Journal of
Gastroenterology 20(14): 3976-3985.
Berman, S., J. Munakata, et al. (2000). "Gender differences in regional brain response to visceral
pressure in IBS patients." European Journal of Pain 4(2): 157-172.
Bhattarai, Y., D. A. Muniz Pedrogo, et al. (2017). "Irritable bowel syndrome: a gut microbiotarelated disorder?" American Journal of Physiology -Gastrointestinal and Liver Physiology
312(1): G52-G62.
Bird, S. T., W. Liu, et al. (2012). "Irritable bowel syndrome and drospirenone-containing oral
contraceptives; a comparative-safety study." Current Drug Safety 7(1): 8-15.
117

Blackshaw, L. A., S. J. Brookes, et al. (2007). "Sensory transmission in the gastrointestinal tract."
Neurogastroenterololgy and Motility 19(1 Suppl): 1-19.
Blurton-Jones, M. and M. H. Tuszynski (2002). "Estrogen receptor-beta colocalizes extensively
with parvalbumin-labeled inhibitory neurons in the cortex, amygdala, basal forebrain,
and hippocampal formation of intact and ovariectomized adult rats." Journal of
Comparative Neurology 452(3): 276-287.
Bouin, M., V. Plourde, et al. (2002). "Rectal distention testing in patients with irritable bowel
syndrome: Sensitivity, specificity, and predictive values of pain sensory thresholds."
Gastroenterology 122(7): 1771-1777.
Bradesi, S., H. Eutamene, et al. (2003). "Stress-induced visceral hypersensitivity in female rats is
estrogen-dependent and involves tachykinin NK1 receptors." Pain 102(3): 227-234.
Bradesi, S., L. Lao, et al. (2007). "Dual role of 5-HT3 receptors in a rat model of delayed stressinduced visceral hyperalgesia." Pain 130(1-2): 56-65.
Bradshaw, H. B., J. L. Temple, et al. (1999). "Estrous variations in behavioral responses to
vaginal and uterine distention in the rat." Pain 82(2): 187-197.
Brookes, S. J., N. J. Spencer, et al. (2013). "Extrinsic primary afferent signalling in the gut."
Nature Reviews Gastroenterology & Hepatology 10(5): 286-296.
Browning, K. N. (2015). "Role of central vagal 5-HT3 receptors in gastrointestinal physiology and
pathophysiology." Frontiers in Neuroscience 9(413).
Buono, J. L., R. T. Carson, et al. (2017). "Health-related quality of life, work productivity, and
indirect costs among patients with irritable bowel syndrome with diarrhea." Health and
Quality of Life Outcomes 15(1): 017-0611.
Buono, J. L., K. Mathur, et al. (2017). "Economic Burden of Irritable Bowel Syndrome with
Diarrhea: Retrospective Analysis of a U.S. Commercially Insured Population." Journal of
Managed Care and Specialty Pharmcy 23(4): 453-460.
Butler, A., J. M. Hill, et al. (1988). "Pharmacological properties of GR38032F, a novel antagonist
at 5-HT3 receptors." British Journal of Pharmacology 94(2): 397-412.
Cain, K. C., P. Headstrom, et al. (2006). "Abdominal pain impacts quality of life in women with
irritable bowel syndrome." The American Journal of Gastroenterology 101(1): 124-132.
Cain, K. C., M. E. Jarrett, et al. (2009). "Gender Differences in Gastrointestinal, Psychological,
and Somatic Symptoms in Irritable Bowel Syndrome." Digestive Diseases and Sciences
54(7): 1542-1549.
118

