University of Iowa
Iowa Research Online
Theses and Dissertations
2013
Elucidating mechanisms by which substance P in
the RVM contributes to the maintenance of pain
following inflammatory injury
Uche Patrick Maduka
University of Iowa
Copyright 2013 Uche Patrick Maduka
This dissertation is available at Iowa Research Online: http://ir.uiowa.edu/etd/5019
Recommended Citation
Maduka, Uche Patrick. "Elucidating mechanisms by which substance P in the RVM contributes to the maintenance of pain following
inflammatory injury." PhD (Doctor of Philosophy) thesis, University of Iowa, 2013.
http://ir.uiowa.edu/etd/5019.
Follow this and additional works at: http://ir.uiowa.edu/etd
Part of the Pharmacology Commons
ELUCIDATING MECHANISMS BY WHICH SUBSTANCE P IN THE RVM
CONTRIBUTES TO THE MAINTENANCE OF PAIN FOLLOWING
INFLAMMATORY INJURY
by
Uche Patrick Maduka
A thesis submitted in partial fulfillment
of the requirements for the
Doctor of Philosophy degree in Pharmacology
in the Graduate College of
The University of Iowa
December 2013
Thesis Supervisor: Professor Donna L. Hammond
Copyright by
UCHE PATRICK MADUKA
2013
All Rights Reserved
Graduate College
The University of Iowa
Iowa City, Iowa
CERTIFICATE OF APPROVAL
_______________________
PH.D. THESIS
_______________
This is to certify that the Ph.D. thesis of
Uche Patrick Maduka
has been approved by the Examining Committee
for the thesis requirement for the Doctor of Philosophy
degree in Pharmacology at the December 2013 graduation.
Thesis Committee: ___________________________________
Donna L. Hammond, Thesis Supervisor
___________________________________
D.P. Mohapatra
___________________________________
Andrew F. Russo
___________________________________
Kathleen A. Sluka
___________________________________
Yuriy M. Usachev
To my family, for the unending support and encouragement I have received
ii
You may encounter many defeats, but you must not be defeated. In fact, it may be
necessary to encounter the defeats, so you can know who you are, what you can rise
from, how you can still come out of it.
-Maya Angelou
iii
ACKNOWLEDGMENTS
When I think of the journey that has led to my being able to complete my doctoral
thesis, I cannot help but be grateful to God. The significance of this opportunity is not
lost on me, especially knowing that I once attended school in a cement brick structure
with a rusted corrugated tin roof and holes in the walls for windows. Where I come from,
not many finish secondary school and fewer still earn any university degree, much less a
doctorate from a world class university. I am truly grateful to have been afforded this
opportunity.
I had the good fortune of completing my thesis research in the company of many
wonderful people. Foremost among them was my mentor, Dr. Donna Hammond, who
was gracious enough to let me join her lab when my original thesis supervisor left the
university. While I have learned a lot from Donna about the rigors of thoroughly testing a
hypothesis, some of the lasting lessons came beyond the bench. I had a front row seat to
watch Donna lead effectively and contribute to the university community, make decisions
based on what was right as opposed to what was convenient, stop to mentor and guide
everyone from fellow students to faculty members and all the while treat people with
respect. To put it succinctly, Donna has been an excellent role model both within and
beyond the laboratory.
Every day in the lab, I worked side by side with Stephanie, Marta, Anne-Sophie,
Frank, Blanca, Roxanne, Chris and Ann Marie Van de Walle Jones. My interactions with
this wonderful group made the long hours seem shorter and they helped cushion the
bumps along the way. I’d like to thank Steph for assistance with various technical aspects
of the project and being the ultimate Solver. I’d also like to thank Marta for being a
iv
willing teacher as we worked to unravel the secrets of substance P and NK1 in the RVM.
I’m indebted to Anne-Sophie for lightening the mood and Frank for being patient with
my million questions about medical school. I’m thankful to Blanca for our many
conversations on the 4th floor island, and to Roxanne for the experience and input she
provided to the project. I am grateful to Chris and Ann for the hundreds to tubes of buffer.
I am also grateful to Drs. Mohapatra, Sluka, Russo and Usachev for serving on my thesis
committee and proving meaningful input as my project progressed.
Beyond the lab, my wife Sara has been a great support, always willing to do
whatever she could to ease my load. Whether I had to work at home in the evenings, or
spend part of the weekend in the lab, never once was she anything but supportive. I was
able to get up every day rested and ready to face new challenges, in large part to the time
I spent with her. I am also grateful to my parents, who despite the physical distance,
always let me know I was in their thoughts and prayers. Their confidence in me, even
before I gave them any reason to believe, emboldened me to dream and hope. I am
especially grateful for their dedication and their sacrifices that paved the way for me to
pursue my goals. I am grateful to my siblings Iheanyi, Liz, Chimezie, Kelechi and
Chimdi for support over the last years, especially Iheanyi for setting a great example and
encouraging the rest of us to follow. I am also grateful to the Van Hemerts, especially
Gary, Mary Beth, John, Hanna and Ben, and the Van Ettens for providing a much
welcome respite from the rigors of graduate work. I am also grateful to friends and well
wishers near and far, especially Bishop U.U. Nmeregini of Item Diocese in Nigeria for
prayers. I am thankful to many teachers and professors who pushed me and encouraged
me not to settle. For all of this and so much more, I am truly grateful.
v
ABSTRACT
Chronic pain is a major healthcare concern that directly affects over one hundred
million people in the United States alone. While current treatment options like opioids
and NSAIDs are effective, they are with significant drawbacks that prevent long term use.
