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NUCLEI-SPECIFIC RESPONSE TO PAIN IN THE BED NUCLEUS OF THE
STRIA TERMINALIS
By
Tania J. Morano
A thesis submitted to the Centre for Neuroscience Studies in conformity with the
requirements for the degree of Master of Science
Queen’s University
Kingston, Ontario, Canada
September, 2007
Copyright  Tania J. Morano, 2007
ABSTRACT
The bed nucleus of the stria terminalis (BST) is a basal forebrain cluster of
several distinct nuclei.
It has been proposed that the BST coordinates
autonomic, neuroendocrine, and behavioral functions through the integration and
organization of homeostatic responses necessary for survival. Dysfunction of the
BST contributes to pathophysiological states such as addiction, anxiety and
aggression. Based on anatomical and behavioral studies, the BST could be a
key contributor to descending modulation of nociception as well as the
physiological responses related to the affective aspect of the pain experience.
The objective of the present study was to further understand the
neurophysiological bases underlying the involvement of the 7 anterior nuclei of
the BST in pain. Using c-Fos as an indicator of neuronal activation, the results
demonstrate that acute noxious stimulation produced an increase in the number
of c-Fos immunoreactive cells (c-Fos-IR) in the dorsal anteromedial (dAM) and
fusiform (FU) nuclei, while non-noxious stimulation did not increase c-Fos-IR in
any of the nuclei examined.
Chronic neuropathic pain induced by chronic
constriction injury (CCI) did not alter basal c-Fos-IR in the FU or dAM. Unlike in
the naïve condition, the number of c-fos-IR cells in the FU induced by acute
noxious stimulation was attenuated in animals with either CCI or sham surgery
compared to naive rats.
In contrast, c-Fos-IR induced by acute noxious
stimulation in the dAM was not affected by CCI or sham surgery. Acute noxious
stimulation in animals that received CCI exhibited increased c-Fos expression in
the ventromedial (vAM) nucleus of the BST, a finding not evident in naïve or
ii
sham control groups. Finally, there was an increase in c-Fos-IR in the oval (OV)
nucleus of sham-operated, but not naive or CCI rats.
This study reveals for the first time that pain induces neuronal activity in
the BST in a nuclei- and condition-specific way. Given the efferent projection
patterns from the BST, this system may relay supraspinal information to the
periphery to produce physiological responses related to the affective pain
experience.
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CO-AUTHOURSHIP
This thesis is based on research conducted by Tania J. Morano under the
co-supervision of Dr. Eric Dumont and Dr. Catherine Cahill.
iv
ACKNOWLEGEMENTS
To Cathy: I lack words to fully express my gratitude. Thank you first for the
opportunity to prove myself and also for your unwavering support. I am eternally
grateful.
To Eric: Thank you for your technical support and patience.
To Mom, Dad, Joey and Andrea: Thank you for always being there for me.
Thank you for putting up with me. Thank you for being you. I love you all dearly.
To Monique: None of this if not for you.
To Sarah: Thank you for answering every one of my stupid questions. Your help
and friendship is appreciated tremendously.
To the Department of Pharmacology & Toxicology and the Centre for
Neuroscience Studies: Thank you for the opportunity to work with such amazing
people and learn amazing things.
To my Friends: Thank you to my friends, old and new. I will truly miss those of
you that I am leaving behind.
To my Rats: Thank you for your sacrifice.
Special thanks to James Reynolds, Nicole Bailey, Linda Plong, Rachel Phelan,
Julian Mackenzie-Feder, Lihua Xiu, Ken Rose and Darren Marks. All of whom
have helped me in different capacities, but all of which are important in their own
right.
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DEDICATION
To everyone and everything that makes life beautiful.
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TABLE OF CONTENTS
ABSTRACT……………………………………………………………………………...ii
CO-AUTHOURSHIP…………………………………………………………………...iv
ACKNOWLEDGEMENTS……………………………………………………………...v
DEDICATION…………………………………………………………………………...vi
TABLE OF CONTENTS………………………………………………………………vii
LIST OF FIGURES……………………………………………………………………..ix
LIST OF ABBREVIATIONS…………………………………………………………..xi
INTRODUCTION………………………………………………………………………..1
1.1
Pain Overview……………………………………………………….…..1
1.2
Affective-Motivational Pain Systems………………………………….4
1.3
Pain Pathways…………………………………………………………..7
1.4
Peripheral and Central Sensitization………………………………...12
1.5
Models of Neuropathic Pain…………………………………………..15
1.6
Molecular Mechanisms of Neuropathic Pain………………………..16
1.7
c-Fos as a Marker of Neuronal Activity……………………………...17
1.8
Objectives and Hypotheses…………………………………………..19
METHODS……………………………………………………………………………...20
2.1
Animals………………………………………………………………….20
2.2
Pain Induction – Acute Nociceptive Stimulus……………………….21
2.3
Pain Induction – Chronic Constriction Injury………………………..21
2.4
Euthanasia and Brain Preparation…………………………………...22
2.5
Immunohistochemical Detection of c-Fos…………………………..23
2.6
Quantitative Analysis………………………………………………….24
2.7
Statistical Analysis……………………………………………………..25
2.8
Experiment 1: Effect of Acute Nociceptive Stimulation on c-Fos-IR
in the BST………………………………………………………………25
2.9
Experiment 2: Effect of Acute Non-Nociceptive Stimulation on cFos-IR in the BST……………………………………………………...26
2.10 Experiments 3 and 4: Effect of Chronic Constriction Injury on Basal
and Noxious Stimulus Induced Neuronal Firing……………………26
RESULTS…...………………………………………………………………………….27
3.1
Experiment 1: Effect of Acute Nociceptive Stimulation on c-Fos-IR
in the BST………………………………………………………………27
3.2
Experiment 2: Effect of Acute Non-Nociceptive Stimulation on cFos-IR in the BST……………………………………………………...31
3.3
Experiment 3: Effect of CCI on Basal c-Fos-IR in the BST………31
3.4
Experiment 4: Effect of CCI on Noxious-Stimulus Induced c-Fos-IR
in the BST………………………………………………………………34
DISCUSSION………………………………………………………………………….46
vii
4.1
4.2
4.3
4.4
4.5
4.6
Experiment 1: Effect of Acute Nociceptive Stimulation on c-Fos-IR
in the BST………………………………………………………………46
Experiment 2: Effect of Acute Non-Noxious Stimulation on c-Fos-IR
in the BST………………………………………………………………53
Experiment 3: Effect of CCI on Basal c-Fos-IR in the BST………54
Experiment 4: Effect of CCI on Noxious-Stimulus Induced c-Fos-IR
in the BST………………………………………………………………55
Reflection and Caveats……………………………………………….60
Future Directions……………………………………………………....61
REFERENCES………………………………………………………………………...64
viii
LIST OF FIGURES
Figure 1
fMRI data showing activation of SI and SII following thermal
noxious stimulation (Bushnell et al., 1999)…..………………………………………3
Figure 2
fMRI analysis of affective-motivational systems activated by thermal
noxious stimulation (Bushnell et al., 1999)……………………………..……………5
Figure 3
Schematic diagram of the projection patterns of the SST. Sensorydiscriminative and affective-motivational components of the SST are depicted
(Dostrovsky & Craig, 2006)……….…………………………………………………..10
Figure 4
Effect of mechanical noxious stimulation on c-Fos-IR in the FU
nucleus of the BST…………………………………………………………………….28
Figure 5
Immunohistochemical representation of c-Fos-IR cells in the FU
nucleus………………………………………………………………………………….28
Figure 6
Effect of mechanical noxious stimulation on c-Fos-IR in the dAM
nucleus of the BST………………………….…………………………………………29
Figure 7
Immunohistochemical representation of c-Fos-IR cells in the dAM
nucleus………………………………………………………………………………….29
Figure 8
Effect of mechanical noxious stimulation on remaining 5 nuclei of
the BST…………………………………………………………………………………30
Figure 9
Comparison of noxious and non-noxious stimulation in the FU and
dAM nuclei of the BST………………………………………………………………...32
Figure 10
Effect of noxious and non-noxious stimulation on c-Fos-IR in the FU
one hour after stimulation……………………………………………………………..32
Figure 11
Analysis of c-Fos-IR in all BST nuclei when comparing nonstimulated control animals and non-noxious stimulated animals…………………33
Figure 12
Comparison of basal c-Fos-IR cells in the FU and dAM nuclei of the
BST 11 days following CCI or sham surgery……………………………………….35
Figure 13
Immunohistochemical representation of basal c-Fos-IR cells in the
FU nucleus of CCI and sham rats……………………………………………………36
Figure 14
Immunohistochemical representation of basal c-Fos-IR cells in the
dAM nucleus of CCI and sham rats………………………………………………….36
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Figure 15
c-Fos-IR in each remaining nuclei when comparing basal firing
levels of CCI and sham animals……………………………………………………..37
Figure 16
Effect of mechanical noxious stimulation on c-Fos-IR in the FU
nucleus of CCI rats……………………………………………………………………38
Figure 17
Immunohistochemical representation of c-Fos-IR cells in the FU
nucleus of non-stimulated and noxious-stimulated CCI rats……………………...38
Figure 18
Effect of mechanical noxious stimulation on c-Fos-IR in the dAM
nucleus of CCI rats…………………………………………………………………….39
Figure 19
Immunohistochemical representation of c-Fos-IR cells in the dAM
nucleus of non-stimulated and noxious-stimulated CCI rats……………………...39
Figure 20
Immunohistochemical representation of c-Fos-IR cells in the vAM
nucleus of non-stimulated and noxious-stimulated CCI and sham rats………….41
Figure 21
Comparison of non-stimulated and noxious-stimulated CCI rats 1
and 2 hours after noxious stimulation……………………………………………….42
Figure 22
Effect of mechanical noxious stimulation on c-Fos-IR in the FU
nucleus of sham rats………………………………………………………………….43
Figure 23
Immunohistochemical representation of c-Fos-IR cells in the FU
nucleus of non-stimulated and noxious-stimulated sham rats……………………43
Figure 24
Effect of mechanical noxious stimulation on c-Fos-IR in the dAM
nucleus of sham rats…………………………………………………………………..44
Figure 25
Immunohistochemical representation of c-Fos-IR cells in the dAM
nucleus of non-stimulated and noxious-stimulated sham rats……………………44
Figure 26
c-Fos-IR scores for each remaining nuclei when comparing basal
versus noxious stimulated evoked firing levels of sham animals…………………45
x
LIST OF ABBREVIATIONS
AC
ACC
ACTH
AM
ANOVA
AP
BG
BLA
BST
BSTa
CCI
CeA
c-Fos-IR
CL
CRH
DAB
dAL
dAM
FU
GHRH
HPA-axis
i.p
JX
LT
MD
mPFC
mpPVH
NAc
NMDA
NP
OV
PAG
Pf
PFA
PFC
PHAL
PI-CPA
SS.E.M
S+
S+1
S+2
S+5
SI
anterior commisure
anterior cingulate cortex
adrenocorticotrophic hormone
anteromedial aspect of the BST
Analysis of Variance
action potential
basal ganglia
basolateral amygdala
bed nucleus of the stria terminalis
anterior bed nucleus of the stria terminalis
chronic constriction injury
central nucleus of the amygdala
c-Fos immunoreactivity
central lateral nucleus of the thalamus
corticotrophin-releasing hormone
1,3-Diaminobenzidine
dorsal anterolateral nucleus of the BST
dorsal anteromedial nucleus of the BST
fusiform nucleus of the BST
growth hormone-releasing hormone
hypothalamic-pituitary-adrenal axis
intraperitoneal
juxtacapsular nucleus of the BST
light touch
medial dorsal nucleus of the thalamus
medial prefrontal cortex
medial parvicellular division of the PVH
nucleus accumbens
N-Methyl-D-Aspartate
neuropathic pain
oval nucleus of the BST
periaqueductal gray
parafascicular nucleus of the thalamus
paraformaldehyde
prefrontal cortex
Phaseolus vulgaris-leucoagglutinin
pain-induced conditioned place aversion
non-stimulated
standard error of the mean
stimulation
sacrificed one hour following noxious stimulation
sacrificed two hours following noxious stimulation
sacrificed 5 hours following noxious stimulation
primary sensory cortex
xi
SII
SMT
SRT
SST
TMEV
TP
TTX
vAL
vAM
VL
VP
VTA
secondary sensory cortex
spinomesecephalaic tract
spinoreticulothalamic tract
spinothalamic tract
Theiler’s murine encephalomyelinitis virus
toe pinch
tetrodotoxin
ventral anterolateral nucleus of the BST
ventral anteromedial nucleus of the BST
ventral lateral nucleus of the thalamus
ventral posterior nucleus of the thalamus
ventral tegmental area
xii
Introduction
1.1
Pain Overview
The International Association for the Study of Pain defines pain as an
unpleasant sensory and emotional experience resulting from actual or potential
tissue damage. Pain is by far the most common reason to seek medical
attention. It is estimated that 80% of doctor visits are for pain-related symptoms.
Of this population, one study estimated that 1.5% of patients are affected by
chronic pain syndromes (Carter & Galer, 2001). The National Population Health
Survey indicated that 3.9 million Canadians (17%) over the age of 15 have
chronic pain. For 70% of these, pain is rated as moderate to severe enough to
interfere with normal daily activities (Meana et al., 2003).
Pain is a complex experience that encompasses sensory features as well
as emotional and motivational components. Sensory and discriminative aspects
of pain encode location and intensity of noxious insult, while affective and
motivational components of pain are responsible for the emotional and
psychological aspects, which act to perpetuate and intensify feelings of
helplessness, stress, aversion and fear associated with pain states (Ohara et al.
2005).
Affective-motivational components of pain are often ignored when
developing methods to address pain (Craig, 2006). For example, approximately
50% of
patients who
develop
chronic pain
also
develop
depression
simultaneously (Romano & Turner, 1985). Depression is not a consequence of
chronic pain, but it can be a symptom.