Camilleri, M. (2004). "Is there a SERT-ain association with IBS?" Gut 53(10): 1396-1399.
Camilleri, M., B. Coulie, et al. (2001). "Visceral hypersensitivity facts sepculations and challenges
camelliri." Gut 48: 7.
Cao, D. Y., Y. Ji, et al. (2012). "Estrogen receptor beta activation is antinociceptive in a model of
visceral pain in the rat." Journal of Pain 13(7): 685-694.
Chang, F. Y., C. L. Lu, et al. (2010). "The current prevalence of irritable bowel syndrome in Asia."
J Neurogastroenterology and Motility 16(4): 389-400.
Chang, L., V. Z. Ameen, et al. (2005). "A dose-ranging, phase II study of the efficacy and safety of
alosetron in men with diarrhea-predominant IBS." The American Journal of
Gastroenterology 100(1): 115-123.
Chen, J. J., Z. Li, et al. (2001). "Maintenance of serotonin in the intestinal mucosa and ganglia of
mice that lack the high-affinity serotonin transporter: Abnormal intestinal motility and
the expression of cation transporters." Journal of Neuroscience 21(16): 6348-6361.
Chen, L., S. J. Ilham, et al. (2017). "Pharmacological Approach for Managing Pain in Irritable
Bowel Syndrome: A Review Article." Anesthesiology and Pain Medicine 7(2).
Cheung, C. K. and J. C. Wu (2014). "Genetic polymorphism in pathogenesis of irritable bowel
syndrome." World Journal of Gastroenterology 20(47): 17693-17698.
Chey, W. D., J. Kurlander, et al. (2015). "Irritable bowel syndrome: a clinical review." Journal of
American Medical Association 313(9): 949-958.
Chiloiro, M., G. Darconza, et al. (2001). "Gastric emptying and orocecal transit time in
pregnancy." Journal of Gastroenterology 36(8): 538-543.
Cho, T. and V. V. Chaban (2012). "Expression of P2X3 and TRPV1 receptors in primary sensory
neurons from estrogen receptors-alpha and estrogen receptor-beta knockout mice."
Neuroreport 23(9): 530-534.
Choghakhori, R., A. Abbasnezhad, et al. (2017). "Sex-Related Differences in Clinical Symptoms,
Quality of Life, and Biochemical Factors in Irritable Bowel Syndrome." Digestive Diseases
and Sciences 62(6): 1550-1560.
Choi, I. S., J. H. Cho, et al. (2013). "5-Hydroxytryptamine 1A receptors inhibit glutamate release
in rat medullary dorsal horn neurons." Neuroreport 24(8): 399-403.
Choi, S., E. Jonak, et al. (2004). "Serotonin reuptake inhibitors do not prevent 5,7dihydroxytryptamine-induced depletion of serotonin in rat brain." Brain Research 8: 1-2.
119

Choi, Y. D., T. S. Sung, et al. (2008). "Increased 5-hydroxytryptamine mediates postinflammatory visceral hypersensitivity via the 5-hydroxytryptamine 3 receptor in rats."
Digestive Diseases and Sciences 53(11): 2909-2916.
Clayton, N. M., R. Sargent, et al. (1999). "The pharmacological properties of the novel selective
5-HT3 receptor antagonist, alosetron, and its effects on normal and perturbed small
intestinal transit in the fasted rat." Neurogastroenterology and Motility 11(3): 207-217.
Coelho, A. M., J. Fioramonti, et al. (1998). "Mast cell degranulation induces delayed rectal
allodynia in rats: role of histamine and 5-HT." Digestive Diseases and Sciences 43(4):
727-737.
Cremon, C., G. Carini, et al. (2011). "Intestinal Serotonin Release, Sensory Neuron Activation,
and Abdominal Pain in Irritable Bowel Syndrome." The American Journal of
Gastroenterology 106(7): 1290-1298.
Cremonini, F., J. P. Nicandro, et al. (2012). "Randomised clinical trial: alosetron improves quality
of life and reduces restriction of daily activities in women with severe diarrhoeapredominant IBS." Alimentary Pharmacology & Therapeutics 36(5): 437-448.
Diaz Brinton, R. (2012). "Minireview: translational animal models of human menopause:
challenges and emerging opportunities." Endocrinology 153(8): 3571-3578.
Drossman, D. A., M. Camilleri, et al. (2002). "AGA technical review on irritable bowel
syndrome." Gastroenterology 123(6): 2108-2131.
Foley, S., K. Garsed, et al. (2011). "Impaired uptake of serotonin by platelets from patients with
irritable bowel syndrome correlates with duodenal immune activation."
Gastroenterology 140(5): 1434-1443 e1431.
Furness, J. B., L. R. Rivera, et al. The gut as a sensory organ, Nature Reviews of
Gastroenterology and Hepatology. 2013 Dec;10(12):729-40. doi:
10.1038/nrgastro.2013.180. Epub 2013 Sep 24.
Galligan, J. J., B. A. Patel, et al. (2013). "Visceral hypersensitivity in female but not in male
serotonin transporter knockout rats." Neurogastroenterology and Motility 25(6): e373381.
Garsed, K., J. Chernova, et al. (2014). "A randomised trial of ondansetron for the treatment of
irritable bowel syndrome with diarrhoea." Gut 63(10): 1617-1625.
Gebhart, G. F. (1999). "Peripheral contributions to visceral hyperalgesia." Canadian Journal of
Gastroenterology & Hepatology 13: 37A-41A.