It is important to identify and understand new druggable targets for the treatment of pain.
Recent findings have demonstrated substance P functions in the RVM to maintain
hypersensitivity to noxious heat stimuli in models of persistent peripheral inflammatory
injury in a manner dependent on presynaptic NMDA receptors. What remains unclear is
how substance P assumes this pronociceptive role following peripheral inflammatory
injury. The experiments detailed in this thesis investigated whether the levels and or
release of substance P in the RVM was altered following peripheral inflammatory injury.
The effect of peripheral inflammatory injury on levels of substance P in the RVM
was tested at several time points. The data show that there were no changes in substance
P levels in the ipsilateral or contralateral RVM of CFA injected rats compared to their
saline controls at any of the time points tested. To assess whether changes in substance P
levels occurred in a subset of neurons within the RVM, computer aided densitometry
analysis was used to measure substance P immunoreactivity in sections from the RVM of
rats treated with CFA or saline. Substance P immunoreactivity was increased in the
ipsilateral RVM of the CFA group compared to the corresponding saline sections at the 4
day, but not the 2 week time point. No other changes were observed.
Electron microscopy was used to demonstrate the presence of the NMDA receptor
and substance P on the same axon terminals within the RVMs of rats treated with either
CFA or saline. This colocalization is significant because it identifies NMDA receptors in
vi
position to regulate the release of substance P from axon terminals in the RVM. There
were no obvious differences in the degree of colocalization between CFA and saline
groups. Functional experiments were devised that tested whether substance P release
(basal and evoked) in the RVM was increased following peripheral inflammatory injury,
and whether said release was regulated by NMDA receptors. The data show that neither
basal nor evoked (potassium or veratridine) release was increased following peripheral
inflammatory injury. NMDA was able to facilitate the release of substance P in both the
CFA and saline treatment groups, but the facilitation was not different between groups. In
the absence of any depolarization stimulus, NMDA was unable to elicit any release of
substance P beyond basal values.
All told, the data show substance P levels in the RVM are not altered by
peripheral inflammatory injury. Additionally, neither basal nor evoked release of
substance P is altered by peripheral inflammatory injury. The data provide functional and
anatomical evidence for modulation of substance P release by glutamate acting at
presynaptic NMDA receptors, but do not support the idea of differential modulation of
substance P release following peripheral inflammatory injury.
vii
TABLE OF CONTENTS
LIST OF FIGURES ........................................................................................................... xi
LIST OF ABBREVIATIONS .......................................................................................... xiii
CHAPTER
I.
INTRODUCTION ............................................................................................1
Pain as a healthcare problem .....................................................................1
Biosynthesis and Metabolism ....................................................................2
Substance P Distribution within the nervous system ................................3
NK1 Receptor Distribution in the CNS .....................................................5
Substance P signaling through the NK1 receptor ......................................6
Substance P as a target for pain; Empirical evidence ................................7
RVM as a major player in modulation of pain: Inhibition ......................12
RVM as a major player in modulation of pain: Facilitation ....................13
Substance P in the RVM..........................................................................14
Interplay between substance P and NMDA receptors .............................16
Hypothesis and study design ...................................................................18
II.
INVESTIGATING THE EFFECT OF UNILATERAL CFAINDUCED INFLAMMATORY INJURY ON SUBSTANCE P
LEVELS OF THE RVM, DRG AND SPINAL CORD OF THE RAT
DORSAL HORN ............................................................................................23
Abstract....................................................................................................23
Introduction .............................................................................................24
Materials and Methods ............................................................................26
Animals ..........................................................................................26
Model of hind paw inflammatory pain ..........................................27
Assessment of localized inflammatory hypersensitivity................27
Tissue Collection for Substance P Level analysis .........................27
Measurement of substance P Levels ..............................................28
Immunohistochemistry ..................................................................29
Quantification of RVM substance P immunoreactivity .................31
Statistical Analysis .........................................................................32
Results .....................................................................................................32
Characterization of CFA induced inflammatory injury .................32
Substance P Levels ........................................................................33
Quantification of RVM substance P Immunoreactivity 4 days
and 2 weeks after peripheral injury ...............................................34
Discussion................................................................................................34
Findings for the RVM ....................................................................35
Findings for the DRG.....................................................................37
viii
Findings for the dorsal horn ...........................................................38
Summary ........................................................................................39
III.