1
Neuropathic pain (NP), one type of chronic pain, is a major health problem
that brings forth major economic burden; not only due to associated medical
costs for treatment, but also due to losses to the work force, worker’s
compensation and pensions paid to individuals suffering from chronic pain and
disability. An estimated 2 to 3% of individuals in developed countries suffer from
some form of chronic neuropathic pain condition (Moulin, 2007). Depression and
anxiety frequently accompany all types of chronic pain including NP. Prevalence
of depression co-morbidly expressed with chronic pain ranges from 31 to 100%
(Romano & Turner, 1985) therefore, developing novel and therapeutic treatments
to alleviate neuropathic pain are of considerable importance.
The advancement of imaging techniques such as functional magnetic
resonance imaging and positron emission tomography allow for clear observation
of brain regions that are activated in response to pain. Various human brainimaging studies have examined cortical areas involved in acute pain processing
(Rainville et al., 1997; Bushnell et al., 1999).
A consistent cortical network
involving sensory, limbic, associative and motor areas has emerged (Bushnell &
Apkarian, 2006). The most commonly activated regions are primary sensory
cortex (SI), secondary sensory cortex (SII), anterior cingulate cortex (ACC),
insular cortex, prefrontal cortex (PFC), thalamus and cerebellum (Casey et al.,
2001).
SI and SII contain neurons that encode intensity, location and temporal
aspects of noxious and non-noxious somatosensory stimuli (Figure 1; Chudler et
al., 1990). Following noxious cutaneous laser stimulation to a patient with
2
Figure 1: fMRI data showing activation of SI and SII following thermal noxious
stimulation (Bushnell et al., 1999).
3
damage to SI and SII following stroke, the patient was unable to localize or
describe the nature of the painful stimulus, but was, however able to describe an
ill-defined unpleasantness associated with the noxious insult (Ploner et al.,
1999). This study proved for the first time in humans that sensory-discriminate
and affective-motivational systems act in concert, but through different
mechanisms to produce a pain experience.
1.2
Affective-Motivational Pain Systems
Studies in rodents and humans have showed that the amygdala, insular
cortex and ACC contribute to negative affective states associated with pain
(Figure 2; Johansen et al., 2001; Rainville et al., 1997; Neugebauer et al., 2004).
It is well established that primary afferents send noxious-stimulus evoked
projections to limbic (amygdala, ACC) and cognitive systems (PFC) therefore
targeting these systems when addressing pain may lead to alternative therapies
to manage pain symptoms. Lesions of the ACC or the amygdala in rodents
occlude somatic noxious stimuli-induced conditioned place aversion behaviors,
an experimental measure of affective pain response in rodents (Johansen, 2004).
Lesions to the ACC also impair the learning of pain-induced conditioned place
aversion, but it does not block the expression of aversive behavior (Johansen,
2004); another example of how the affective system and the sensory systems are
separate when creating the overall experience of pain.
The amygdala is an almond shape structure in the temporal lobe that
plays a key role in the emotional evaluation of sensory stimuli, anxiety and
4
Figure 2: fMRI analysis of affective-motivational systems activated by thermal
noxious stimulation (Bushnell et al., 1999).
5
depression (Neugebauer et al., 2004). Highly processed affective and cognitive
information enters the amygdala and through an intricate internal connection,
these signals (including noxious information) are processed and filtered in the
basolateral (BLA) and central nucleus (CeA) of the amygdala.
Directly
connected to the amygdala through a highly specific fibre tract is the bed nucleus
of the stria terminalis (BST).
The BST is a limbic forebrain structure consisting of approximately 12
highly interconnected nuclei that is connected directly to the BLA and CeA of the
amygdala through the stria terminalis. The BST can be divided into anterior and
posterior divisions, each of which has been identified based on their projection
pattern and neurochemical identity: The fusiform (FU), oval (OV), juxtacapsular
(JX), dorsal and ventral anterolateral nuclei (dAL and vAL, respectively) and
dorsal and ventral anteromedial nuclei (dAM and vAM, respectively) comprise the
anterior portion of the BST (BSTa).
BSTa neurons all receive and send
projections to the CeA and most neurons in this area express corticotropinreleasing hormone (CRH; Ju et al., 1989).
While very little is known about the
function of the BST, there is agreement that it probably coordinates
neuroendocrine, autonomic and somatomotor responses to disruptions in
homeostasis, including anxiety and stress (Dong et al., 2001; Dong & Swanson,
2004).
The BST has been implicated in generating anxiogenic-induced relapse
in drug seeking in a model of opiate withdrawal-induced condition place aversion
(Delfs et al., 2000) and synaptic plasticity in this area is critical in food and
cocaine reward-seeking behaviours (Dumont et al., 2005). In 2005, Braz and
6
colleagues developed a transgenic mouse that self-expressed a wheat germ
agglutinin tracer driven by the Nav1.8 sodium channel gene. This type of channel
is expressed exclusively on non-peptidergic C-fibres (Julius & McCleskey, 2006).
Because the tracer was expressed only in non-peptidergic C-fibres and was able
to transmit across synapses, Braz and colleagues (2005) were able to determine
the transmission pathway of this subset of pain fibres to the cortex. Accordingly,
non-peptidergic C-fibres were found to project to the amygdala, hypothalamus,
globus pallidus and the BST (Braz et al., 2005). This was the first study to
implicate the BST in pain transmission. More recently, the BST (more specifically
the BSTa) was shown to be involved in the development of noxious-visceral and
noxious-chemically induced condition place aversion (Deyama et al., 2007). It is
unclear, however, as to how the BST contributes to pain-induced condition place
aversion or which nuclei in the BST are responsible for modulating affect.
Understanding how BST function is altered as a result of chronic pain has never
been explored.
1.3
Pain Pathways
Sensory information including pain is transmitted from the periphery to the
spinal cord via primary afferent neurons; A fibres and C-fibres. Of the 3 subtypes
of A-fibre (α, β and δ) the Aδ nociceptors encode mechanical, thermal
(Silberstein, 2003) and chemical noxious insult (Caterina et al., 1999). Aδ-fibres
have myelinated axons and are responsible for evoking pricking pain, sharpness
and aching pain (Meyer et al., 2006). It should be noted that while Aβ and Aα are
7
not considered to relay noxious transmission, recent evidence has determined
that approximately 20% of Aα and Aβ fibres in rat also transmit noxious
information to the spinal cord (Djouhri & Lawson, 2004). C-fibres, the second
class of nociceptor, represent 70% of all primary afferents (Nagy et al., 1983). Cfibres evoke burning pain sensations and are also responsive to noxious
mechanical stimulation. C-fibres are small, unmyelinated fibres with receptive
fields spanning approximately 100 mm2 in humans (Schmidt et al., 1997). Cfibres can be subdivided into peptidergic and non-peptidergic neurons, with
peptidergic neurons expressing nociceptive peptides such as substance P and
calcitonin gene-related peptide. Approximately 50% of C-fibres and 20% of Aδ
fibres are classified as peptidergic (Lawson et al., 2002). Peptidergic C-fibres
terminate in the superficial layers of the spinal cord, targeting primarily lamina I
neurons that project to the thalamus and brainstem as well as interneurons of
outer lamina II (Braz et al., 2005). Non-peptide C-fibres are positive for the
isolectin IB4 and project mostly to inner lamina II interneurons (Braz et al., 2005),
indicating that different sub-populations of C-fibres likely transmit signals to
different areas of the brain for processing.
The cell bodies of all primary afferent neurons that transmit sensory
information from the periphery (excluding the head and neck) have their cell
bodies located in the dorsal root ganglia. The dorsal horn of the spinal cord
receives most noxious information directly from primary afferent neurons. From
the dorsal horn, pain signals are transmitted to the brain through several tract
systems via the anterolateral system - the most documented being the
8
spinothalamic tract (STT). The STT originates predominately in lamina I and also
in laminas IV and V of the dorsal horn and transmits nociceptive information
through a synapse at the ventral white commisure to the contralateral ventral
gray matter. Fibre tracts of the STT project through the brainstem and terminate
in 5 regions of the lateral and medial thalamus (Willis & Westlund, 1997); the
ventral posterior nuclei (VP), the ventral lateral nucleus (VL), and central lateral
nucleus (CL), the parafascicular nucleus (Pf) and the medial dorsal nucleus (MD;
Dostrovsky & Craig, 2006). Afferent projections to the VP arise from laminas I,
IV and V of the spinal cord, whereas efferent projections from VP terminate in
superficial layers of the sensorimotor cortex.
They are thought to mediate
sensory integration or motor responses to pain (Rausell & Jones, 1991). VL
innervation,
resulting
in
projections
from
lamina
V
neurons
provides
somatosensory responsiveness to pain (Mackel et al., 1992). The CL receives
input from lamina V neurons.
The majority of CL cells project to the basal
ganglia (BG) and motor and parietal corticies. CL projections may be involved in
attention and control of orientation to withdrawal or avoidance of prolonged
painful stimuli (Dostrovsky & Craig, 2006).
Projections to the Pf arise from
Laminas I and V, however this innervation is weak. Pf connections, given their
projections to the BG, substantia nigra and motor cortex indicate that these
afferents are related to motor systems (Sadikot et al., 1992). The MD nucleus
projects to area 24c in the cortex at the fundus of the ACC (Craig, 2003) as well
as the orbital and prefrontal cortex (Ray & Price, 1993).
This latter STT
component contributes to affective and motivational aspects of pain (Figure 3).
9
Figure 3: Schematic diagram of the projection patterns of the STT. Sensorydiscriminative and affective-motivational components of the STT are depicted
(Dostrovsky & Craig, 2006).
10
The spinomesecephalaic tract (SMT) is another tract within the
anterolateral system that sends nociceptive information to the brain. Similar to
the STT, the SMT projects from primary afferents to neurons in lamina I and
lamina V of the spinal cord. From here, the axons decussate in the ventral white
commisure, ascend to the midbrain and terminate in midbrain nuclei including the
periaqueductal gray (PAG) and the superior colliculus (Willis & Westlund, 1997).
Projections to the PAG could serve different functions. Firstly, termination in the
PAG may contribute to aversive behaviour (Nashold et al., 1969), but may also
be responsible for modulating descending analgesia (Reynolds, 1969).
Projections
to the superior colliculus are likely to play a role in orienting to noxious stimuli
(Willis & Westlund, 1997).
A spino-limbic tract, referred to as the spinoreticulothalamic tract (SRT)
also sends afferent noxious information from the spinal cord to the brain (Willis &
Westlund, 1997). These projections originate in lamina I and V of the SC, but are
also found in deeper layers (laminae VII and VIII). These signals cross the spinal
cord to the contralateral ventral gray matter and travel to the reticular formation
(Willis & Coggeshall, 1991).
From the reticular formation, SRT information
terminates in the thalamus, hypothalamus, amygdala and septal nucleus.
In
addition to these systems, tracing evidence indicates that direct spinohypothalamic (Burstein et al., 1990) and spino-amygdalar (Burstein & Potrebic,
1993) projections exist that send noxious information directly to limbic structures
11
in the brain that are proposed to mediate affective and motivational components
of pain.
1.4
Peripheral and Central Sensitization
Pain signaling results in acute or lasting changes to primary afferents that
increase the nociceptors responsiveness to stimulation – a phenomenon known
as sensitization. Sensitization is characterized by “a decrease in firing threshold,
an augmented response to suprathreshold stimuli and ongoing spontaneous
activity” of central and peripheral nociceptive neurons (Meyer et al., 2006; p. 14).
These changes in nociceptor function are translated to a physical exacerbation in
the pain experience reported by humans, a symptom known as hyperalgesia
(Meyer et al., 2006). Hyperalgesia directly at the site of injury is defined as
primary hyperalgesia, however in some cases, an increased sensitivity to
innocuous stimulation of tissue surrounding the site of injury also becomes
sensitized, a condition known as secondary hyperalgesia (Lewis, 1935).
Primary hyperalgesia results from the sensitization of peripheral afferent
nociceptors, while secondary hyperalgesia is often caused by decreased firing
thresholds of centrally-located mechanoreceptors, a system that in normal
conditions blunts afferent signals to the central nervous system (Meyer et al.,
2006).
Acute or chronic injury results in the release of several chemicals that
facilitate the pain response and lead to hyperalgesia.
In the case of an
inflammatory response, injury results in the local release of substances such as
12
bradykinin, substance P, histamine, ATP and a variety of other pro-inflammatory
cytokines, all of which act to either directly activate nociceptors or indirectly
through the reorganization of the nervous system initiated by invading
inflammatory cells (Mayer et al., 2006).
Bradykinin is released from plasma following tissue injury and leads to
afferent sensitization (Reeh & Sauer, 1997). Its mechanism of action involves
the activation of second messengers that modulate the activity of vanilloid
receptors (transient receptor potential ion channels – TRPV). Vanilloid receptors
transmit heat information to the central nervous system in normal conditions,
however the release of bradykinin acts on this receptor to lower their threshold,
making them more sensitive to heat stimuli (Sugiura et al., 2002; Carlton &
Coggeshall, 2001).
Following noxious insult, increased levels of ATP activate nociceptors by
binding to P2X3 receptors in dorsal root ganglion neurons, enhancing pain
behaviours (Bland-Ward & Humphrey, 1997; Lewis et al., 1995).
Substance P is released from nociceptor terminals and results in the
release of histamine from mast cells. Histamine causes vasodilation and when
applied directly to the skin produces itching sensations (Simone et al., 1991).
Histamine has also been shown to sensitize the response of nociceptors to
bradykinin (Mizumura et al., 1995).
Primary afferents express more sodium channels than any other neuron in
the mammalian nervous system. As a result, primary afferents produce unique
action potentials (APs) that allow for variation in signals transmitted to the spinal
13
cord. This differentiation in firing patterns may be what allows sensory neurons
to transmit variability in the intensity and duration of both noxious and innocuous
stimulation. Primary afferents that propagate noxious information are unique in
two ways. First, nociceptors produce long duration APs (Djouhri et al., 1998).