120

Gebhart, G. F. and K. Bielefeldt (2016). "Physiology of Visceral Pain." Comprehensive Physiology
6(4): 1609-1633.
Gershon, M. D. (2004). "Review article: serotonin receptors and transporters -- roles in normal
and abnormal gastrointestinal motility." Alimentary Pharmacology & Therapeutics 7: 314.
Gershon, M. D. and J. Tack (2007). "The serotonin signaling system: from basic understanding to
drug development for functional GI disorders." Gastroenterology 132(1): 397-414.
Giamberardino, M. A., G. Affaitati, et al. (1997). "Changes in visceral pain reactivity as a function
of estrous cycle in female rats with artificial ureteral calculosis." Brain Research 774(12): 234-238.
Gintzler, A. R. and N. J. Liu (2012). "Importance of sex to pain and its amelioration; relevance of
spinal estrogens and its membrane receptors." Frontiers in Neuroendocrinology 33(4):
412-424.
Glatzle, J., C. Sternini, et al. (2002). "Expression of 5-HT3 receptors in the rat gastrointestinal
tract." Gastroenterology 123(1): 217-226.
Glaum, S. R., H. K. Proudfit, et al. (1990). "5-HT3 receptors modulate spinal nociceptive
reflexes." Brain Research 510(1): 12-16.
Goldberg, P. A., M. A. Kamm, et al. (1996). "Modification of visceral sensitivity and pain in
irritable bowel syndrome by 5-HT3 antagonism (ondansetron)." Digestion 57(6): 478483.
Goldman, J. M., A. S. Murr, et al. (2007). "The rodent estrous cycle: characterization of vaginal
cytology and its utility in toxicological studies." Birth Defects Research Part B:
Developmental and Reproductive Toxicology 80(2): 84-97.
Gralnek, I. M., R. D. Hays, et al. (2000). "The impact of irritable bowel syndrome on healthrelated quality of life." Gastroenterology 119(3): 654-660.
Greenwood-Van Meerveld, B., E. Mohammadi, et al. (2014). "Synergistic effect of 5hydroxytryptamine 3 and neurokinin 1 receptor antagonism in rodent models of somatic
and visceral pain." Journal of Pharmacology and Experimental Therapeutics 351(1): 146152.
Gregersen, H. and G. Kassab (1996). "Biomechanics of the gastrointestinal tract."
Neurogastroenterology and Motility 8(4): 277-297.

121

Grover, M. and M. Camilleri Ramosetron in irritable bowel syndrome with diarrhea: new hope
or the same old story?, Clinical Gastroenterology and Hepatology. 2014 Jun;12(6):960-2.
doi: 10.1016/j.cgh.2013.12.025. Epub 2014 Jan 3.
Grzesiak, M., J. A. Beszlej, et al. (2014). "The lifetime prevalence of anxiety disorders among
patients with irritable bowel syndrome." Advances in Clinical and Experimental
Medicine 23(6): 987-992.
Gu, Q. Y., J. Zhang, et al. (2015). "Association of genetic polymorphisms in HTR3A and HTR3E
with diarrhea predominant irritable bowel syndrome." International Journal of Clinical
and Experimental Medicine 8(3): 4581-4585.
Gunawardene, A. R., B. M. Corfe, et al. (2011). "Classification and functions of enteroendocrine
cells of the lower gastrointestinal tract." International Journal of Experimental Pathology
92(4): 219-231.
Gupta, S., K. E. McCarson, et al. (2011). "Mechanisms of pain modulation by sex hormones in
migraine." Headache 51(6): 905-922.
Gustafsson, J. K. and B. Greenwood-Van Meerveld (2011). "Amygdala activation by
corticosterone alters visceral and somatic pain in cycling female rats." American Journal
of Physiology-Gastrointestinal and Liver Physiology 300(6): 31.
Halac, U., A. Noble, et al. (2010). "Rectal sensory threshold for pain is a diagnostic marker of
irritable bowel syndrome and functional abdominal pain in children." Journal of
Pediatrics 156(1): 60-65 e61.
Hall, F. S., J. M. Schwarzbaum, et al. (2011). "A greater role for the norepinephrine transporter
than the serotonin transporter in murine nociception." Neuroscience 175: 315-327.
Hall, J. D., C. DeWitte, et al. (2015). "Serotonin enhances urinary bladder nociceptive processing
via a 5-HT3 receptor mechanism." Neuroscience Letters 604: 97-102.
Harris, L. A., S. B. Umar, et al. (2016). "Irritable Bowel Syndrome and Female Patients."
Gastroenterol Clin North Am 45(2): 179-204.
Heitkemper, M. M. and L. Chang (2009). "Do fluctuations in ovarian hormones affect
gastrointestinal symptoms in women with irritable bowel syndrome?" Gender Medicine
6 Suppl 2: 152-167.
Heitkemper, M. M. and L. Chang (2009). "Do fluctuations in ovarian hormones affect
gastrointestinal symptoms in women with irritable bowel syndrome?" Gender Medicine
2: 152-167.