ULTRASTRUCTURAL IDENTIFICATION OF AXON TERMINALS
CONTAINING BOTH NMDA RECEPTORS AND SUBSTANCE P .........50
Abstract ....................................................................................................50
Introduction..............................................................................................51
Materials and Methods ............................................................................53
Animals ..........................................................................................53
Model of hind paw inflammatory pain ..........................................54
Tissue Preparation ..........................................................................54
Western Blotting ............................................................................58
Knockdown of GluN1 in the RVM ................................................59
Antibody Characterization .............................................................60
Results..…................................................................................................61
Characterization of Substance P antibody .....................................61
Characterization of the GluN1 primary antibody ..........................61
Ultrastructural Morphology of the RVM .......................................62
Ultrastructural GluN1 labeling .....................................................64
Ultrastructural Substance P labeling ..............................................64
GluN1 and substance P colocalization in both CFA and
saline-treated rats ...........................................................................65
Discussion ................................................................................................65
Technical Considerations ...............................................................66
Summary ........................................................................................68
IV.
RELEASE OF SUBSTANCE P IN THE RAT RVM: MODULATION BY
PERIPHERAL INFLAMMATORY INJURY AND PRESYNAPTIC NMDA
RECEPTORS………………… ………………………………..………….. 81
Abstract ....................................................................................................81
Introduction..............................................................................................83
Materials and Methods ............................................................................85
Animals ..........................................................................................85
Model of hind paw inflammatory pain ..........................................86
Substance P Release from RVM Tissue Prisms ............................86
Drugs and Depolarization Buffers .................................................87
Prism Protein Quantification..........................................................88
Experimental Design ......................................................................89
Statistical Analysis .........................................................................89
Results…..................................................................................................90
Evoked, but not Basal Release is Calcium-Dependent ..................90
Basal Release of Substance P is Similar in Saline and CFAtreated Rats.....................................................................................91
Potassium Evoked Release of Substance P is Similar in
RVM Prisms of Saline and CFA-treated Rats ...............................91
NMDA Alone Does Not Evoke Substance P Release in the
Absence of Depolarization .............................................................91
NMDA Facilitates Evoked Release of Substance P from
Prisms of Saline- and CFA-treated Rats ........................................92
Veratridine Evoked substance P Release .......................................94
ix
Discussion ................................................................................................95
Basal Release .................................................................................95
Evoked Release ..............................................................................95
NMDA Mediated Facilitation of Substance P release ...................97
Sufficient Obligate Co-Agonist .....................................................99
Conclusions ....................................................................................99
V.
CONCLUSIONS ..........................................................................................113
Substance P Levels within the RVM following peripheral
Inflammatory Injury .....................................................................113
Colocalization of NMDA receptors and substance P in the
same presynaptic terminals ..........................................................114
Substance P release (basal and evoked) and its regulation by
NMDA receptors ..........................................................................115
Present Findings relative to NK1 antagonist failures as
analgesics .....................................................................................117
Disclosure Regarding Kits used to Measure Substance P ...........118
Concluding Remarks ....................................................................119
REFERENCES……………………………………………………………................120
x
LIST OF FIGURES
Figure
1.
Hypothesis Schematic 1 ............................................................................................21
2.
Hypothesis Schematic 2 ...........................................................................................22
3.
Tissue Analysis Schematic ......................................................................................40
4.
Representative image of substance P in the RVM and corresponding binary
image .........................................................................................................................41
5.
Characterization of CFA-induced heat hyperalgesia 4 hours, 4 days and 2
weeks after intraplantar injection. .............................................................................42
6.
Characterization of CFA-induced inflammation 4 hours, 4 days and 2 weeks
after intraplantar injection .........................................................................................43
7.
Time course of substance P levels in the RVM ipsilateral (A) and contralateral
(B) to peripheral inflammatory injury ......................................................................44
8.
Time course of substance P levels in the DRG ipsilateral (A) and contralateral
(B) to peripheral inflammatory injury ......................................................................45
9.
Time course of substance P levels in the dorsal horn ipsilateral (A) and
contralateral (B) to peripheral inflammatory injury. ................................................46
10.
Representative photomicrographs of substance P immunoreactivity in the
RVM 4 days after intra-plantar saline (A) or CFA (B) ............................................47
11.
Representative images of substance P immunoreactivity in the RVM 2 weeks
after intra-plantar saline (A) or CFA (B). .................................................................48
12.
Quantification of substance P Immunoreactivity in the RVM 4 days (A) and 2
weeks (B) following intra-plantar CFA or saline .....................................................49
13.
Representative photomicrographs of substance P immunolabeling in the
diencephalon of (A,B) a wildtype mouse and (C) tac1 null mouse. .........................69
14.
Representative photomicrographs of GluN1 immunoreactivity in the (A,C,D)
RVM and (B) facial motor nucleus...........................................................................70
15.
Western blots labeled with the GluN1 antibodies. ...................................................71
16.
Representative high magnification photomicrographs of GluN1
immunoreactivity in the RVM of rats microinjected with FIV-miRNA to
knock down GluN1 or empty vector virus................................................................72
17.