These APs are slow (10 per second) and erratic when compared to AP of other
neurons. Secondly, nociceptors phenotypically display sodium channels that are
resistant to tetrodotoxin (TTX) and as a result have distinct kinetic features such
as longer duration APs (Julius & McCleskey, 2006). Hyperalgesic agents have
been shown to increase TTX-resistant sodium currents in nociceptors, which may
indicate a role for these unique receptors in pain states (Gold et al., 1996).
Calcium channels are responsible for translating electrical signals into
chemical signals in order for neurons to generate neurotransmitter release.
There are several types of calcium channels, some of which are found in higher
concentrations on sensory neurons. N-type calcium channels are responsible for
transmitting most of the calcium current from sensory neurons and blocking
these receptors essentially eliminates rapid neurotransmission (Gruner & Silva,
1994). N-type calcium channels are found most commonly on small diameter
primary afferents and antagonists of N channels are effective in achieving
analgesia (Taddese et al., 1995).
The role of potassium channels in nociception is far less clear than those
of sodium and calcium channels. Voltage-gated potassium channels modify the
timing and shape of APs by brining neurons back to their resting potential.
Potassium channels may be useful in suppressing hyperactive sensory neurons,
14
and consequently blunting the nocifensive signal that reaches the spinal cord to
evoke pain states (Julius & McCleskey, 2006).
1.5
Models of Neuropathic Pain
Several animal models have been developed that display various
symptoms of NP observed clinically.
Some NP pain symptoms arise from
disease, which is usually a result of an inflammatory and immune mediated
response against the nervous system, while others are a result of traumatic
nerve damage. One example of an immune mediated neuropathic pain state is
Theiler’s murine encephalomyelinitis virus (TMEV; Mestre et al., 2005). TMEV is
a common picornavirus found in mice however when injected into susceptible
strains of mice it induces a demyelinating disease that serves as a highly
relevant model for human multiple sclerosis. Injection of TMEV results in a lifelong infection of macrophages, microglia and astrocytes.
Progressive
demyelination begins 30-60 days post infection where myelin damage is
mediated by phagocyte cells that have been activated by inflammatory cytokines.
During the chronic phase of the disease, the virus causes a T cell mediated
response that targets the destruction of myelin. Mice infected with TMEV exhibit
progressive impaired motor coordination, NP, incontinence and paralysis
associated with axonal loss and spinal cord atrophy (Mestre et al., 2005).
The Chronic Constriction Injury model (CCI) was developed to study the
effects of peripherally mediated nerve damage in laboratory animals (Bennet &
Xie, 1988), and has been widely used as an animal model that displays
15
symptoms of NP observed clinically such as hyperalgesia and allodynia (Bridges
et al., 2001). Briefly, a CCI is achieved by loose ligation of the sciatic nerve.
Animals with this injury display ipsilateral hind paw deformities, thermal and
chemical hyperalgesia and thermal and mechanical allodynia (Bennet & Xie,
1988). Taken together, the CCI is a reliable animal model of traumatic nerve
injury observed in human and can thus be used to study the effects of NP
observed clinically.
1.6
Molecular Mechanisms of Neuropathic Pain
Mechanisms of NP are relatively well understood, however treatments for
this condition are difficult to elucidate because drug therapies that may work for
one condition are not affective for NP associated with other disease states.
Additionally, current therapeutics used to treat NP typically exhibit limited efficacy
and/or dose-limiting side effects (Gilron et al., 2006). Individual susceptibility to
NP is not well understood, because NP does not consistently occur following
nerve injury. NP is characterized by pathological changes in the central and
peripheral nervous system that include (but are not limited to) anatomical
reorganization of the nervous system, phenotypic changes in sensory neurons
and afferent ectopic discharge where subtype-selective abnormalities in
trafficking and expression of sodium channels causes hyperexcitability in
neurons (Devor, 2006). This hyperexcitability of primary afferent nociceptors as
well as increased activity of spinal cord neurons leads to peripheral and central
16
sensitization that ultimately results in hyperalgesia, allodynia, paresthesias and
other NP pain symptoms.
NP is also associated with allodynia, a condition where following nerve
injury, non-noxious stimulation is perceived as noxious. Allodynia is a debilitating
condition given that simple light touch to an affected area can result in
unbearable pain.
Aβ fibres may acquire the ability to express substance P
following nerve injury. This release of substance P, a nociceptive agent may be
the mechanism by which these fibres evoke touch-mediated pain (Malcangio et
al., 2000).
1.7
c-Fos as a Marker of Neuronal Activity
c-Fos is an immediate early gene that is induced in the central nervous
system following neuron depolarization (Morgan & Curran, 1986) and as such is
used widely in neuroscience as a histological marker of neuronal activation. cFos is used in the study of Alzheimer’s disease, stroke and learning and memory
formation (Herrera & Robertson, 1996). c-Fos immunoreactivity is also used to
examine the effects of acute, inflammatory and NP on cell firing.
Following noxious mechanical stimulation (foot pinch with a toothed clamp
every 1-5 minutes for 4 hours) c-Fos is expressed in laminae I-V of the spinal
cord with the most robust staining found in lamina I (Bullitt, 1990). c-Fos is
expressed in the anterior medial preoptic area, the BLA, the lateral habenula and
the ventral tegmental area (VTA) of young rats following toe pinch, but to a lesser
extent in adult rats (Smith et al., 1997). c-Fos expression in response to repeated
17
toe pinch with toothed forceps has also been found in the rat brainstem (Pinto et
al., 2003).
Persistent c-Fos expression is also found as a result of chronic
inflammatory pain states.
Complete Freund’s adjuvant injected into the
tempomandibular joint of the rat produces deep orofacial and cutaneous tissue
inflammation. This noxious insult induces c-Fos expression at the C1 level of the
spinal cord as well as in parts of the medulla 2 hours following injection (Zhou et
al., 1999).
c-Fos is used to detect basal and stimulus-evoked changes in neuronal
activity following neuropathic injury. Long-term changes in c-Fos expression are
detected in lumbar segments 4, 5 and 6 of the spinal cord 1, 3, 7 and 21 days
following CCI of the sciatic nerve (Jergova & Cizkova, 2005). Catheline et al
(1999) observed that two weeks following CCI in non-stimulated rats there was
no change in the number of c-Fos positive cells in the lumbar region of the spinal
cord as compared to sham or naïve controls. However, when CCI animals were
stimulated with either noxious-cold, noxious mechanical or noxious chemical
stimulation, CCI expression was elevated in lamina I and II of the dorsal horn 2
hours following stimulus onset (Catheline et al., 1999).
Changes in c-Fos
expression following neuropathic injury also occur in the brain.
Four days
following partial ligation of the sciatic nerve, Narita and colleagues (2003) used
immunoblot analysis of c-Fos to determine neuropathy-induced changes in
protein expression.
An increase in c-Fos expression was seen in the PFC,
thalamus and PAG, while decreased levels of c-Fos were found in the nucleus
18
accumbens (NAc) and VTA when comparing sham and neuropathic rats. No
changes in c-Fos expression were seen in the hypothalamus, hippocampus or
amygdala (Narita et al., 2003). Following one hour of restraint stress to CCI
animals, increased levels of c-Fos expression are found in the paraventricular
nucleus of the hypothalamus (PVH) one hour after cessation of stressor. Similar
results were found in sham controls (Bomholt et al., 2005)
Together, these studies indicate that neuronal activation induced by NP
causes lasting, but specific changes in supraspinal brain regions associated with
cognitive, sensory and affective systems. Understanding how these changes
affect an individual afflicted by neuropathy would allow for more specific
therapies to treat this condition.
1.8
Objectives and Hypotheses
This research project was conducted to test 3 hypotheses: Primarily, given
the location and known function of the BST, I hypothesized that acute noxious
mechanical stimulation would cause neuronal activation in the BST. To address
this hypothesis, I determined if noxious stimulation evokes c-Fos expression in
specific nuclei of the BST.
It was hypothesized that chronic pain would increase basal neuronal
activity in the BST. To address this hypothesis, I aimed to elucidate whether
chronic pain associated with peripheral nerve injury lead to an increase in c-Fos
expression when compared to controls.
19
Finally, it was hypothesized that noxious mechanical stimulation would
cause an increase in c-Fos immunoreactivity (c-Fos-IR) in sub-nuclei of the BST
in neuropathic animals when compared to either naïve or sham controls. To
address this hypothesis, I determined whether noxious toe pinch to neuropathic
animals augmented c-Fos-IR in the BST when compared to sham and naïve
controls.
METHODS
2.1
Animals
Adult male Sprague Dawley rats (approx. 8 weeks old) weighing 180-220
grams at the beginning of the experiments (Charles River Canada, Montreal,
Quebec) were housed in pairs on a reverse 12-hour light/dark cycle in a
temperature controlled setting with free access to standard rat chow and water.
All experiments were conducted in the afternoon and early evening. Animals
were customized to the animal facility for no less than 3 days prior to any
experimental procedures or surgical manipulation.
Animal protocols were
approved by the Queen’s University Animal Care Committee in accordance with
the guidelines set by the Canadian Council on Animal Care. All efforts were
made to ensure that the fewest number of animals were used and that animal
suffering was kept to a minimum.
On testing day, animals were taken from the animal care facility and
brought into the laboratory where they remained in their home cage for a
minimum of 1.5 hours prior to testing or experimental procedures. Lights were
20
turned off in the laboratory as to not affect circadian rhythms established by the
reversed light/dark cycle.
One experimenter completed all experiments and
surgeries in an effort to maintain consistency across experiments.
2.2
Pain Induction – Acute Nociceptive Stimulus
A non-toothed, fixed-gauge alligator clip applied to the 4th knuckle of the
left hind paw was used to evoke an acute noxious injury in this experiment. Prior
to the onset of the noxious insult, animals were wrapped in a towel with their left
hind limb exposed. The stimulus was applied for approximately two seconds, or
until vocalization.
2.3
Pain Induction – Chronic Constriction Injury (CCI)
Neuropathic pain was induced by CCI of the sciatic nerve as outlined by
Bennett and Xie (1988). Rats were deeply anesthetized with isoflourane (2.5%,
by inhalation) and induction of sleep was determined by a tail pinch. When tail
pinch no longer produced a withdrawal response, a small incision was made at
the mid-thigh of the left hind limb to expose the underlying muscle tissue and
blunt dissection was used to expose the sciatic nerve. Connective tissue was
carefully removed from the sciatic nerve and 4 loose ligatures of chromic gut
suture (CP Medical, Portland, OR) were knotted around the nerve proximal to its
trifurcation point. Sutures were ligated approximately 1 millimeter apart and care
was used to ensure that sutures were not disrupting perineural blood flow.
Muscle and skin were sutured shut with monocryl (Ethicon, Somerville, NJ).
21
Sham animals received the same surgical treatment, but the sciatic nerve was
not manipulated. Pre-anesthesia, rats were orally administered 0.5 ml of liquid
children’s Tylenol (1.7 mg/kg; McNeil, Fort Washington, PA). Pre-operatively,
animals received subcutaneous injection of 5 ml of lactated Ringer’s solution,
0.013 ml/100g Tribissen antibiotic and eye gel (Novartis, Mississauga, Ontario,
Canada).
On the following day, animals were again orally administered
children’s Tylenol (1.7 mg/kg) as well as lactated Ringer’s solution if signs of
dehydration were evident. Animals received fruit for 4 days following surgery.
Rats were allowed to recover for 10 days to allow neuropathy to produce
significant allodynia and were used for experimental testing on post-surgical day
11.
All animals displayed ipsilateral hind paw deformities and contralateral paw
favouring on day 11 when they were sacrificed.
2.4
Euthanasia and Brain Preparation
For sacrifice, animals were deeply anesthetized with sodium pentobarbital
(70 mg/kg) and perfused via the aortic arch with 500 ml of 4% paraformaldehyde
(PFA) in 0.1M phosphate buffer (PB), pH 7.4 at 4°C. Rat brains were extracted
and post-fixed for 24 hours at 4°C in the same 4% PFA solution. Following postfixing, brains were cryoprotected in 30% sucrose made in 0.1M PB for 48 hours.
Brains were mounted on a freezing sledge microtome and 30-micron
transverse slices were obtained starting at the most rostral point of the BST (as
outlined in the Swanson Atlas, 2003) and concluded when no more BST was
22
evident. Every second slice was collected in 0.1M TBS for immunohistochemical
protocols.
2.5
Immunohistochemical Detection of c-Fos
Neuronal activity was visualized with antibodies directed against c-Fos, an
early oncogene that is used to detect neuronal activity. Following sectioning,
free-floating brain slices were incubated in a solution consisting of 0.3% H2O2
and TBS for 10 minutes to reduce endogenous peroxidase activity. Following 3
five-minute washes with TBS, sections were incubated in solution containing 5%
BSA and 0.1% H2O2 in TBS-T (0.1M TBS with triton X-100) at 4°C for 2 hours to
reduce non-specific immuno-labeling.
Following incubation with blocking
solution, sections were washed 3 times for five minutes with 1% BSA in 0.1 M
TBS-T. Sections were incubated at 4°C, overnight, in a 1:5000 dilution of rabbit
anti-c-Fos (Sigma, St. Louis, MO) prepared in TBS-T and 1% BSA. Following 3
ten-minute washes in TBS-T, sections were incubated in biotinylated goat antirabbit IgG (1:1500; Vector Laboratories, Burlingame, CA) prepared in 1% BSA
and TBS-T for 2 hours at room temperature.
c-Fos labeling was magnified
according to the avidin-biotinylated-horseradish-peroxidase complex (ABC;
Vector Laboratories, Burlingame, CA) for 1 hour at room temperature. Sections
were again washed 3 times for 5 minutes in TBS prior to a 5-minute incubation in
1,3-Diaminobenzidine (DAB) solution (0.15mg/ml) in TBS. Antigenic sites were
precipitated by adding 1% H2O2 to the DAB solution. Washing sections in TB
stopped this reaction. Sections were mounted on 1% gelatinated super frost
23
glass slides (Fisher Scientific, Ottawa, ON, Canada), air dried, dehydrated with
graded alcohol concentrations (50%, 70%, 95%, 100% ethanol in double distilled
water, respectively), cleared with a xylene substitute (Histoclear) and cover
slipped with Permount (Fischer Scientific, Ottawa, ON, Canada).