122

Herman, J., V. Pokkunuri, et al. (2010). "Gender distribution in irritable bowel syndrome is
proportional to the severity of constipation relative to diarrhea." Gender Medicine 7(3):
240-246.
Hoekman, D. R., J. M. Rutten, et al. (2015). "Annual Costs of Care for Pediatric Irritable Bowel
Syndrome, Functional Abdominal Pain, and Functional Abdominal Pain Syndrome."
Journal of Pediatrics 167(5): 1103-1108.
Homberg, J. R., J. D. Olivier, et al. (2007). "Characterization of the serotonin transporter
knockout rat: a selective change in the functioning of the serotonergic system."
Neuroscience 146(4): 1662-1676.
Hornung, J. P. (2003). "The human raphe nuclei and the serotonergic system." Journal of
Chemical Neuroanatomy 26(4): 331-343.
Houghton, L. A., W. Atkinson, et al. (2003). "Increased platelet depleted plasma 5hydroxytryptamine concentration following meal ingestion in symptomatic female
subjects with diarrhoea predominant irritable bowel syndrome." Gut 52(5): 663-670.
Houghton, L. A., H. Brown, et al. (2009). "5-hydroxytryptamine signalling in irritable bowel
syndrome with diarrhoea: effects of gender and menstrual status." Alimentary
Pharmacology & Therapeutics 30(9): 919-929.
Houghton, L. A., J. M. Foster, et al. (2000). "Alosetron, a 5-HT3 receptor antagonist, delays
colonic transit in patients with irritable bowel syndrome and healthy volunteers."
Alimentary Pharmacology & Therapeutics 14(6): 775-782.
Houghton, L. A., R. Lea, et al. (2002). "The menstrual cycle affects rectal sensitivity in patients
with irritable bowel syndrome but not healthy volunteers." Gut 50(4): 471-474.
Hyland, N. P., S. M. O'Mahony, et al. (2015). "Early-life stress selectively affects gastrointestinal
but not behavioral responses in a genetic model of brain-gut axis dysfunction."
Neurogastroenterol Motil 27(1): 105-113.
Iyengar, S., M. H. Ossipov, et al. (2017). "The role of calcitonin gene-related peptide in
peripheral and central pain mechanisms including migraine." Pain 158(4): 543-559.
Jensen, A. A., P. A. Davies, et al. (2008). "3B but which 3B and that's just one of the questions:
the heterogeneity of human 5-HT3 receptors." Trends in Pharmacological Sciences
29(9): 437-444.
Jeong, H. J., V. A. Mitchell, et al. (2012). "Role of 5-HT(1) receptor subtypes in the modulation of
pain and synaptic transmission in rat spinal superficial dorsal horn." British Journal of
Pharmacology 165(6): 1956-1965.
123