General morphology of the RVM at the ultrastructural level ...................................73
18.
Electron micrographs of symmetrical and asymmetrical synapses within the
RVM. ........................................................................................................................74
xi
19.
Examples of GluN1 labeling with silver enhanced 6 nm gold particles in axon
terminals within the RVM. .......................................................................................75
20.
Examples of post-synaptic GluN1 labeling with gold particles within the
RVM. ........................................................................................................................76
21.
GluN1 labeling on or around mitochondria within the RVM ...................................77
22.
Substance P labeling within the RVM. .....................................................................78
23.
GluN1 and substance P immunoreactivity colocalize in axon terminals in the
RVM of CFA treated rats ..........................................................................................79
24.
GluN1 and substance P immunolabeling colocalize to axon terminals in the
RVM of SALINE treated rats ....................................................................................80
25.
Evoked, but not basal release of substance P is calcium dependent .......................101
26.
Substance P release in response to 50 mM K+ Krebs Ringer is similar in
prisms from saline and CFA treated rats ................................................................102
27.
10 µM NMDA alone does not increase release of Substance P from RVM
prisms of saline or CFA treated rats .......................................................................103
28.
100 µM NMDA alone does not increase release of Substance P from RVM
prisms of saline or CFA treated rats .......................................................................104
29.
Addition of D-Serine does not enable either 10 or 100 µM NMDA to release
Substance P from RVM prisms of naive rats ..........................................................105
30.
Addition of 10 µM NMDA increases substance P release evoked by 50 mM
K+ Krebs Ringer from RVM prisms from saline and CFA treated rats ..................106
31.
Substance P release in response to 25 mM K+ Krebs Ringer is different in
prisms from saline and CFA treated rats ................................................................107
32.
Addition of 10 µM NMDA increases substance P release evoked by 25 mM
K+ Krebs Ringer from RVM prisms from saline and CFA treated rats ..................108
33.
Addition of 100 µM NMDA increases substance P release evoked by 25 mM
K+ Krebs Ringer from RVM prisms from saline, but not CFA treated rats ...........109
34.
Substance P measured from RVM prism releasates depolarized with 25 mM
K+ Krebs Ringer, 10 µM D-Serine and 10 µM NMDA during period 3 ................110
35.
10 µM NMDA facilitates substance P release induced by 50 µM veratridine
in RVM prisms from saline, but not CFA treated rats ............................................111
36.
10 µM NMDA facilitates substance P release induced by 25 µM veratridine
in RVM prisms from saline, but not CFA treated rats ............................................112
xii
LIST OF ABBREVIATIONS
ABC
Avidin Biotin Complex
ACC
Anterior Cingulate Cortex
ACE
Angiotensin 1 Converting Enzyme
AMPA
(2-amino-3-(3-hydroxy-5-methyl-isoxazol-4-yl)propanoic acid)
ANOVA
Analysis of Variance
BCA
Bicinchoninic Acid
CaCl
Calcium Chloride
CaMKII
Calcium Calmodulin Dependent Protein Kinase II
CFA
Complete Freund’s Adjuvant
CGRP
Calcitonin Gene Related Peptide
CNS
Central Nervous System
DAB
Diaminobenzidine
DRG
Dorsal Root Ganglion (Ganglia)
DTT
Dithiothreitol
ECL
Enhanced Chemiluminescence
EDTA
Ethylenediaminetetraacetic Acid
EIA
Enzyme Immuno-Assay
xiii
FIV
Feline Immunodeficiency Virus
GABA
Gamma-Aminobutyric Acid
GFP
Green Fluorescent Protein
GluN1
NMDA receptor subunit 1
GluN2B
NMDA receptor subunit 2B
GPCR
G-protein Coupled Receptor
HK-1
Hemokinin-1
HRP
Horse Radish Peroxidase
IHC
Immunohistochemistry
KCl
Potassium Chloride
kDa
Kilodaltons
LDS
Lithium Dodecyl Sulfate
MgCl
Magnesium Chloride
miRNA
Micro-Ribonucleic Acid
mRNA
Messenger Ribonucleic Acid
NaCl
Sodium Chloride
NaHCO3
Sodium Bicarbonate
xiv
NaH2PO4
Monosodium Phosphate
NEP
Neutral Endopeptidase
NGC
Nucleus Gigantocellularis
NK1
Neurokinin-1 Receptor
NK2
Neurokinin-2 Receptor
NK3
Neurokinin-3 Receptor
NMDA
N-methyl-D-Aspartate
NP-γ
Neuropeptide Gamma
NRM
Nucleus Raphe Magnus
NSAIDs
Non-Steroidal Anti-Inflammatory Drugs
OCT
Optimum Cutting Temperature
PAG
Periaqueductal Gray
PKC
Protein Kinase C
PPT-A
Preprotachykinin-A Gene
PVDF
Polyvinylidene Difluoride
PWL
Paw Withdrawal Latency
RVM
Rostral Ventromedial Medulla
xv
SEM
Standard Error of the Mean
SM-SP
(Sar9,Met(O2)11)-Substance P
TBS-T
Tris Buffered Saline - Tween
USP
United States Pharmacopeia
WDR
Wide Dynamic Range
xvi
1
CHAPTER I
INTRODUCTION
Pain as a healthcare problem
Chronic pain is a significant public health challenge, affecting at least 100 million
people in the United States (US) and an estimated 1.5 billion people worldwide. A study
commissioned by the US Department of Health and Human Services estimated the annual
economic toll of chronic pain to be as much as $635 billion in the US alone in direct
medical costs and lost productivity [1]. Beyond its myriad physical burdens, chronic pain
also exerts an emotional and psychological toll on patients, as evidenced by the increased
comorbidity of chronic pain and various other psychological phenomena like depression
and anxiety, among others [2, 3]. Unfortunately, management of chronic pain is likely to
become even more important. As a result of advances in medicine, an ever increasing
percentage of the population is comprised of elderly individuals (a segment most likely to
suffer from chronic pain). The obesity epidemic is associated with the proliferation of
diseases like diabetes, of which neuropathic pain is a major comorbidity. It is therefore
imperative to find new treatments for and achieve a greater understanding of the
mechanistic bases for chronic pain.