2.6
Quantitative Analysis
Three rostral sections of BST were counted for c-Fos-IR.
Sections
correlated to levels 18 and 19 in the Swanson Atlas (Swanson, 2003), plus one
section between levels 18 and 19 that is not explained in the Atlas, which we
have named level 18.5.
c-Fos immunoreactive labeling was quantified using
bright field microscopy examined under a Leica DMRE microscope (Leica
Microsystems, Cambridge, ON) equipped with a digital camera (Qimaging Retiga
EXi fast 1394, Surrey, BC) and image capturing software (Q Capture, Lukas
Microscope Service, Skokie, IL). All BSTa nuclei were analyzed: FU, OV, JX,
dAM, vAM, dAL and vAL. Nuclei location and perimeter were determined by size
and shape of cells in each given nuclei, as well as distance from consistent
anatomical markers such as fibre tracts. Quantification of c-Fos-IR cells was
performed blind to all experimental conditions.
Only c-Fos-IR cells in which the cell’s nucleus was entirely stained were
considered c-Fos positive. Cells were counted at 20x objective and questionable
cells were verified at 40x objective. Because of the variability in the number of
sections obtained per nucleus, per rat, only the most c-Fos-IR section (the
section with the highest number of c-Fos positive cells) was used. Scores from
24
each hemisphere for each nucleus were summed to give a total of all c-Fos-IR
cells per BST nuclei per rat brain. Scores from each rat were then averaged for
statistical analysis.
2.7
Statistical Analysis
GraphPad Prizm software 4.0 (San Diego, CA) was used for statistical
analyses and graph generation. Results were presented as mean ± Standard
Error of the Mean (S.E.M). A series of one-way Analysis of variance (ANOVA)
and Newman-Keuls Multiple Comparison Tests were performed within each
experimental group to generate parametric data for all immunohistochemical
experiments. A two-way between subjects ANOVA was performed to determine
if an interaction effect was observed between subjects in Experiments 3 and 4.
For all experiments, p < 0.05 was considered statistically significant.
2.8
Experiment 1: Effect of Acute Nociceptive Stimulation on c-Fos-IR in
the BST
To determine if acute noxious mechanical stimulation (S+) caused
neuronal activation in the BST, rats were randomly assigned to one of 4
conditions. Condition 1 served as a control group, where rats did not receive the
stimulus (S-). Group delegation was dependent on time of sacrifice following toe
pinch. Toe pinch S+ 1 hour (TP S+1) received a toe pinch, were returned to their
home cage and sacrificed 1 hour following pinch. TP S+ 2 and TP S+ 5 were
sacrificed 2 and 5 hours following toe pinch, respectively. All groups contained
25
an n of 6 with the exception of TP S+ 1 where n=9. All rats were anesthetized
with sodium pentobarbital (70 mg/kg) and sacrificed by cardiac aortic perfusion
with 4% paraformaldehyde (PFA).
2.9
Experiment 2: Effect of Acute Non-Nociceptive Stimulation on c-Fos-
IR in the BST
In order to confirm that neuronal activation evident in the BST was in fact
a result of nociceptive stimulation rather than non-noxious sensory stimuli, a
series of experiments was performed to determine if light touch (LT) stimulation
and/or animal handling produced neuronal activation in the BST. Twelve rats
were divided into two groups; LT S+1 and LT S+2 (n=6 per group). Using the
same protocol as in Experiment 1, each rat was taken from the home cage,
wrapped in a towel and the alligator clip set in the open position was lightly
rubbed against the 4th knuckle of the left hind paw for 2 seconds. Animals were
then returned to their home cage. One or two hours following this stimulation,
depending on grouping condition, the animals were sacrificed. All rats were
anesthetized with sodium pentobarbital (70 mg/kg) and sacrificed by cardiac
aortic perfusion with 4% PFA.
2.10
Experiments 3 and 4: Effect of Chronic Constriction Injury on Basal
and Noxious Stimulus Induced Neuronal Firing
The next series of experiments were conducted to determine the effect of
chronic constriction injury on basal and noxious stimulus-induced neuronal firing
26
in the BST. Seventeen rats received a CCI of the left common sciatic nerve and
were randomly divided into 3 groups following the same protocol as experiment
1; CCI S- (n=6), CCI S+1 (n=5) and CCI S+2 (n=6).
Additionally, 20 sham
operated rats were divided into 3 groups where Sham S- (n=5) received a sham
surgery and no stimulation, Sham S+1 (n=9) received a toe pinch and were
sacrificed one hour following S+, and Sham S+ 2 (n=6) received a toe pinch and
were sacrificed two hours following mechanical nociceptive stimulation. Again, all
rats were anesthetized with sodium pentobarbital (70 mg/kg) and sacrificed by
cardiac aortic perfusion with 4% paraformaldehyde.
RESULTS
3.1
Experiment 1: Effect of Acute Nociceptive Stimulation on c-Fos-IR in
the BST
A series of One-Way within subjects ANOVAs were conducted to
determine if noxious mechanical toe pinch produced changes in the level of cFos-IR in all 7 nuclei of the BSTa. Pain-induced c-Fos-IR was compared to
naïve animals one, two and five hours following noxious insult.
Noxious toe pinch significantly increased c-Fos-IR in the FU (Figures 4 & 5) and
dAM (Figures 6 & 7) one and two hours following noxious insult, while the
remaining 5 nuclei did not exhibit a change in neuronal activation to noxious toe
pinch (Figure 8). Five hours after stimulation, noxious-induced c-Fos-IR was not
significantly different from baseline c-Fos-IR in the FU and was significantly lower
27
TP Fusiform
**
20
*
15
10
5
0
S-
TP S+1
TP S+2
TP S+5
Time
Figure 4: Effect of mechanical noxious stimulation on c-Fos-IR in the FU
nucleus of the BST. Toe pinch significantly increased c-Fos-IR one and two
hours following stimulation.
Mean number of cells expressing c-Fos is
presented. Data are presented as means ± S.E.M. n = 6 for each group.
Statistical analysis performed using One-Way ANOVA [F(3,20) = 11.05, p =
0.0002]. Newman-Keuls Multiple Comparison post-hoc tests were used to
determine differences between groups where * = p < 0.05 and ** = p < 0.01 as
compared to baseline (S-).
Figure 5: Immunohistochemical representation of c-Fos-IR cells in the FU
nucleus. (a) naïve animals (b) one hour following noxious mechanical
stimulation (b). Scale bars, 250 µm.
28
TP dorsal AM
40
30
***
**
20
10
*
0
S-
TP S+ 1
TP S+ 2
TP S+ 5
Time
Figure 6: Effect of mechanical noxious stimulation on c-Fos-IR in the dAM
nucleus of the BST. Toe pinch significantly increased c-Fos-IR one and two
hours following stimulation.
Mean number of cells expressing c-Fos is
presented. Data are presented as means ± S.E.M. n = 6 (S-, S+2 and S+5), n =
9 (S+1). Statistical analysis performed using One-Way ANOVA [F(3,23) = 17.91,
p < 0.0001]. Newman-Keuls Multiple Comparison post-hoc tests were used to
determine differences between groups where * = p < 0.05, ** = p < 0.01 and *** =
p < 0.001 as compared to baseline (S-).
Figure 7: Immunohistochemical representation of c-Fos-IR cells in the dAM
nucleus. (a) naïve animals (b) one hour following noxious mechanical
stimulation. Scale bars, 250 µm.
29
Figure 8: Effect of mechanical noxious stimulation on remaining 5 nuclei
of the BST. Data are presented as means ± S.E.M. Red bars indicate nonstimulated controls, blue bars represent mean c-Fos-IR cells one hour following
noxious insult. p > 0.05 in all groups. n = 6 in all groups except in dAM and vAL
S+1 where n = 5.
30
in the dAM (Figures 4 and 6, respectively). The 5 hour time point was included in
the study to assess the time course of pain-induced c-Fos-IR in these nuclei.
3.2
Experiment 2: Effect of Acute Non-Nociceptive Stimulation on c-Fos-
IR in the BST
To control for effects of animal handling and other non-noxious stimuli a
second control group was included in addition to the naïve animals to ensure that
c-Fos-IR was due to noxious stimulation.
The protocol was identical to the
noxious toe pinch group with the exception that animals received a stimulus
consisting of brushing of the toe rather than a toe pinch. Light touch stimulation
did not increase c-Fos-IR in either the FU or dAM nuclei one hour following
stimulation (Figure 9). Figure 10 shows the difference in c-Fos-IR in noxiousstimulus induced c-Fos-IR as compared to non-noxious induced c-Fos-IR in the
FU. Similar results were found in the dAM (data not shown). One hour following
LT stimulation, c-Fos expression in the FU, dAM and vAM was significantly lower
than naïve controls (Figure 11). This observation confirms that increased levels
of c-Fos-IR cells are a direct result of mechanical noxious stimulation. T-tests
were used to determine statistical significance between noxious and non-noxious
stimulated conditions.
3.3
Experiment 3: Effect of CCI on Basal c-Fos-IR in the BST
Upon discovering that pain-induced c-Fos-IR is evident in specific nuclei of the
BST, the next series of experiments were conducted to determine if painful
31
dAM
FU
20
40
15
30
10
20
***
0
***
10
5
TP S+1
0
LT S+ 1
TP S+ 1
LT S+ 1
Condition
Condition
Figure 9: Comparison of noxious and non-noxious stimulation in the FU
and dAM nuclei of the BST. T-test was used to determine if one hour following
stimulation, changes in c-Fos-IR were evident when comparing noxious versus
non-noxious stimulation. Left: FU nucleus where t = 4.208 (10) p < 0.001. Right:
dAM nucleus where t = 6.934 (13) p < 0.001. n = 6 in all groups except TP S+1
where n = 9. Data are presented as means ± S.E.M. *** = p < 0.001.
Figure 10: Effect of noxious and non-noxious stimulation on c-Fos-IR in
the FU one hour after stimulation. (a) c-Fos-IR one hour following noxious toe
pinch (b) c-Fos-IR one hour following light tough stimulation. Scale bars, 250 µm.
Similar results were obtained in dAM (data not shown).
32
Figure 11: Analysis of c-Fos-IR in all BST nuclei when comparing nonstimulated control animals and non-noxious stimulated animals. T tests
were used to determine if non-noxious stimulation caused an increase in fos-IR
in each nucleus of the BST when compared to naïve controls. c-Fos expression
in the FU, dAM and vAM was significantly lower than naïve controls in the LT
condition. FU: t = 2.348 (10), p < 0.05. dAM: t = 2.326 (10), p < 0.05. vAM: t =
2.500 (10), p < 0.05. dAL: t = 1.146 (10), n.s. vAL: t = 1.180 (10), n.s. OV: t =
1.052 (10), n.s. JX: t = 0.955 (10), n.s. Data are presented as means ± S.E.M. n
= 6 in each group. * = p < 0.05.
33
neuropathy associated with chronic constriction of the sciatic nerve increased
basal firing levels of neurons in the FU and dAM nuclei of the BST. Contrary to
expectation, basal firing levels of neurons in the FU (Figure 13) and dAM (Figure
14) did not change significantly 11 days following induction of CCI when
compared sham controls (Figure 12). No significance was found in the remaining
5 nuclei when comparing CCI S- to sham S- conditions (Figure 15).
3.4
Experiment 4: Effect of CCI on Noxious-Stimulus Induced c-Fos-IR
in the BST
The next series of experiments were conducted to determine is c-Fos-IR
in the BST was increased following noxious stimulation of the injured hind paw in
CCI rats when compared to sham controls. Since Experiment 1 demonstrated
that c-Fos-IR was increased in the FU and dAM, it was hypothesized that
stimulation of neuropathic animals would exceed the levels of c-Fos-IR seen in
acute animals. A series of One-Way within subjects ANOVAs were conducted to
compare CCI non-stimulated controls to CCI animals who received noxious toe
pinch one or two hours prior to sacrifice and these scores were compared to
sham animals in similar conditions. Contrary to the hypothesis, noxious evoked
c-Fos-IR was not increased in the FU (Figures 16 and 17) or the dAM (Figures
18 and 19) nuclei of the BST compared to non-stimulated CCI or sham animals
(data not shown).
While CCI did not cause an increase in c-Fos-IR in expected nuclei, a
significant increase in c-Fos labeling was found in the vAM of CCI rats 2 hours
following noxious stimulation, a result that was not found in sham animals (Figure
34
FU
dAM
20
40
15
30
10
20
5
10
0
CCI S-
SHAM S-
0
Condition
CCI S-
SHAM S-
Condition
Figure 12: Comparison of basal c-Fos-IR cells in the FU and dAM nuclei of
the BST 11 days following CCI or sham surgery. T-test was used to
determine if basal levels of c-Fos expression increased in CCI animals when
compared to shams. Left: FU nucleus where t = 1.106 (9), n.s. Right: dAM
nucleus where t = 0.788 (9), n.s. CCI S- n = 6 per group, sham S- n = 5. Data
are presented as means ± S.E.M.
35
Figure 13: Immunohistochemical representation of basal c-Fos-IR cells in
the FU nucleus of CCI and sham rats. (a) CCI non-stimulated (b) sham nonstimulated. Scale bars, 250 µm.
Figure 14: Immunohistochemical representation of basal c-Fos-IR cells in
the dAM nucleus of CCI and sham rats. (a) CCI non-stimulated (b) sham nonstimulated. Scale bars, 250 µm.
36
Figure 15: c-Fos-IR scores for each remaining nuclei when comparing
basal firing of CCI and sham animals. T tests were used to determine if basal
levels of c-fos expression were higher in CCI animals. No increase in c-Fos-IR
was observed across any sample tested. vAM: t = 1.886 (10), n.s. dAL: t = 2.059
(10), n,s. vAL: t = 0.353 (9), n.s. OV: t = 0.692 (9), n.s. JX: t = 0.000 (9), n.s.