Ji, Y., A. Z. Murphy, et al. (2003). "Estrogen modulates the visceromotor reflex and responses of
spinal dorsal horn neurons to colorectal stimulation in the rat." Journal of Neuroscience
23(9): 3908-3915.
Ji, Y., B. Tang, et al. (2005). "Modulatory effects of estrogen and progesterone on colorectal
hyperalgesia in the rat." Pain 117(3): 433-442.
Ji, Y., B. Tang, et al. (2008). "The visceromotor response to colorectal distention fluctuates with
the estrous cycle in rats." Neuroscience 154(4): 1562-1567.
Ji, Y., B. Tang, et al. (2011). "Spinal estrogen receptor alpha mediates estradiol-induced
pronociception in a visceral pain model in the rat." Pain 152(5): 1182-1191.
Jin, D.-C., H.-L. Cao, et al. (2016). "Regulation of the serotonin transporter in the pathogenesis
of irritable bowel syndrome." World Journal of Gastroenterology 22(36): 8137.
Jorgensen, H. S. (2007). "Studies on the neuroendocrine role of serotonin." Danish Medical
Bulletin 54(4): 266-288.
Kanazawa, M., M. Hongo, et al. (2011). "Visceral hypersensitivity in irritable bowel syndrome."
Journal of Gastroenterology and Hepatology 26: 119-121.
Kapeller, J., L. A. Houghton, et al. (2008). "First evidence for an association of a functional
variant in the microRNA-510 target site of the serotonin receptor-type 3E gene with
diarrhea predominant irritable bowel syndrome." Human Molecular Genetics 17(19):
2967-2977.
Kawamata, T., K. Omote, et al. (2003). "The activation of 5-HT(3) receptors evokes GABA release
in the spinal cord." Brain Research 978(1-2): 250-255.
Kim, J. J. and W. I. Khan (2014). "5-HT7 receptor signaling: improved therapeutic strategy in gut
disorders." Frontiers in Behavioral Neuroscience 8(396).
Kirkup, A. J., A. M. Brunsden, et al. (2001). "Receptors and transmission in the brain-gut axis:
potential for novel therapies. I. Receptors on visceral afferents." American Journal of
Physiology-Gastrointestinal and Liver Physiology 280(5): G787-794.
Koch, K. M., B. W. Corrigan, et al. (2004). "Alosetron repeat dose pharmacokinetics, effects on
enzyme activities, and influence of demographic factors." Alimentary Pharmacology &
Therapeutics 20(2): 223-230.
Koch, K. M., J. L. Palmer, et al. (2002). "Sex and age differences in the pharmacokinetics of
alosetron." British Journal of Clinical Pharmacology 53(3): 238-242.

124

Kohen, R., M. E. Jarrett, et al. (2009). "The Serotonin Transporter Polymorphism rs25531 Is
Associated with Irritable Bowel Syndrome." Digestive Diseases and Sciences 54(12):
2663-2670.
Kovac, A. L. (2016). "Comparative Pharmacology and Guide to the Use of the Serotonin 5-HT3
Receptor Antagonists for Postoperative Nausea and Vomiting." Drugs 76(18): 17191735.
Kumar, S., P. Ranjan, et al. (2012). "Serotonin transporter gene (SLC6A4) polymorphism in
patients with irritable bowel syndrome and healthy controls." Journal of
Gastrointestinal Liver Disease 21(1): 31-38.
Langlois, A., X. Pascaud, et al. (1996). "Response heterogeneity of 5-HT3 receptor antagonists in
a rat visceral hypersensitivity model." European Journal of Pharmacology 318(1): 141144.
Layer, P., J. Keller, et al. (2007). "Tegaserod in the treatment of irritable bowel syndrome (IBS)
with constipation as the prime symptom." Therapeutics and Clinical Risk Management
3(1): 107-118.
Lee, K. N. and O. Y. Lee (2016). "The Role of Mast Cells in Irritable Bowel Syndrome."
Gastroenterology Research and Practice 2031480(10): 28.
Li, T. J., B. P. Yu, et al. (2004). "Ovarian hormone modulates 5-hydroxytryptamine 3 receptors
mRNA expression in rat colon with restraint stress-induced bowel dysfunction." World
Journal of Gastroenterology 10(18): 2723-2726.
Llorca-Torralba, M., G. Borges, et al. (2016). "Noradrenergic Locus Coeruleus pathways in pain
modulation." Neuroscience 338: 93-113.
Lu, C. L., J. C. Hsieh, et al. (2009). "Estrogen rapidly modulates 5-hydroxytrytophan-induced
visceral hypersensitivity via GPR30 in rats." Gastroenterology 137(3): 1040-1050.
Lu, C. L., J. C. Hsieh, et al. (2007). "Estrogen rapidly modulates mustard oil-induced visceral
hypersensitivity in conscious female rats: A role of CREB phosphorylation in spinal dorsal
horn neurons." American Journal of Physiology-Gastrointestinal and Liver Physiology
292(1): 14.
Lu, C. R., H. H. Willcockson, et al. (2005). "Ionotropic glutamate receptors are expressed in
GABAergic terminals in the rat superficial dorsal horn." Journal of Comparative
Neurology 486(2): 169-178.
Lummis, S. C. (2012). "5-HT(3) receptors." J Biol Chem 287(48): 40239-40245.