The pain signaling system exists to warn of potential or actual tissue damage.
When this system goes awry, the perception of pain can persist in the absence of potential
or actual tissue damage. This pain state can last for months and years beyond the initial
injury, the very definition of chronic pain. Treatments for chronic pain vary depending on
pain severity, ranging from acetaminophen and non-steroidal antiinflammatory drugs
(NSAIDs) for mild and moderate pain to opioids for severe pain conditions. While
2
effective, these drugs are not without their drawbacks. Prolonged use of NSAIDs can
lead to ulcers and other gastrointestinal problems. Opioids have a high probability of
abuse and their efficacy may decrease upon prolonged use. Over the last several decades,
research efforts have focused on identifying novel non-opioid or non-NSAID “druggable
targets” for pain relief. One of the earliest identified was the 11 amino acid peptide,
substance P and its receptor, neurokinin-1 (NK1).
Biosynthesis and Metabolism
Substance P is a member of a group of neuroactive peptides known as tachykinins.
First isolated as an unknown substance from horse intestines and found to produce
vasodilation after intravenous administration to a rabbit [4], substance P was
subsequently purified as a powdery substance [5]; hence its name: substance P. The
amino acid sequence of substance P was determined to be Arg-Pro-Lys-Pro-Gln-GlnPhe-Phe-Gly-Leu-Met. As such, it is a member of the tachykinin family of peptides, all
of which share a common carboxy terminal sequence of Phe-X-Gly-Leu-Met, where the
X residue is either a Phe or Val. [6, 7]. Substance P is coded for by a single gene,
preprotachykinin-A (PPT-A). The same gene also codes for neuropeptide gamma (NPγ),
neurokinin A and neuropeptide K, among others [8, 9]. As a result of alternative splicing,
this gene encodes three separate transcription products, αPPT-A, βPPT-A and γPPT-A,
all of which can yield substance P and other tachykinins through a series of enzymatic
cleavage steps [9]. Although the three transcription products of PPT-A can each yield
substance P following cleavage by convertases, they differ based on where they are
expressed. Analysis of central nervous system (CNS) and peripheral tissue found
significantly more expression of αPPT-A mRNA in the CNS, whereas there was
3
significantly more expression of βPPT-A and γPPT-A mRNAs in the peripheral tissues
[8]. Precursor forms of substance P protein are made on ribosomes located within the
neuronal soma, where it is packaged into vesicles for axonal transport to nerve terminals
[10, 11]. Within the nerve terminal, it undergoes further enzymatic processing to yield the
final form of substance P stored in large granular synaptic vesicles (large dense core
vesicles) until release is evoked by terminal depolarization [12-14]
Upon release into the synaptic cleft, substance P is susceptible to enzyme
degradation. While a number of enzymes can degrade substance P in vitro, the most
likely candidates based on localization in vivo are neutral endopeptidase (NEP), also
known as neprilysin, and angiotensin 1 converting enzyme (ACE) [15], especially since
substance P and NEP expression overlaps in the brain [16]. NEP derived from human
synaptic membranes, as well as from pig striatum and kidney, was able to degrade
substance P [15, 17, 18]. Neuronal and endothelial derived ACEs were also able to
degrade substance P to peptide fragments [15, 19, 20].
Substance P Distribution within the nervous system
The distribution of substance P within the CNS was the subject of intense
investigation. Early studies identified relatively high concentrations in the dorsal root
ganglia (DRG), spinal cord and brain [21, 22]. Based on this localization, substance P
was hypothesized to be important for pain neurotransmission. Its robust expression within
these regions was confirmed by many subsequent studies [23-26]. Radioimmunoassay
and immunohistochemical surveys of DRG suggest that about 20% of neurons express
substance P and substance P expression seems to be confined to small neurons [27, 28].