Data are presented as means ± S.E.M. n = 6 in each group with the exception of
vAL, OV and JX sham S- where n = 5.
37
CCI fusiform
20
15
10
5
0
CCI S-
CCI S+ 1
CCI S+ 2
Time
Figure 16: Effect of mechanical noxious stimulation on c-Fos-IR in the FU
nucleus of CCI rats. Toe pinch did not significantly increased c-Fos-IR one or
two hours following stimulation. Mean number of cells expressing c-Fos is
presented. Data are presented as means ± S.E.M. CCI S- and CCI S+2: n = 6
per group. S+1 is n = 5. Statistical analysis performed using One-Way ANOVA
[F(2,14) = 2.242, n.s].
Figure 17: Immunohistochemical representation of c-Fos-IR cells in the FU
nucleus of non-stimulated and noxious-stimulated CCI rats. (a) CCI nonstimulated (b) CCI one hour following noxious mechanical stimulation. Scale
bars, 250 µm.
38
CCI dorsal am
40
30
20
10
0
CCI S-
CCI S+ 1
CCI S+ 2
Time
Figure 18: Effect of mechanical noxious stimulation on c-Fos-IR in the dAM
nucleus of CCI rats. Toe pinch did not significantly increased c-Fos-IR one or
two hours following stimulation. Mean number of cells expressing c-Fos is
presented. Data are presented as means ± S.E.M. CCI S- and CCI S+2: n = 6
per group. CCI S+1 is n = 5. Statistical analysis performed using One-Way
ANOVA [F(2,14) = 0.803, n.s].
Figure 19: Immunohistochemical representation of c-Fos-IR cells in the
dAM nucleus of non-stimulated and noxious-stimulated CCI rats. (a) CCI
non-stimulated (b) CCI one hour following noxious mechanical stimulation. Scale
bars, 250 µm.
39
20). c-Fos-IR did not increase significantly in CCI animals one hour following
stimulation in the vAM or one and two hours following stimulation in the
remaining 4 nuclei examined (Figure 21).
While CCI did not cause an increase in c-Fos-IR in expected nuclei, a
significant increase in c-Fos labeling was found in the vAM of CCI rats 2 hours
following noxious stimulation, a result that was not found in sham animals (Figure
20). c-Fos-IR did not increase significantly in CCI animals one hour following
stimulation in the vAM or one and two hours following stimulation in the
remaining 4 nuclei examined (Figure 21).
A series of One-Way within subjects ANOVAs were used to determine
whether c-Fos-IR in the FU and dAM nuclei of sham animals increased
significantly following noxious stimulation when compared to non-stimulated
controls. Unlike in naïve animals, the FU of stimulated sham animals was not
responsive to noxious insult (Figures 22 and 23), while the dAM displays similar
activity to stimulated naïve animals (Figures 24 and 25). c-Fos-IR increased
significantly in the OV nucleus of sham animals following stimulation. There
were no increases in the vAM, vAL, dAL or JX nuclei (Figure 26).
To determine if there was an interaction effect between CCI and Sham
animals in the FU and dAM, a two-way ANOVA was used to examine the
relationship between time (S- & S+1) and treatment condition (CCI & Sham). In
the FU, there was no significant interaction effect [F (1,18)=1.854, n.s]. In the
dAM, there was a significant interaction effect between treatment condition and
time: [F (1,21)= 4.543, p = 0.05].
40
Figure 20: Immunohistochemical representation of c-Fos-IR cells in the
vAM nucleus of non-stimulated and noxious-stimulated CCI and sham rats.
(a) CCI non-stimulated (b) CCI two hours following noxious mechanical
stimulation (c) sham non-stimulated (d) sham 2 hours following toe pinch. Scale
bars, 250 µm.
41
Figure 21: Comparison of non-stimulated and noxious-stimulated CCI rats
1 and 2 hours after noxious stimulation. Newman-Keuls Multiple Comparison
post-hoc tests were used to determine if noxious stimulation increased c-Fos
expression in CCI rats when compared to basal c-Fos-IR levels. * = p < 0.05
when compared to baseline and S+1 hour group. Data are presented as means
± S.E.M.
42
SHAM fusiform
20
15
10
5
0
SHAM S-
SHAM S+ 1
SHAM S+ 2
Time
Figure 22: Effect of mechanical noxious stimulation on c-Fos-IR in the FU
nucleus of sham rats. Toe pinch did not significantly increase c-Fos-IR one or
two hours following stimulation. Mean number of cells expressing c-Fos is
presented. Data are presented as means ± S.E.M. Sham S-: n = 5. Sham S+1
and S+2: n = 6 per group. Statistical analysis performed using One-Way ANOVA
[F(2,14) = 1.362, n.s].
Figure 23: Immunohistochemical representation of c-Fos-IR cells in the FU
nucleus of non-stimulated and noxious-stimulated sham rats. (a) Sham nonstimulated (b) Sham one hour following noxious mechanical stimulation. Scale
bars, 250 µm.
43
SHAM dAM
40
*
30
20
10
0
SHAM S-
SHAM S+ 1
SHAM S+ 2
Time
Figure 24: Effect of mechanical noxious stimulation on c-Fos-IR in the dAM
nucleus of sham rats. Toe pinch significantly increased c-Fos-IR one hour
following stimulation. Mean number of cells expressing c-Fos is presented. Data
are presented as means ± S.E.M. CCI S- and CCI S+2: n = 6 per group. Sham
S+1 is n = 5. Statistical analysis performed using One-Way ANOVA [F(2,17) =
5.856, p < 0.05]. Newman-Keuls Multiple Comparison post-hoc tests were used
to determine differences between groups where * = p < 0.05 as compared to
baseline (sham S-).
Figure 25: Immunohistochemical representation of c-Fos-IR cells in the
dAM nucleus of non-stimulated and noxious-stimulated sham rats. (a)
Sham non-stimulated (b) Sham one hour following noxious mechanical
stimulation. Scale bars, 250 µm.
44
Figure 26: c-Fos-IR scores for each remaining nuclei when comparing
basal versus noxious stimulus evoked firing levels of sham animals.
Newman-Keuls Multiple Comparison post-hoc tests revealed a significant
increase in c-Fos positive cells in the OV when comparing non-stimulated and
noxious stimulated sham animals. * = p < 0.05 when compared to S-. Data are
presented as means ± S.E.M. n = 5 in sham S-, n = 6 for sham S+1. * = p <
0.05.
45
DISCUSSION
4.1
Experiment 1: Effect of Acute Nociceptive Stimulation on c-Fos-IR in
the BST
This study reveals for the first time that there is a nuclei-specific response
to acute noxious pain in the BST. Recently, Deyama and colleagues (2007)
elucidated a role of the BST in pain-induced conditioned place aversions (PICPA), a technique that is used to study the affective component of pain. Briefly,
animals were introduced to a two-compartment apparatus with distinct tactile and
visual cues in each compartment. The noxious stimuli used in the study included
2% acetic acid (i.p) or intraplantar injection of 2% formalin into the right hind paw.
As predicted, animals spent less time in the noxious-paired compartment after
training than they had during habituation. Once the PI-CPA was established, all
rats were then given excitotoxic lesions to the anterior portion of the BST.
Deyama et al (2007) hypothesized that lesions to the BST would ablate PI-CPA.
One week following lesion, animals were once again placed in the twocompartment apparatus and were allowed to roam freely.
As predicted, in
lesioned animals, time spent in each compartment returned to pre-testing
baseline, while animals with sham lesions continued to show a preference to the
non-pain paired compartment, indicating that the BST contributes to exacerbating
affective dimensions of pain.
While this research was certainly seminal in
elucidating a role for the BST in pain states, the study could not determine which
of the 7 anterior nuclei contribute to this phenomenon. Using c-Fos as a marker
for neuronal activation, the current research project demonstrates that only two of
46
the 7 anterior nuclei – the FU and the dAM are activated in response to acute
noxious insult.
When compared to naïve animals, noxious toe pinch caused a 3-fold
increase in c-Fos-IR in the FU. It is important, then to understand how increased
neuronal activity in the FU affects pain systems. Within the BST, the FU projects
almost exclusively to dorsal aspects of the AL and AM (Dong et al., 2001).
Relatively little is known about the specific functions of the different neuronal
populations within the FU, however projection patterns originating in this nucleus
have been characterized with the Phaseolus vulgaris-leucoagglutinin (PHAL)
anterograde tracing methods in male adult rats (Dong et al., 2001). The FU
nucleus projects along approximately 4 distinct pathways that terminate in the
amygdala, hypothalamus and lower brainstem (Dong et al., 2001). Two distinct
fibre tracts, the ansa peduncularis and the stria terminalis, connect the FU to the
amygdala. Within the amygdala, FU projections terminate almost exclusively in
the medial region of the CeA, with little or no PHAL expressed in other areas of
this region. The CeA is responsible for mediating many aspects of stress and
anxiety, particularly through this structure’s connection to the hypothalamus,
basal forebrain and brainstem (Kalin et al., 2004). Noxious-encoded projections
from the FU to the CeA may initiate down-stream cognitive responses to
disruptions in homeostasis. Fibre tracts originating in the FU also travel via the
medial forebrain bundle where some travel through the VTA and terminate in the
PAG and the pons.
Functions of the PAG are relatively well understood as
activation of neurons in this area contributes to modulating several biological
47
functions such as defense, reproductive behaviour, pain, anxiety and
cardiovascular responses (Rossi et al., 1994). Specifically, the dorsal nucleus of
the raphé and the laterodorsal tegmental nucleus in the PAG are heavily
innervated by FU fibres.
Both of which regulate various aspects of arousal
(Satoh & Fibiger, 1986, Vertes, 1991). Pons systems are well understood to
mediate various aspects of autonomic breathings such as inspiratory duration
and frequency of ventilation (Dick & Coles, 2000). Because the FU projects to
the PAG, this pathway may be involved in modulating descending analgesia
(Reynolds, 1969) while also mediating changes in cardiovascular function
following noxious insult (Van Der Plas et al., 1995). Projections from the medial
forebrain bundle also send rich projections to the medial parvicellular division of
the paraventricular nucleus of the hypothalamus (mpPVH). The remaining fibre
bundle that originates in the FU, the periventricular pathway is the major pathway
from the FU. This fibre tract sends very dense projections almost exclusively to
the PVH (Dong et al., 2001).
Perhaps the most interesting and predominant efferent projections
originating in the FU are those that terminate in the PVH. The FU receives
dense projections from the CeA, locus coeruleus, nucleus of the solitary tract
(Dong et al., 2001) the ventral subicular nucleus of the hippocampus (Cullinan et
al., 1993) and other forebrain structures such as the infralimbic cortex (Choi et
al., 2007). None of these structures (with the exception of the CeA) has a direct
projection to the PVH.
In the case of the CeA, only some axons from this cell
group are synaptically linked directly to the PVH (Gray et al., 1989). Most CeA
48
projections synapse in the FU nucleus first and from there are sent to the PVH
(Prewitt & Herman, 1998, Dong et al., 2001). The PVH (specifically the mpPVH)
is the origin of a final pathway involved in activation of the hypothalamic-pituitaryadrenal axis (HPA axis). Neurons in the PVH synthesize and release a plethora
of hormones involved in the physiological response to disruptions of
homeostasis, including CRH and vasopressin (Cullinan et al., 1993).
These
neuropeptides act on the pituitary gland where they regulate the release of
adrenocorticotrophic hormone (ACTH). ACTH acts upon the adrenal cortex to
mediate the release of glucocorticoids (Bomholt et al., 2005). Several studies
have shown that acute stressors shape behavioural responses through the
activation of the HPA axis. Glucocorticoids exert their effects throughout the
brain during disruptions to homeostasis (such as stress) to alter behaviour and
cognition in an adaptive manner (Radley & Morrison, 2005). Glucocorticoids also
maintain a negative feedback function that acts on the hippocampus to terminate
defensive stress responses following the cessation of a stressor (Radley &
Morrison, 2005). There is an increase in circulating levels of glucocorticoids
following the induction of restraint stress in rats (Choi et al., 2007) and elevated
levels of circulating glucocorticoids are consistently seen in rats employing
defensive aggressive behavioural strategies (Brain & Haug, 1992). Activation of
the HPA axis, and the subsequent release of glucocorticoids induce major
physiological responses that work in concert to return disrupted systems back to
homeostasis. HPA axis activation causes a diversion of energy to exercising
muscles, enhanced cardiovascular tone, immune function stimulation and
49
activation, inhibition in reproductive behaviour, decreased appetite and feeding
and sharpened cognitive function (Sapolsky et al., 2000). The present study is
the first to show that acute mechanical noxious stimulation causes an increased
response in cell firing in the FU nucleus of the BST. Given this direct projection
from limbic and cognitive systems, acute pain most likely causes activation of the
HPA axis via the FU’s connections to the PVH to create a behavioural and
affective response to acute pain. Hence, activation of the FU is responsible for
mediating physiological-affective responses to acute noxious stimulation. The
discovery that higher-level cognitive and limbic structures do not maintain direct
connections to the PVH to activate the HPA axis in response to stressors
indicates that the FU nucleus is critical to relay cognitive and affective information
to the PVH to produce a hormonal response to noxious insult.
In addition to increased c-Fos-IR in the FU, the dAM nucleus of the BST
displayed a 4-fold increase in c-Fos positive cells when comparing toe-pinch
animals to naïve controls. The anteromedial (AM) nucleus of the BST is the
largest sub-nuclei of the BST. This area spans across the anterior commisure
(AC) and as such is divided into a dorsal and ventral aspect (with dorsal laying
above the AC and ventral below). With the exception of 3 connections originating
from the vAM that are absent in the dAM, these structures share almost identical
projection patterns (Dong & Swanson, 2006). While little is known about specific
functions of the FU, less still is known about functions of the AM.