125

Machu, T. K. (2011). "Therapeutics of 5-HT3 receptor antagonists: current uses and future
directions." Pharmacology and Therapeutics 130(3): 338-347.
Mangel, A. W. and A. R. Northcutt (1999). "Review article: the safety and efficacy of alosetron, a
5-HT3 receptor antagonist, in female irritable bowel syndrome patients." Alimentary
Pharmacology & Therapeutics 2: 77-82.
Marino, M., P. Galluzzo, et al. (2006). "Estrogen signaling multiple pathways to impact gene
transcription." Current Genomics 7(8): 497-508.
Matsumoto, K., M. W. Lo, et al. (2012). "Experimental colitis alters expression of 5-HT receptors
and transient receptor potential vanilloid 1 leading to visceral hypersensitivity in mice."
Laboratory Investigation 92(5): 769-782.
Mayer, E. A., S. Berman, et al. (2002). "The effect of the 5-HT3 receptor antagonist, alosetron,
on brain responses to visceral stimulation in irritable bowel syndrome patients."
Alimentary Pharmacology & Therapeutics 16(7): 1357-1366.
McEwen, B. S. and S. E. Alves (1999). "Estrogen actions in the central nervous system."
Endocrine Reviews 20(3): 279-307.
Meleine, M. and J. Matricon (2014). "Gender-related differences in irritable bowel syndrome:
potential mechanisms of sex hormones." World Journal of Gastroenterology 20(22):
6725-6743.
Millan, M. J. (2002). "Descending control of pain." Progress Neurobiology 66(6): 355-474.
Miquel, M. C., M. B. Emerit, et al. (1995). "Developmental changes in the differential expression
of two serotonin 5-HT3 receptor splice variants in the rat." Journal of Neurochemistry
65(2): 475-483.
Miranda, A., S. Peles, et al. (2006). "Effects of the 5-HT3 receptor antagonist, alosetron, in a rat
model of somatic and visceral hyperalgesia." Pain 126(1-3): 54-63.
Moloney, R. D., J. Sajjad, et al. (2016). "Estrous cycle influences excitatory amino acid transport
and visceral pain sensitivity in the rat: effects of early-life stress." Biology of Sex
Differences 7(33): 016-0086.
Morales, M., E. Battenberg, et al. (1998). "Distribution of neurons expressing immunoreactivity
for the 5HT3 receptor subtype in the rat brain and spinal cord." Journal of Comparative
Neurology 402(3): 385-401.
Morales, M. and S. D. Wang (2002). "Differential composition of 5-hydroxytryptamine3
receptors synthesized in the rat CNS and peripheral nervous system." Journal of
126