Within the DRG, substance P was detected in all ganglia tested and its expression
4
appeared to correlate with ganglia weight. Highest expression was in the C4-T2 and L4L6 regions [26], a distribution pattern that also applies to calcitonin gene related peptide
(CGRP). This observation was significant because substance P appears to co-localize
with other neuropeptides in the DRG, especially CGRP [27-29]. It follows that upon
depolarization, both peptides could be released in response to the same stimuli, especially
since they might be stored in the same synaptic vesicles [30].
Substance P is also highly expressed in the spinal cord [24, 31-33]. It is widely
accepted that its expression is higher in the dorsal horn compared to the ventral horn,
especially in laminae I and II [21, 31, 32]. While a significant portion of substance P
within the spinal cord is from axon terminals of primary afferent neurons that terminate
in the superficial laminae [34], substance P is also synthesized by neurons originating
within the spinal cord [32, 35]. Quantitative measurements of substance P within the
spinal cord suggest that levels of substance P are higher in caudal segments of the spinal
cord compared to rostral segments [36, 37]. They also confirm immunohistochemical
and radioimmunoassay data indicating that levels of substance P are higher in the dorsal
horn compared to the ventral horn [38].
Substance P is also highly expressed in various supraspinal nuclei [23]. Its
localization in the rostral ventromedial medulla (RVM), the source of bulbospinal
neurons that can facilitate or inhibit nociceptive transmission in the spinal cord, is of
particular interest. Immunohistochemical survey of the RVM revealed robust substance P
staining in puncta along neuronal processes [24, 35]. Cell bodies that are immunoreactive
for substance P were only visible when animals are treated with colchicine [31], a toxin
that blocks axonal transport. This observation suggests that substance P is not stored
5
within cell bodies but is quickly transported to terminals after synthesis in the
endoplasmic reticulum. The majority of substance P immunoreactivity within the RVM is
concentrated medially or para-medially [39]. It is worth noting that while neurons within
the RVM synthesize substance P, substance P in the RVM also derives from the
projections of other brainstem nuclei to the RVM (see figures 1 and 2). Interestingly, the
chief sources of substance P-containing afferents in the RVM are the cuneiform nucleus
and the periaqueductal gray [7, 40], both of which are implicated in the supraspinal
regulation of nociception. Although substance P containing neurons in the RVM project
to the spinal cord [7, 39], it appears that these projections are largely to the ventral horn
and unlikely to play a significant role in nociception. Put together, the data suggest that
substance P within the RVM has the potential to be an important signaling molecule in
the processing of pain within the CNS.
NK1 Receptor Distribution in the CNS
NK1 is widely distributed within the CNS and the periphery. In the spinal cord,
NK1 distribution is greatest in the dorsal regions of the spinal cord, compared to ventral
regions. Its distribution is considered discrete because while the dorsal laminae I, III and
IV contain high levels of receptor, expression in lamina II is relatively low. This has been
shown through electrophysiology experiments as only 10.5% of neurons in lamina II
responded to NK1 receptor agonists, compared to 48.3 % in other laminae [41]. This
finding is bolstered by results from immunohistochemical studies that showed greater
expression of NK1 in laminae I, III and IV compared to lamina II [41, 42]. Additionally,
NK1 did not co-localize with gamma aminobutyric acid (GABA) or glycine
immunoreactive neurons, indicating NK1 was found mainly on excitatory neurons [42].
6
Finally, NK1 receptor expression on spino-parabrachial neurons in lamina I is considered
a phenotypic marker of their nociceptive nature [43, 44].
NK1 is also highly expressed in the supraspinal areas of the CNS, many of which
are implicated in pain [45, 46]. These areas include the anterior cingulate and insular
cortex, pons, thalamus, periaqueductal gray and amygdala among others. Of particular
importance to this study is that NK1 is also highly expressed in the RVM [46, 47]. Of the
various medullary pain control areas tested, the RVM had the highest proportion of
spinally projecting NK1 expressing neurons [46], a finding that underscores the
importance of the RVM and substance P / NK1 signaling in descending control of pain.
It is worth noting that while the distribution pattern of NK1 closely mirrors that of
substance P, there are instances where post synaptic NK1 is not apposed by presynaptic
substance P [48, 49]. These instances of incongruity between neurotransmitter and
receptor, though rare, suggest that substance P may reach its receptor by diffusion from
relatively distant release sites, as seen with the neuropeptide NKA [50]. It is also
possible that other tachykinins capable of binding NK1 may be present in high enough
concentrations to activate the receptor. While the majority of NK1 is expressed in postsynaptic terminals, there is growing evidence that NK1 receptors are also situated
presynaptically in both the central nervous system and peripheral organs, where they may
act as an auto receptor [51-53].