Using
anterograde PHAL tracing techniques with injections specific to the AM,
projection patterns from this nuclei have been elucidated. The AM receives
50
massive inputs from the CeA (Sun et al., 1991), the ventral subiculum of the
hippocampus (Canteras & Swanson, 1992), the lateral septal nucleus (Risold &
Swanson, 1977) and the infralimbic area of the PFC (Hurley et al., 1991). Three
fibre tracts originate in the AM. The rostral-ascending pathway innervates the
shell of the nucleus accumbens, the medial olfactory tubercle, substantia
innominata and infralimbic cortex of the PFC (Dong & Swanson, 2006),
structures important in motivation, reward-perception and higher order cognitive
function (Barrot et al., 2002, Chudasama & Robbins, 2003). The stria terminalis
and ansa peduncularis pathway transmit information primarily to the CeA and to
a lesser extent the BLA (Dong & Swanson, 2006). Several descending systems
also originate in the AM, which, collectively innervate essentially all regions of the
hypothalamus. The paraventricular propriohypothalamic pathway innervates the
periventricular, paraventricular and arcuate regions of the hypothalamus. The
medial propriohypothalamic pathway synapses in the medial preoptic nucleus
and the nucleus circularis of the hypothalamus. The ventral propriohypothalamic
pathway terminates in both the lateral and posterior hypothalamus and finally, the
medial forebrain bundle that innervates the paraventricular and lateral areas of
the hypothalamus (Dong & Swanson, 2006). The periventricular nucleus of the
hypothalamus (consisting of the arcuate nucleus and the posterior periventricular
nucleus) is abundant in dopamine neuroendocrine motorneurons that are
responsible for inhibiting the release of prolactin (Markakis & Swanson, 1997) as
well as growth hormone-releasing hormone (GHRH; Sawchenko et al., 1985).
Inhibition of prolactin, among other functions is responsible for the inhibition of
51
sexual arousal (Levin & Meston, 2006) while GHRH stimulates the release of
growth hormone from the adrenal glands to stimulate cell growth and
reproduction (Strobl & Thomas, 1994). Terminations to the anterior regions of
the hypothalamus, notably the periventricular nucleus, the periventricular aspect
of the PVH and the dorsalmedial parvicellular region of the PVH synthesize and
release somatostatin, thyrotropin-releasing hormone and CRH (Dong &
Swanson, 2006), all of which act to control metabolic processes, collectively.
The release of these hormones by activation on the AM in response to noxiousmechanical insult is likely an effort to restore peripheral hormone-mediated
homeostasis following noxious stimulus onset. Finally, the AM densely innervates
the magnocellular area of the PVH – an area rich in oxytocinergenic neurons
(Dong & Swanson, 2006). Oxytocin is involved in social recognition and bonding
(Uvnas-Moberg, 1998) as well as the regulation of wakefulness, body
temperature and circadian homeostasis (Argiolas & Gessa, 1991).
Daily
injections of oxytocin have been shown to decrease stress levels of chronically
stressed rats in the laboratory (Uvnas-Moberg, 1998). Finally, the AM projects to
the brainstem where axons terminate in the PAG and retrorubral area (Dong &
Swanson, 2006). Activation of the retrorubral nucleus is involved in orofacial
motor control. Following noxious insult, arousal, attention and facial expression
in response to stimulus is at least in part, mediated by connections originating in
the AM nucleus of the BST. The current project, for the first time reveals that an
acute mechanical noxious stimulus causes a 4-fold increase of c-Fos-IR in the
dAM nucleus of the BST.
Considering the brain regions innervated by cell
52
populations originating in the dAM, this extremely complex information system
seems to respond to noxious threat by initiating neuroendocrine, autonomic and
behavioural systems that collectively restore homeostasis in response to external
stressors.
The discovery of a nuclei-specific response to acute noxious-insult in the
BST allows for a clear interpretation of how higher order brain structures connect
to more primitive structures to create an affective and physiological response to
acute pain. The BST, through the FU and dAM is responsible for relaying and
converting cortical, highly processed information into basic neuroendocrine
signals that result in an affective and autonomic peripheral response to noxious
insult.
4.2
Experiment 2: Effect of Acute Non-Noxious Stimulation on c-Fos-IR
in the BST
LT stimulation did not increase c-Fos-IR in any nuclei of the BST. There
was, however a significant decrease in c-Fos-IR in the vAM, dAL and vAL nuclei
of the BST. This finding is likely the result of time spent in the laboratory prior to
sacrifice. c-Fos is highly sensitive to stress (Kovacs, 1998) and is up-regulated
in several brain areas following novel stressors (Briski & Gillen, 2001).
Significantly lower levels of c-Fos-IR seen in LT S+1 rats are likely the result of
habituation to the novel environment. Because LT S+1 animals have been in the
laboratory for upwards of 2.5 hours without any additional stressors other than
brief handling it is likely that only the introduction into the laboratory induced c-
53
Fos expression.
Stress-induced c-Fos-IR peaks at 30 and 60 minutes post-
stressor then begins to decline (Cullinan et al., 1995).
Therefore, it is not
surprising that these animals maintained much lower c-Fos-IR levels at the time
of sacrifice when compared to noxiously manipulated rats who were given a
noxious toe pinch, consequently adding to, or reinitiating a c-Fos response just
one hour prior to sacrifice.
4.3
Experiment 3: Effect of CCI on Basal c-Fos-IR in the BST
It is well documented in the literature that chronic neuropathic pain results
in changes to basal firing rates of neurons in the spinal cord, both using a c-Fos
immunohistochemical markers
electrophysiological techniques.
(Jergova & Cizkova,
2005) as
well as
CCI of the sciatic nerve causes increased
spontaneous firing rates, increased after-discharge firing and increases in the
receptive field size of wide dynamic range neurons in the ipsilateral dorsal horn
of the spinal cord (Lui & Walker, 2006).
Changes in activity of neurons in
supraspinal regions are less clearly understood and varies dramatically between
regions.
For example, chronic pain has been shown to decrease NMDA-
mediated activity in the medial PFC (mPFC) and amygdala. Direct infusion of dcycloserine, an NMDA partial agonist into the mPFC and amygdala decreased
cold and tactile hyperalgesia and increased motor activity in rats who received a
spared nerve injury when compared to sham controls (Millecamps et al., 2007).
The cuneate nucleus, a somatosensory relay centre shows increased c-Fos-IR
following medial nerve transection (Lue et al., 2002).
54
Also, 4 days following
partial ligation of the sciatic nerve, c-Fos expression is increased in the PFC,
thalamus and PAG, where there is a marked decrease in c-Fos-IR in the VTA
and the NAc (Narita et al., 2003).
Protein expression in the hypothalamus,
hippocampus and amygdala are not affected (Narita et ah, 2003). Surprisingly,
very little research has been conducted to elucidate the role of supraspinal sites
in the contribution or manifestation of chronic pain states. We therefore wanted
to determine if the BST is involved in exacerbating NP pain conditions. Our
research showed that 11 days following chronic constriction of the sciatic nerve,
there is no increase in c-Fos expression in any of the 7 nuclei examined.
C-
Fos-IR was similar in the FU and dAM in NP, sham and naïve rats, indicating
that, at this time point, there is no effect on basal c-Fos expression in the BST
following nerve injury. This study reveals that the BSTa does not endure lasting
changes to basal firing levels following chronic neuropathic injury as might be
expected due to ongoing spontaneous pain that such an insult induces. This
study, however cannot rule out earlier changes in c-Fos-IR that may have
occurred in the 10 days prior to sacrifice. As discovered in Experiment 4 (below),
functional changes may have occurred in the anterior aspect of the BST following
CCI.
4.4
Experiment 4: Effect of CCI on Noxious-Stimulus Induced c-Fos-IR
in the BST
Given that there was a 3-fold increase in noxious-stimulus evoked
neuronal firing in the FU of the BST following acute stimulation, it was anticipated
55
that chronic neuropathic injury would also produce a significant increase in cell
firing following mechanical noxious stimulation.
One hour following noxious
stimulation of 11-day neuropathic rats, we found that there was no increase in cFos-IR in the FU nucleus of the BST. Considering that the FU is an important
relay centre for limbic information that is then transmitted to the hypothalamus to
produce a neuroendocrine response to stress and noxious-threat, this finding
solidifies the conclusion that the BST may indeed be responsible for mediating
affective components of pain.
It is well documented in the literature that chronic pain, including NP is
often accompanied by affective disorders such as sleep disturbances, depression
and anxiety (Argoff, 2007). Aside from traditional analgesics that only produce
minimal efficacy in response to NP (Max et al., 1988), antidepressants are highly
efficacious in producing analgesia for chronic NP symptoms, with one study
reporting a 50-85% decrease in reported pain when compared to placebo
controls (McQuay et al., 1996). The novel discovery that noxious-stimulation
following CCI does not increase c-Fos-IR in the FU nucleus may have important
implications in the blunted affect observed in individuals suffering from chronic
neuropathic injuries. Given that the FU is the relay centre for all cognitive and
limbic information arriving at the PVH (Choi et al., 2007), the FU may be the brain
region responsible for blunting neuroendocrine responses during chronic pain to
affect the physiological response to noxious-stimulation. In 2003, Vissers and
colleagues observed that following CCI in rats, levels of circulating ACTH and
corticosterone did not increase significantly from baseline following 5% formalin-
56
induced stimulation of either the injured or non-injured hind paw, while formalin
injections into naïve rats significantly increased circulating hormone levels
(Vissers et al., 2003). It is also reported that CCI increases the expression of
CRH mRNA in the amygdala, but not the PVH or the BST (Ulrich-Lai et al.,
2006). This indicates that fibre tracts originating in other limbic structures are not
affected by CCI until the signal reaches the BST where it is blunted. Our study
reveals for the first time that the FU nucleus of the BST may be the relay centre
that interrupts information traveling to the PVH to produce an HPA axis response
to chronic nerve injury.
This blockade may be a contributing factor to the
depression, anxiety and blunted physiological responses that often co-exist with
NP.
Similar to the FU, the dAM also displayed no increase in c-Fos-IR one
hour following noxious stimulation in CCI animals when compared to sham
controls. Projections from the dAM are more complex; however as discussed
previously, several of these projections terminate in structures important in
motivation, reward perception and higher order cognitive function (Dong &
Swanson, 2006; Barrott et al., 2002; Chudasama & Robbins, 2003).
Neuroendocrine systems activated by dAM also seemingly mediate reward and
social aspects of behaviour, such as the release of oxytocin and prolactin. Given
these projection systems, a decrease in neuronal activity in the dAM nucleus of
the BST may be responsible for manifesting psychological and cognitive
disturbances commonly found in individuals afflicted with chronic NP conditions,
such as depression and anhedonia.
57
Taken together, decreased responses in the FU seem to blunt
physiological and endocrine responses following chronic nerve injury, while the
dAM nucleus is responsible for psychological maladaptations that arise in
response to sustained NP. In acute pain conditions, activation of the FU and
dAM increase the release of stress hormones and activate downstream systems
that cause animals to react to disruptions in homeostasis. Following chronic
nerve injury, the FU and dAM no longer respond normally to acute noxious
stressors, indicating that these nuclei are contributing players in the deleterious
effects of chronic NP conditions.
Two hours following noxious-stimulation to CCI animals revealed a
significant increase in c-Fos-IR in the vAM of the BST. This was not seen in
sham or naïve subjects. Anatomical studies that discuss AM projections state
that the dorsal and ventral aspects of the AM are similar with only a few
exceptions: The vAM terminates much more densely in the magnocellular and
medial parvicellular parts of the PVH (Dong & Swanson, 2006). Also, while the
dAM terminates lightly in the nucleus circularis, projections from the vAM seem to
do so more robustly (Dong & Swanson, 2006). Importantly, the discovery that
the dorsal and ventral aspects of the AM have slight differences is the result of a
chance injection isolated to the vAM and as such this data is extrapolated from
an N of 1. PHAL injections isolated to the vAM and dAM separately are required
to clarify these results.
Considering that the dAM becomes unresponsive to
noxious stimulation following neuropathic injury, and since the projection systems
of the dorsal and ventral nuclei are strikingly similar, a possible explanation for
58
this finding is that vAM activity is increased as a compensatory mechanism to
adjust to decreases in systemic function following neuropathic injury.
Data from sham animals revealed interesting results.
Most interesting
was the discovery that the FU nucleus of sham operated animals also showed a
blunted response one hour following noxious stimulation. It was expected that
sham animals would serve as a control for CCI subjects and consequently would
display increases in noxious stimulus induced c-Fos-IR similar to those found in
naïve animals.
Instead, the result obtained in the FU of sham animals was
similar to those of CCIs. The dAM nucleus of sham animals one hour following
noxious stimulation displayed c-Fos-IR similar to naïve controls; therefore, the
discrepancy in results was only seen in the FU nucleus.
This finding is
somewhat perplexing, however no effect in c-Fos-IR in the FU may result from a
mechanistic decrease of signaling to the HPA-axis as a result of post-operative
pain. Inflammatory pain, as well as simple surgical incision has been shown to
alter c-Fos expression in the spinal cord (Bereiter & Bereiter, 1996), therefore it is
also likely that sham surgery alone is sufficient to produce lasting changes to cFos expression in supraspinal areas, including the BST.
Unlike CCI and naïve animals, sham rats showed increased c-Fos-IR in
the OV nucleus one hour following noxious stimulation. The OV receives inputs
from several association areas of the cortex such as the postpiriform area, a
region that receives massive inputs from the olfactory bulb (McDonald, 1998) as
well as the insular region. The insular region of the cortex includes visceral,
gustatory and viscerolfactory association areas of the cortex. PHAL anterograde
59
tracers indicate that the OV projects very densely and almost exclusively to the
FU (Dong et al., 2001). Outside the BST, the OV projects to the retrorubral area,
the caudal substantia innominata and the CeA (Dong et al., 2001). Very little is
known about the OV nucleus, however, much like the FU, the OV is suggested to
coordinate autonomic responses to disruptions in homeostasis as a result of
stress. Since the FU loops back to the OV (Dong & Swanson), the decrease in
neuronal activity in the FU may result in increased activity in the OV to maintain
endocrine homeostatic function in the presence of post-operative tissue damage.