Neuroscience 22(15): 6732-6741.
Mulak, A., Y. Tache, et al. (2014). "Sex hormones in the modulation of irritable bowel
syndrome." World Journal of Gastroenterology 20(10): 2433-2448.
Myers, B., J. Schulkin, et al. (2011). "Sex steroids localized to the amygdala increase pain
responses to visceral stimulation in rats." Journal of Pain 12(4): 486-494.
Nakai, A., M. Diksic, et al. (2005). "The effects of the 5-HT3 antagonist, alosetron, on brain
serotonin synthesis in patients with irritable bowel syndrome." Neurogastroenterology
and Motility 17(2): 212-221.
Naliboff, B. D., S. Berman, et al. (2003). "Sex-related differences in IBS patients: central
processing of visceral stimuli." Gastroenterology 124(7): 1738-1747.
Ness, T. J. and G. F. Gebhart (1988). "Colorectal distension as a noxious visceral stimulus:
physiologic and pharmacologic characterization of pseudaffective reflexes in the rat."
Brain Research 450(1-2): 153-169.
Olivier, J. D., M. G. Van Der Hart, et al. (2008). "A study in male and female 5-HT transporter
knockout rats: an animal model for anxiety and depression disorders." Neuroscience
152(3): 573-584.
Oyama, T., M. Ueda, et al. (1996). "Dual effect of serotonin on formalin-induced nociception in
the rat spinal cord." Neuroscience Research 25(2): 129-135.
Palecek, J., V. Paleckova, et al. (2002). "The roles of pathways in the spinal cord lateral and
dorsal funiculi in signaling nociceptive somatic and visceral stimuli in rats." Pain 96(3):
297-307.
Papka, R. E. and M. Storey-Workley (2002). "Estrogen receptor-alpha and -beta coexist in a
subpopulation of sensory neurons of female rat dorsal root ganglia." Neuroscience
Letters 319(2): 71-74.
Papka, R. E., M. Storey-Workley, et al. (2001). "Estrogen receptor-alpha and betaimmunoreactivity and mRNA in neurons of sensory and autonomic ganglia and spinal
cord." Cell and Tissue Research 304(2): 193-214.
Park, J. M., Choi, et al. (2006). "Serotonin transporter gene polymorphism and irritable bowel
syndrome." Neurogastroenterology and Motility 18(11): 995-1000.
Rahimi, R., S. Nikfar, et al. (2008). "Efficacy and tolerability of alosetron for the treatment of
irritable bowel syndrome in women and men: a meta-analysis of eight randomized,

127

placebo-controlled, 12-week trials." Clinical Therapeutics 30(5): 884-901.
Rahman, W., K. Bannister, et al. (2011). "A pronociceptive role for the 5-HT2 receptor on spinal
nociceptive transmission: an in vivo electrophysiological study in the rat." Brain
Research 25: 29-36.
Rybaczyk, L. A., M. J. Bashaw, et al. (2005). "An overlooked connection: serotonergic mediation
of estrogen-related physiology and pathology." BMC Women's Health 5: 12.
Sanoja, R. and F. Cervero (2010). "Estrogen-dependent changes in visceral afferent sensitivity."
Autonomic Neuroscience: Basic and Clinical 153(1-2): 84-89.
Saria, A., F. Javorsky, et al. (1990). "5-HT3 receptor antagonists inhibit sensory neuropeptide
release from the rat spinal cord." Neuroreport 1(2): 104-106.
Sawynok, J. and A. Reid (1996). "Interactions of descending serotonergic systems with other
neurotransmitters in the modulation of nociception." Behavioural Brain Research 73(12): 63-68.
Schmitt, A., R. Mossner, et al. (2003). "Organic cation transporter capable of transporting
serotonin is up-regulated in serotonin transporter-deficient mice." Journal of
Neuroscience Research 71(5): 701-709.
Sengupta, J. N. (2009). "Visceral Pain: The Neurophysiological Mechanism." 194: 31-74.
Simino, G. P., L. P. Marra, et al. (2016). "Efficacy, safety and effectiveness of ondansetron
compared to other serotonin-3 receptor antagonists (5-HT3RAs) used to control
chemotherapy-induced nausea and vomiting: systematic review and meta-analysis."
Expert Review of Clinical Pharmacology 9(9): 1183-1194.
Simren, M., O. S. Palsson, et al. (2017). "Update on Rome IV Criteria for Colorectal Disorders:
Implications for Clinical Practice." Current Gastroenterology Reports 19(4): 017-0554.
Sinchak, K. and E. J. Wagner (2012). "Estradiol signaling in the regulation of reproduction and
energy balance." Frontiers in Neuroendocrinology 33(4): 342-363.
Song, Z., B. A. Meyerson, et al. (2011). "Spinal 5-HT receptors that contribute to the painrelieving effects of spinal cord stimulation in a rat model of neuropathy." Pain 152(7):
1666-1673.
Spohn, S. N. and G. M. Mawe (2017). "Non-conventional features of peripheral serotonin
signalling - the gut and beyond." Nature Reviews Gastroenterology & Hepatology 14(7):
412-420.