Substance P signaling through the NK1 receptor
Substance P, along with neurokinin A and neurokinin B, form a group of
tachykinins that are all able to bind neurokinin receptors NK1, NK2 and NK3 with
varying affinities by virtue of their common carboxy terminal amino acid sequence [54,
7
55]. Substance P binds NK1 with the highest affinity and potency. Therefore, NK1 is
thought to be the main receptor for substance P.
The NK1 receptor is a rhodopsin like G-protein coupled receptor (GPCR). Upon
receptor activation, NK1 is able to activate various second messenger cascades within
mammalian systems by activation of G-proteins. Recruitment of G-proteins and signaling
cascades are thought to be dependent on the tissue. Recent data in the RVM suggests that
NK1 signaling is dependent on protein kinase C (PKC) activation [56]. Following
receptor activation by substance P, the NK1 receptor rapidly desensitizes and is
subsequently internalized into endosomes along with its ligand [57, 58]. Receptor
desensitization is thought to be dependent on receptor phosphorylation. Once in the
endosomes, the receptor can be recycled back to the membrane and dephosphorylated by
phosphatases, a step which is responsible for receptor re-sensitization while substance P
is degraded in the acidified endosome [57, 59]. This receptor recycling pathway is very
well characterized and is readily observable by fluorescent immunohistochemistry. As
such, internalization of NK1 has been the basis of many studies examining substance P
signaling through its receptor within the context of pain.
Substance P as a target for pain; Empirical evidence
Based on the distribution of substance P and its receptor, a concerted research
effort began to determine whether substance P played a role in pain and analgesia. Some
of the first functional data implicating substance P in pain transmission were produced by
Kuraishi and colleagues, when they evoked a 10-fold increase in substance P release in
the dorsal horn in response to a noxious pinch [60]. Subsequent experiments confirmed
release of substance P in response to noxious mechanical stimuli [61], noxious thermal
8
stimuli [61-63] and in response to topically applied capsaicin or methylene chloride, both
chemical irritants [61, 63]. Further evidence that substance P was important for pain
transmission came from studies showing that depletion of spinal cord substance P by
intrathecal injection of capsaicin resulted in reduced responsiveness to noxious thermal
stimuli [64, 65]. Additionally, exogenous substance P injected directly into the spinal
cord induced significant pain-like behaviors in both mice [66, 67] and rats [68].
The role of substance P has also been explored in established models of pain. Formalin
injection into the hind paw yields a reproducible biphasic pain response in conscious
animals and increases in the firing of dorsal horn neurons. Of note, intra-plantar injection
of formalin caused substance P release in the spinal cord dorsal horn in the same biphasic
pattern as the pain behaviors, and the release persisted even beyond pain behaviors [6971]. Interestingly, the pain response as well as the increase in neuronal firing could both
be significantly reduced by drugs effective in human pain conditions [72, 73].
Further insight into the role of endogenous substance P has been provided by
studies using genetically altered mice that lack PPT-A, the gene that codes for substance
P. In these mice (neurokinin A is also absent as both peptides are coded for by the same
gene), responsiveness to mild thermal stimuli was not different compared to wild type
mice, but responsiveness to mildly noxious thermal stimuli was reduced in the PPT-A
knockouts. Responsiveness to mild mechanical stimuli was not different between wild
type and knockouts, but responsiveness to noxious mechanical stimuli was diminished in
the knockout mice. In models of chemical pain (intraplantar capsaicin and formalin), pain
behaviors were reduced in the knock out compared to wild type mice, but it is worth
noting that there was no difference between groups when injected with the highest
9
concentration of formalin. In two models of visceral pain, knockout mice exhibited
reduced pain behaviors compared to the wild type control [74]. The data from these mice
indicate that substance P (and or neurokinin A) is necessary for responsiveness to a wide
range of noxious stimuli, although other mechanisms exist to respond to overwhelmingly
noxious stimuli.
Other studies have also probed the role of endogenous substance P. In the
complete Freund’s adjuvant (CFA) model of localized inflammatory pain, adjuvant
injection into the hind paw induces hypersensitivity of rapid onset (4 hours) and long
duration (up to 2 weeks following the initial injection). The development of pain
associated with this model is accompanied by changes in substance P. For example,
expression of PPT-A, the substance P precursor mRNA, was increased in the DRG and
the lumbar spinal cord 8 hours, 4 days and 2 weeks following adjuvant injection [75, 76].
Levels of substance P were also increased in the lumbar spinal cord 1 week and 2 months
after hind paw adjuvant injection [77, 78]. Additionally, substance P release was
significantly increased in the spinal cord following adjuvant injection compared to
control injection [79, 80]. Similarly, levels of NK1 receptor transcript and protein were
increased in the spinal cord in the adjuvant model of inflammatory pain [71, 81-83] as
well as in the formalin model [71, 84]. In the formalin model, this upregulation is
dependent on the initial activation of NK1 receptors as it was blocked by NK1 receptor
antagonists [84]. Cycling of already synthesized receptor to the membrane does not seem
to be an adequate explanation for this finding as it takes 4 hours for the upregulation to
occur. A better explanation is that following activation and internalization of the NK1
receptors in response to noxious stimuli, a signaling cascade is initiated to synthesize new
10
NK1 receptors. The CFA model also resulted in an initial decrease (6 hours after intraplantar CFA injection) in substance P binding sites in the spinal cord, shown by studies
using iodinated Bolton-Hunter substance P [125]BH-SP [85]. This initial decrease was
attributed to internalization and desensitization of NK1. At later time points (2-8 days
after intra-plantar CFA injection), there was a substantial increase in substance P binding,
which the authors attributed not to increased receptor numbers, but rather to increased
affinity for substance P. This finding suggests possible post-translational modifications to
NK1 following inflammatory injury.