4.5
Reflection and Caveats
The most evident setback to the current study was perhaps the use of c-
Fos as a marker for neuronal activation following neuropathic injury. It is clear
that as a noxious-acute neural marker, c-Fos is excellent to detect changes in
cell firing resulting from noxious insult (Jergova & Cizkova, 2005; Catheline et al.,
1999).
As a marker for neuronal activation following chronic nerve injury,
however, questions have been raised in regards to the temporal relationship
between pain behaviours and neuronal activation resulting from neuropathic
injury.
For example, Jergova and Cizkova (2005) observed a correlation
between c-Fos activation in the spinal cord following tactile allodynia and thermal
hyperalgesia 1 to 7 days following CCI, however when tested at days 7 and 14,
the incidence of c-Fos-IR did not correlate with noxious stimulation. Finally, at 28
days following CCI or sham surgery, c-Fos expression in the spinal cord was
similar in both subject groups, indicating that long-duration tracking of the c-Fos
60
protein as a nociceptive marker may not be sufficient to understand NP-induced
neuronal activity. Given that c-Fos is an early immediate gene, it may be
responsive to spinal and supraspinal modifications directly following neuropathic
injury, however other techniques should be employed to track the progression
and longer duration adaptations to neurological systems. Additionally, c-Fos is
only expressed in select populations of neurons therefore it may be an
incomplete marker for nociresponsive activity (Bullitt, 1990).
4.6
Future Directions
Future studies must be used to address two important issues. First, it is
imperative to understand what specific cell populations exist in the BST and how
these cell groups are affecting termination points. The FU consists of small to
medium sized oval-to-spindle shaped neurons (Ju & Swanson, 1989). However,
there is currently much controversy regarding cell populations in the FU. GAD65
immunolabeling reveals a small subpopulation of GABAergic neurons in this
region, however this labeling is minimal when compared to other posterior BST
nuclei (Choi et al., 2007).
Autoradiographic studies using [H3] D-aspartate
retrograde tracers injected into the PVH conclude that glutamatergic neurons
also inhabit areas of the anteroventral BST that include the FU nucleus (Csáki et
al., 2000). CRH neurons are the most abundant cell type in the FU.
Other
neuropeptide-releasing neurons such as substance P, neurotensin, galanin and
cholecystokinin are essentially absent in this nucleus but are found in higher
concentrations in surrounding BST nuclei (Ju et al., 1989).
61
To date, it is unclear as to whether neurons originating in the FU nucleus
that synapse at the PVH are excitatory or inhibitory in nature (Choi et al., 2007).
Considering what is known about cell populations originating in higher order
limbic structures, Choi and colleagues (2007) hypothesize that excitatory
glutamatergic signals are sent to the FU from the PFC while a combination of
excitatory CRH and inhibitory GABAergic signals are sent from the CeA. These
projections synapse at the FU and from there are transmitted to the PVH as
either an excitatory, glutamatergic input or an inhibitory GABAergic signal to
activate, inhibit or disinhibit activation of the HPA axis via the PVH.
Unfortunately, the current study cannot confirm or refute this hypothesis. Future
studies must be conducted using double-immunolabeling to determine exactly
what types of cell populations arrive at the BST as well as how those neurons
influence descending systems.
To study what types of cells are responsive to
noxious stimulation, a future study could combine immunolabeling for c-Fos with
GAD65 in an acute-noxious stimulation paradigm to elucidate if signals
originating in the FU, which project to the PVH, are excitatory or inhibitory in
nature.
Second, to address concerns regarding the functional modification of the
BST following CCI, an electrophysiological approach may be more conducive to
elucidate changes that occur over time to the FU and dAM nuclei. Following the
initiation of a CCI, extracellular electrodes should be implanted into these nuclei
daily to track changes in basal and noxious-stimulus evoked neuronal firing. This
future study would address two issues. First, it would allow us to validate the
62
results obtained from our c-Fos experiment, and secondly, it would give us a
clear representation of the synaptic reconfiguration of BST nuclei following
chronic neuropathic injury.
63
REFERENCES
Argiolas A & Gessa G.L (1991). Central functions of oxytocin. Neuroscience
and Biobehavioural Reviews, 15:217-231.
Argoff C.E (2007). The co-existence of neuropathic pain, sleep and psychiatric
disturbances. Clinical Journal of Pain, 23:15-22.
Barrot M, Olivier J.D.A., Perrotti L.I, DiLeone R.J, Berton O, Eisch A.J, Impey S,
Storm D.R, Neve R.L, Yin J.C, Zachariou V & Nestler E.J (2002). CREB
activity in the nucleus accumbens shell controls gating of behavioural
responses to emotional stimuli. PNAS, 99:11435-11440.
Bennet G. J & Xie Y.K (1988). A peripheral mononeuropathy in rat that produces
disorders of pain sensation like those seen in man. Pain, 33: 87-107.
Bereiter D.A, Bereiter D.F (1996). NMDA and non-NMDA receptor antagonism
reduces fos-like immunoreactivity in central trigeminal neurons after
corneal stimulation in the rat. Neuroscience, 73:249-258.
Bernard J.F, Alden M & Besson J.M (1993). The organization of the efferent
projections from the pontine parabrachial area to the amygdaloid complex:
a PHAL study in the rat. Journal of Comparative Neurology, 329:201-229.
Bland-Ward P.A & Humphrey P.P.A (1997). Acute nociception mediated by
hindpaw P2X receptor activation in the rat.
Brittish Journal of
Pharmacology, 122:365-371.
Bomholt S.F, Mikkelsen J.D & Blackburn-Munro G (2005). Normal hypothalamopituitary-adrenal axis function in a rat model of peripheral neuropathic
pain. Brain Research, 1044: 216-226.
Brain P.F & Haug M (1992). Hormonal and neurochemical correlates of various
forms of animal “aggression”. Psychoneuroendrocrinology, 17:537-551.
Braz J.M, Nassar M.A, Wood J.N & Basbaum A.I (2005). Parallel “pain”
pathways arise from subpopulations of primary afferent nociceptor.
Neuron, 47: 787-793.
Bridges D, Thompson S.W.N & Rice A.S.C (2001). Mechanisms of neuropathic
pain. British Journal of Anesthesia, 87: 12-26.
Briski K & Gillen E (2001). Differential distribution of fos expression within the rat
preoptic area and hypothalamus in response to physical vs. psychological
stress. Brain Research Bulletin, 55:401-408.
64
Bullitt E (1990). Expression of c-Fos-like protein as a marker for neuronal activity
following noxious stimulation in the rat.
Journal of Comparative
Neurology, 296: 517-530.
Burstein R & Potrebic S (1993). Retrograde labeling of neurons in the spinal cord
that project directly to the amygdala or the orbital cortex in the rat. Journal
of Comparative Neurology, 335: 469-485.
Burstein R, Cliffer K.D, Geisler G.J (1990). Cells of origin of the
spinohypothalamictract in the rat Journal of Comparative Neurology, 291:
329-344.
Bushnell M.C & Apkarian A.V (2006). Representation of pain in the brain. In:
Wall & Melzack’s Textbook of Pain. 5th Edition. pp: 107-124. London:
Elsevier Academic Press.
Bushnell M.C, Duncan G.H, Hofbauer R.K, Ha B, Chen J.I & Carrier B (1999).
Pain perception: is there a role for primary somatosensory cortex? PNAS,
96:7705-7709.
Canteras N.S & Swanson L.W (1992). Projections of the ventral subiculum to the
amygdala, septum and hypothalamus: a PHAL anterograde tract-tracing
study in the rat. Journal of Comparative Neurology, 324:195-212.
Carlton S.M & Coggeshall R.E (2001). Periperal capsaicin receptors increase in
the inflamed rat hindpaw: a possible mechanism for peripheral
sensitization. Neuroscience Letters, 310:53-56.
Casey K.L, Morrow T.J, Lorenz J & Minoshima S (2001). Temporal and spatial
dynamics of human forebrain activity during heat pain: analysis by
positron emission tomography. Journal of Neurophysiology, 85: 951-959.
Caterina M.J, Rosen T.A, Tominga M, Brake A.J & Julius D (1999). A capsaicinreceptor homologue with a high threshold for noxious heat. Nature, 398:
436-441.
Catheline G, Le Guen S, Honore P & Besson J.M (1999). Are there long term
changes in basal or evoked fos expression in the dorsal horn of the
mononeuropathic rat? Pain, 80: 347-357.
Chudasama Y & Robbins T.W (2003). Dissociable contributions of the
orbitofrontal and infralimbic cortex to pavlovian autoshaping and
discrimination reversal learning: further evidence for functional
heterogeneity of the rodent frontal cortex. Journal of Neuroscience,
24:8771-8780.
65
Chudler E.H, Anton F, Dubner R & Kenshalo (1990). Responses of nociceptive
SI neurons in monkeys and pain sensations in humans elicited by noxious
thermal stimulation: effect of interstimulus interval. Journal of
Neurophysiology, 63: 559-569.
Craig A.D (2003). Pain mechanisms: labeled lines versus convergence in central
processing. Annual Review of Neuroscience, 26: 1-30.
Craig K.D (2006). Emotions and psychobiology. In: Wall & Melzack’s Textbook
of Pain. 5th Edition. pp: 231-239. London: Elsevier Academic Press.
Csáki Á, Kocsis K, Halász B & Kiss J (2000).
Localization of
glutamatergic/aspartergic neurons projecting to the hypothalamic
paraventricular nucleus studied by retrograde transport of [H3] Daspartate autoradiography. Neuroscience, 101:637–655.
Cullinan W.E, Herman J.P & Watson S.J (1993). Ventral subicular interaction
with the hypothalamic paraventricular nucleus: evidence for a relay in the
bed nucleus of the stria terminalis. Journal of Comparative Neurology,
332:1-20.
Cullinan W.E, Herman J.P, Battalgia D.F, Akil H & Watson S.J (1995). Patten
and time course of immediate early gene expression in rat brain following
acute stress. Neuroscience, 64:477-505.
Delfs J.M, Zhu Y, Druhan J.P & Aston-Jones G (2000). Noradrenaline in the
ventral forebrain is critical for opiate withdrawal-induced aversion. Nature,
403: 430-434.
Devor M (2006). Sodium channels and mechanisms of neuropathic pain. Journal
of Pain, 7: s3-s12.
Deyama S, Nakagawa T, Kaneko S, Uehara T & Masabuni M (2007).
Involvement of the bed nucleus of the stria terminalis in the negative
affective component of visceral and somatic pain in rats. Behavioural
Brain Research, 176: 367-371.
Dick T.E & Coles S.K (2000). Ventrolateral pons mediates short-term depression
of respiratory frequency after brief hypoxia. Respiration Physiology, 121:
87-100.
Djouhri L & Lawson S.N (2004). Aβ-fibre nociceptive primary afferent neurons. A
review of incidence and properties in relation to other afferent A-fibre
neurons in mammals. Brain Research Reviews, 46: 131-145.
Djouhri L, Bleazard L & Lawson S.N (1998). Association of somatic action
66
potential shape with sensory receptive properties in guinea-pig dorsal root
ganglion neurones. Journal of Physiology, 513: 857-872.
Dong H.W & Swanson S.W (2004). Organization of axonal projections from the
anterolateral area of the bed nucleus of the stria terminalis. The Journal
of Comparative Neurology, 468: 277-298.
Dong H.W, Petrovich G.D & Swanson L.W (2000). Organization of projections
from the juxtacapsular nucleus of the BST: a PHAL study in the rat. Brain
Research, 859:1-14.
Dong H.W, Petrovich G.D, Watts A.G & Swanson S.W (2001). Basic organization
of projections from the oval and fusiform nuclei of the bed nucleus of the
stria terminalis in adult rat brain. The Journal of Comparative Neurology,
436: 430-455.
Dong H-W & Swanson L.W (2006). Projections from the bed nuclei of the stria
terminalis, anteromedial area: cerebral hemisphere integration of
neuroendocrine, autonomic, and behavioural aspects of energy balance.
The Journal of Comparative Neurology, 494:142-178.
Dostrovsky J.O & Craig A.D (2006). Ascending projection systems. In: Wall &
Melzack’s Textbook of Pain. 5th Edition. pp: 187-203. London: Elsevier
Academic Press.
Dumont E.C, Mark G.P, Mader S & Williams J.T (2005). Self-administration
enhances excitatory synaptic transmission in the bed nucleus of the stria
terminalis. Nature Neuroscience, 8: 413-414.
Forray M.I & Gysling K (2004). Role of noradrenergic projections to the bed
nucleus of the stria terminalis in the regulation of the hypothalamicpituitary-adrenal axis. Brain Research Reviews, 47:145-160.
Gilron I, Watson C.P, Cahill C.M & Moulin D.E (2006). Neuropathic pain: a
practical guide for the clinician. CMAJ, 175: 265-275.
Gold M.S, Reichling D.B, Shuster M.J & Levine D.J (1996). Hyperalgesic
agents increase a tetrodotoxin-resistant sodium current in nociceptors.
PNAS, 93: 1108-1112.
Gray T.S, Carney M.E & Magnuson D.J (1989). Direct projections from the
contral amygdaloid nucleus to the hypothalamic paraventricular nucleus:
possible
role
in
stress-induced
adrenocorticotropin
release.
Neuroedocrinology, 50:433-446.
Gruner W & Silva L.R (1994). Omega-conotoxin sensitivity and presynaptic
67
inhibition of glutaminergic sensory neurotransmission in vitro. Journal of
Neuroscience, 14: 2800-2808.
Herrera D.G & Robertson H.A (1996). Activatin of c-Fos in the brain. Progress
in Neurobiology, 50: 83-107.