128

Su, X. and G. F. Gebhart (1998). "Mechanosensitive pelvic nerve afferent fibers innervating the
colon of the rat are polymodal in character." Journal of Neurophysiology 80(5): 26322644.
Sumner, B. E., K. E. Grant, et al. (2007). "Raloxifene blocks estradiol induction of the serotonin
transporter and 5-hydroxytryptamine2A receptor in female rat brain." Neuroscience
Letters 417(1): 95-99.
Tang, B., Y. Ji, et al. (2008). "Estrogen alters spinal NMDA receptor activity via a PKA signaling
pathway in a visceral pain model in the rat." Pain 137(3): 540-549.
Thompson, A. J. and S. C. Lummis (2007). "The 5-HT3 receptor as a therapeutic target." Expert
Opinion in Therapeutic Targets 11(4): 527-540.
Thompson, A. J., M. H. Verheij, et al. (2013). "Structure-activity relationships of quinoxalinebased 5-HT3A and 5-HT3AB receptor-selective ligands." ChemMedChem 8(6): 946-955.
Todd, A. J. (2010). "Neuronal circuitry for pain processing in the dorsal horn." Nature Reviews of
Neuroscience 11(12): 823-836.
Tran, M., J. A. Kuhn, et al. (2017). "Neuronal aromatase expression in pain processing regions of
the medullary and spinal cord dorsal horn." Journal of Comparative Neurology 525(16):
3414-3428.
Vanderhorst, V. G., J. A. Gustafsson, et al. (2005). "Estrogen receptor-alpha and -beta
immunoreactive neurons in the brainstem and spinal cord of male and female mice:
relationships to monoaminergic, cholinergic, and spinal projection systems." Journal of
Comparative Neurology 488(2): 152-179.
Viramontes, B. E., M. Camilleri, et al. (2001). "Gender-related differences in slowing colonic
transit by a 5-HT3 antagonist in subjects with diarrhea-predominant irritable bowel
syndrome." The American Journal of Gastroenterology 96(9): 2671-2676.
Vogel, C., R. Mossner, et al. (2003). "Absence of thermal hyperalgesia in serotonin transporterdeficient mice." Journal of Neuroscience 23(2): 708-715.
Wierman, M. E. (2007). "Sex steroid effects at target tissues: mechanisms of action." Advances
in Clinical and Experimental Medicine 31(1): 26-33.
Wolf, H. (2000). "Preclinical and clinical pharmacology of the 5-HT3 receptor antagonists."
Scandinavian Journal of Rheumatology Suppl 113: 37-45.

129

Xu, D., X. Wu, et al. (2013). "Butyrate-induced colonic hypersensitivity is mediated by mitogenactivated protein kinase activation in rat dorsal root ganglia." Gut 62(10): 1466-1474.
Xu, X. J., D. Roberts, et al. (2016). "Competitive antagonists facilitate the recovery from
desensitization of alpha1beta2gamma2 GABAA receptors expressed in Xenopus
oocytes." Acta Pharmacologica Sinca 37(8): 1020-1030.
Yamamoto, C., H. Murakami, et al. (2002). "Contribution of P-glycoprotein to efflux of
ramosetron, a 5-HT3 receptor antagonist, across the blood-brain barrier." Journal of
Pharmacy and Pharmacology 54(8): 1055-1063.
Yeo, A. (2004). "Association between a functional polymorphism in the serotonin transporter
gene and diarrhoea predominant irritable bowel syndrome in women." Gut 53(10):
1452-1458.
Yoshimura, M. (2006). "Plastic changes of the descending serotonergic system in the spinal
dorsal horn." Current Anaesthesia & Critical Care 17(1): 3-8.
Zagorodnyuk, V. P., M. Kyloh, et al. (2011). "Loss of visceral pain following colorectal distension
in an endothelin-3 deficient mouse model of Hirschsprung's disease." Journal of
Physiology 589(Pt 7): 1691-1706.
Zeitz, K. P., N. Guy, et al. (2002). "The 5-HT3 subtype of serotonin receptor contributes to
nociceptive processing via a novel subset of myelinated and unmyelinated nociceptors."
Journal of Neuroscience 22(3): 1010-1019.
Zheng, Y., T. Yu, et al. (2017). "Efficacy and safety of 5-hydroxytryptamine 3 receptor
antagonists in irritable bowel syndrome: A systematic review and meta-analysis of
randomized controlled trials." PLoS One 12(3).

130