Hunt S.P & Rossi J (1985). Peptide- and non-peptide-containing unmyelinated
primary afferents: The parallel processing of nociceptive information.
Philosophical Transactions of the Royal Society of London. Series B,
Biological Sciences, 308: 283-289.
Hurley K.M, Herbert H, Moga M.M & Saper C.B (1991). Efferent projections of
the infralimbic cortex of the rat. Journal of Comparative Neurology,
308:249-276.
International Association for the Study of Pain Press, 1994.
Jergova S & Cizkova D (2005). Long-term changes in c-Fos expression in the
rat spinal cord following chronic constriction injury. European Journal of
Pain, 9: 345-354.
Johansen J.P & Fields H.L (2004). Glutamatergic activation of anterior cingulate
cortex produces and aversive teaching signal. Nature Neuroscience, 7:
398-403.
Johansen J.P, Fields H.L & Manning B.H (2001). The affective component of
pain in rodents: direct evidence for a contribution of the anterior cingulate
cortex. PNAS, 98: 8077-8082.
Ju G & Swanson L.W (1989). Studies on the cellular architecture of the bed
nuclei of the stria terminalis in the rat. I. cytoarchitecture. Journal of
Comparative Neurology, 280:587-602.
Ju G, Swanson L.W & Simerly R.B (1989). Studies on the cellular architecture of
the bed nuclei of the stria terminalis in the rat. II. chemoarchitecture.
Journal of Comparative Neurology, 280:603-621.
Ju G, Swanson L.W, Simerly R.B (1989). Studies on the cellular architecture of
the bed nucleus of the stria terminalis in the rat: II. chemoarchitecture.
The Journal of Comparative Neurology, 280: 603-621.
Julius D & McCleskey E.W (2006). Cellular and molecular properties of primary
afferent neurons. In: Wall & Melzack’s Textbook of Pain. 5th Edition. pp:
35-48. London: Elsevier Academic Press.
Kalin N.H, Shelton S.E & Davidson R.J (2004). The role of the central nucleus of
68
the amygdala in mediating fear and anxiety in the primate. The Journal of
Neuroscience, 24: 5506-5515.
Karakiewicz P.I, Perrotte P, Valiquette L, Benard F, McCormack M, Menared C,
McNaughton Collins M & Nickel JC (2005). French-Canadian linguistic
validation of the NIH Chronic Prostatitis Symptom Index. Can J Urol,12:
2816-2823.
Kovacs K.J (1998). C-Fos as a transcription factor: a stressful (re)view from a
functional map. Neurochemistry International, 33:287-297.
Lawson S.N, Crepps B & Perl E.R (2002). Calcitonin gene related peptide
immunoreactivity and afferent receptive properties of dorsal root ganglion
neurons in guinea-pigs. Journal of Physiology, 540: 989-1002.
Levin R & Meston C (2006). Nipple/breast stimulation and sexual arousal in
young men and women. Journal of Sexual Medicine, 3:450-454.
Lewis T (1935). Experiments relating to cutaneous hyperalgesia and its spread
through somatic fibres. Clinical Science, 2: 373-423.
Lue J.H, Leong S.M, Day A.S, Tsai Y.J, Shieh J.Y & Wen C.Y (2002). Changes
in c-Fos protein expression in the rat cuneate nucleus after electrical
stimulation of the transected medial nerve. Journal of Neurotrauma,
19:897-907.
Lui C & Walker J.M (2006). Effects of a canabinoid agonist on spinal nociceptive
neurons in a rodent model of neuropathic pain.
Journal of
Neurophysiology, 96:2284-2294.
Mackel R, Iriki A, Brink E.E (1992). Spinal input to thalamic VL neurons:
Evidence for direct spinothalamic effects. Journal of Neurophysiology, 67:
132-144.
Malcangio M, Ramer M.S, Jones M.G & McMahon S.B (2000). Abnormal
substance P release from the spinal cord following injury to primary
sensory neurons. European Journal of Neuroscience, 12: 397-399.
Markakis E.A & Swanson L.W (1997). Spatiotemporal patterns of secretomotor
neuron generation in the parvicellular neuroendocrine system. Brain
Research Review, 24:255-291.
Max M.B, Schafer S.C & Culnane M (1988). Association of pain relief with drug
side effects in postherpetic neuralgia: a single-dose study of clonidine,
codeine, ibuprofen and placebo. Clinical Pharmacology and Therapeutics,
43:363-371.
69
McDonald A.J (1983). Neurons of the bed nucleus of the stria terminalis: a golgi
study in the rat. Brain Res. Bull., 10:111-120.
McDonald A.J (1998). Cortical pathways to the mammalian amygdala.
Progressive Neurobiology, 55:257-332.
McQuay H.J, Tramer M, Nye B.A, Carroll D, Wiffen P.J & Moore R.A (1996). A
systematic review of antidepressants in neuropathic pain. Pain, 68:217227.
Meana M, Cho R & DesMeules M (2003). Chronic pain- the extra burden on
Canadian women. Women’s Health Surveillance Report. 1-19.
Mestre L, et al (2005). Pharmacological modulation of the endocannabinoid
system in a viral model of MS. J. Neurochem, 92: 1327-1339.
Meyer R.A, Ringkamp M, Campbell J.N & Raja S.N (2006). Peripheral
mechanisms of cutaneous nociception. In: Wall & Melzack’s Textbook of
Pain. 5th Edition. pp: 3-34. London: Elsevier Academic Press.
Millecamps M, Centeno M.V, Berra H.H, Rudick C.N, Lavarello S, Tkatch T &
Apkarian A.V (2007). D-Cycloserine reduces neuropathic pain behaviour
through limbic NMDA-mediated circuitry. Pain, [Epub ahead of print].
Mizumura K, Minagawa M, Koda H (1995). Influence of histamine on the
bradykinin response of canine testicular polymodal receptors in vitro.
Inflammation Research, 44:376-378.
Moga M.M, Weis R.P & Moore R.Y (1995). Efferent projections of the
paraventricular thalamic nucleus in the rat. Journal of Comparative
Neurology, 359:221-238.
Morgan J.I & Curran T (1986). Role of ion flux in the control of c-Fos expression.
Nature, 322: 552-555.
Moulin D.E, Clark A.J, Gilron I, Ware M.A, Watson C.P, Sessle B.J, Coderre T,
Morley-Forster P.K, Stinson J, Boulanger A, Peng P, Finley G.A, Taezner
P, Squire P, Dion D, Cholkan A, Gilani A, Gordon A, Henry J, Jovey R,
Lynch M, Mailis-Gagnon A, Panju A, Rollman G.B, Velly A, Canadian Pain
Society (2007). Pharmacological management of chronic neuropathic
pain – consensus statement and guidelines from the Canadian Pain
Society. Pain Research and Management, 12:13-21.
Nagy J.I, Iversen L.L, Godert M, Chapman D & Hunt S.P (1983). Dose
70
dependent effects of capsaicin on primary sensory neurons in the
neonatal rat. Journal of Neuroscience, 3: 399-406.
Narita M, Ozaki S, Narita M, Ise Y, Yajima Y & Suzuki T (2003). Change in the
expression of c-Fos in the rat brain following sciatic nerve ligation.
Neuroscience Letters, 352: 231-233.
Nashold B.S, Wilson W.P & Slaughter D.G (1969). Sensations evoked by
stimulation in the midbrain of man. Journal of Neurosurgery, 30: 14-24.
Neugebauer V, Li W, Bird G.C & Han J.S (2004). The amygdala and persistent
pain. The Neuroscientist, 10: 221-234.
Ohara P.T, Vit J.P & Jasmin L (2005). Cortical modulation of pain. Cellular and
Molecular Life Sciences, 62: 44-52.
Ottersen O.P (1982). Connections of the amygdala of the rat: corticoamygdaloid
and intraamygdaloid connections as studied with axonal transport of
horseradish peroxidase. Journal of Comparative Neurology, 205:30-48.
Pinto M, Deolinda L, Castro-Lopes J & Tavares I (2003). Noxious-evoked c-Fos
expression in brainstem neurons immunoreactive for GABAB, mu-opioid
and NK-1 receptors. European Journal of Neuroscience, 17: 1393-1402.
Ploner M, Freundt H.J & Schnitzler A (1999). Pain affect without pain sensation
in a patient with postcentral lesion. Pain, 81: 211-214.
Prewitt C.M & Herman J.P (1998). Anatomical interactions between they central
amygdaloid nucleus and the hypothalamic paraventricular nucleus of the
rat: a dual tract-tracing analysis. Chemical Neuroanatomy, 15:173-185.
Radley J.J & Morrison J.H (2005). Repeated stress and structural plasticity in
the brain. Ageing research Reviews, 4:271-287.
Rainville P, Duncan G.H, Price D.D, Carrier B & Bushnell M.C (1997). Pain
affect encoded in human anterior cingulate but not somatosensory cortex.
Science, 277: 968-971.
Rausell E & Jones E.G (1991). Chemically distinct compartments of the thalamic
VPM nucleus in monkeys relay principal and spinal trigeminal pathways to
different layers of the somatosensory cortex. Journal of Neuroscience, 11:
226-237.
Ray J.P & Price J.L (1993). The organization of projections from the
71
medialdorsal nucleus of the thalamus to orbital and medial prefrontal
cortex in macaque monkeys. Journal of Comparative Neurology. 337: 131.
Reynolds D.V (1969). Surgery in the rat during electrical analgesia induced by
focal brain stimulation. Science, 164: 444-445.
Risold P.Y & Swanson L.W (1997). Connections of the rat lateral septal
complex. Brain Research Review, 24:115-195.
Romano J.M & Turner J.A (1985). Chronic pain and depression: does the
evidence support a relationship? Psychological Bulletin, 97:18-34.
Rossi F, Maione S & Berrino L (1994). Periaqueductal gray area and
cardiovascular function. Pharmacological Research, 29: 27-36.
Sadikot A.F, Parent A, François C (1992). Efferent connections from the
centromedian and parafascicular thalamic nuclei in the squirrel monkey: a
PHA-L study of subcortical projections. Journal of Comparative Neurology,
315: 137-159.
Sapolsky R.M, Romero L.M & Munck A.U (2000). How do glucocorticoids
influence stress responses? Integrating permissive, suppressive,
stimulatory and preparative actions. Endocrine Reviews, 21:55-89.
Satoh K & Fibiger H.C (1986). Cholinergic neurons of the laterodorsal tegmental
nucleus: efferent and afferent connections. Journal of Comparative
Neurology, 253:277-302.
Sawchenko P.E, Swanson L.W, Rivier J & Vale W.W (1985). The distribution of
growth-hormone-releasing factor (GRF) immunoreactivity in the central
nervous system of the rat: an immunohistochemical study using antisera
directed against rat hypothalamic GRF.
Journal of Comparative
Neurology, 237:100-115.
Schmidt R, Schmelz M, Matthias R, Harmann H.O & Torebjork H.E (1997).
Innervation territories of mechanically activated C nociceptor units in
human skin. Journal of Neurophysiology, 78: 2641-2648.
Silberstein S.D (2003). Neurotoxins in the neurobiology of pain. Headache: The
Journal of Head and Face Pain, 43: 2-8.
Simone D.A, Alreja M & LaMotte R.H (1991). Psychophysical studies of the itch
sensation and itchy skin (‘alloknesis’) produced by intracutaneous injection
of histamine. Somatosensory and Motor Research, 8:271-279.
72
Smith W.J, Stewart J & Pfaus J.G (1997). Tail pinch induces fos
immunoreactivity within several regions of the male rat brain: effects of
age. Physiology and Behaviour, 61: 717-723.
Strobl J.S & Thomas M.J (1994). Human growth hormone. Pharmacological
Review, 46:1-34.
Sugiura T, Tominaga M, Kaysuya H & Mizumura K (2002). Bradykinin lowers the
threshold temperature for heat activation of vanilloid receptor 1. Journal of
Neurophysiology, 88:544-548.
Sun N, Roberts L & Cassell M.D (1991). Rat central amygdaloid nucleus
projections to the bed nucleus of the stria terminalis. Brain Research
Bulletin, 27:651-662.
Swanson L.W (2005). Brain Maps: Structure of the rat brain. San Diego:
Elesvier.
Tadesse A, Nah S.Y & McCleskey E.W (1995). Selective opioid inhibition of
small nociceptive neurons. Science, 270: 1366-1369.
Ulrich-Lai Y.M, Xie W, Meij J.T.A, Dolgas C.M, Yu L & Herman J.P (2006).
Limbic and HPA axis function in an animal model of chronic neuropathic
pain. Physiology & Behaviour, 88:67-76.
Unvas-Moberg K (1998). Oxytocin may mediate the benefits of positive social
interations and emotions. Psychoneuroendocrinology, 23:819-835.
Van Der Plas J, Wiersigna-Post J.E.C, Maes F.W & Bohus B (1995).
Cardiovascular effects and changes in midbrain periaqueductal grey
neuronal activity induced by electrical stimulation of the hypothalamus in
rat. Brain Research Bulletin, 37: 645-656.
Vertes R.P (1991). A PHA-L analysis of ascending projections of the dorsal
raphe nucleus in the rat. Journal of Comparative Neurology, 313:643-668.
Vissers K, Adriaensen H, De Coster R, De Deyna C & Meert T.F (2003). A
chronic-constriction injury of the sciatic nerve reduces bilaterally the
responsiveness to formalin in rats: a behavioural and hormonal evaluation.
Anesth Analg, 97:520-525.
Willis W.D & Coggelshall R.E (1991). Sensory Mechanisms of the Spinal Cord,
2nd Ed. New York: Plenum Press.
Willis W.D & Westlund K.N (1997). Neuroanatomy of the pain system and the
73
pathways that modulate pain. Journal of Clinical Neurophysiology, 14: 231.
Zhou Q, Imbe H, Dubner R & Ren K (1999). Persistent fos protein expression
after orofacial deep or cutaneous tissue inflammation in rats: implications
for persistent orofacial pain. Journal of Comparative Neurology, 412: 276291.
74