The development of non-peptide NK1 receptor antagonists allowed for further
investigation into substance P’s involvement in various pain models. These drugs prevent
NK1 signaling, thus blunting substance P’s effects. One of the first of these compounds
was CP-99,345, developed by Pfizer in 1991. Initial investigations using CP-99,345 were
promising, as intravenous application blocked dorsal horn neuron responses to
iontophoretic application of substance P as well as responsiveness to noxious thermal
stimuli [86, 87]. The same compound was successful in reducing pain behaviors in other
models, like acetic acid induced abdominal constriction [88], intra-plantar capsaicin
injection [89], as well as during the second phase of the formalin model [87, 89, 90]. The
compound was also effective in longer lasting models of inflammatory pain like
carrageenan induced hypersensitivity [91, 92] and adjuvant induced hyperalgesia [91].
Other NK1 receptor antagonists were effective in animal models of postoperative pain
[93, 94]. These drugs were generally successful, however, there are several instances
where they were with limited or no effect [92, 95, 96].
11
More evidence implicating NK1 in pain was obtained in experiments using NK1-/knockout mice. For example, electrophysiological studies showed that sensitization that
occurs in spinal cord neurons of wild type mice in response to noxious mechanical and
electrical stimuli does not occur in NK1-/- mice. However, basal responsiveness to
noxious mechanical or electrical stimulation of the tail or hind paw was not different
between NK1-/- and wild type control mice. Mechanical hypersensitivity in the CFA
model of inflammatory pain developed similarly in NK1-/- and wild type controls.
Interestingly, the NK1-/- mice developed bilateral mechanical hypersensitivity, even
though the injection of CFA was to just one hind paw. Pain behaviors were reduced in the
second phase of the formalin test in NK1-/- compared to wild type mice. Additionally,
stress induced analgesia measured by the hot plate and tail flick assays were deficient in
the NK1-/- mice compared to wild type controls [97]. A subsequent study also using these
mice characterized the wide dynamic range (WDR) neurons in the spinal cord [98]. The
authors found that responsiveness to various noxious mechanical, thermal and chemical
stimuli were not different between NK1-/- and wild type mice. What was different was the
degree of sensitization that occurred in WDR neurons, as it was greatly reduced in the
NK1-/- mice. This result would suggest that while substance P may be released in
response to various noxious stimuli, it is not necessary for acute responsiveness to the
insult, but rather for the sensitization that occurs after the noxious stimulus.
All told, the preclinical evidence for substance P signaling being important in pain
transmission was strong. Substance P had been identified in various areas of the nervous
system important for pain. It was released in the spinal cord in response to noxious
stimuli and its receptor was activated in various models of pain. Further, receptor
12
antagonists blocked pain behaviors in various established pain models and knockout of
either the peptide or the receptor resulted in deficits in pain transmission. Another
appealing aspect of targeting the substance P signaling with small molecule antagonists
was that overt motor or other side effects were not observed at analgesic concentrations
[87, 89, 90, 93, 96]. It is worth noting that the evidence for a role of substance P was not
limited to laboratory models of pain, as substance P increases have been reported in the
serum of patients suffering from arthritis, fibromyalgia and vaso-occlusive crisis
associated with sickle cell [99-101]. These and other findings led to consensus within the
field that substance P signaling through its receptor was a promising target for new drugs
to treat pain conditions in humans.
The vast majority of the studies of substance P function in the context of pain
focused solely on primary afferents and the dorsal horn, and its role in central and
peripheral sensitization (see [102] for review). The role of substance P in supraspinal
sites was largely overlooked despite the data demonstrating that substance P and NK1 are
highly expressed in supraspinal sites and particularly in the RVM.
RVM as a major player in modulation of pain: Inhibition
The RVM, which is comprised of the nucleus raphe magnus (NRM) and adjacent
nucleus gigantocellularis (NGC) pars alpha, has long been established as an important
component of supraspinal pain processing pathways. Initial work examining the RVM’s
role in pain processing focused on its pain-inhibitory actions. Fields and colleagues found
that electrical stimulation of the nucleus raphe magnus was sufficient to inhibit pain
transmitting spinal neurons receiving high threshold inputs [103]. Further, the NRM
receives inputs from the opioid receptor rich PAG, stimulation of which resulted in
13
analgesia through inhibition of spinal cord neurons [104]. Lesions destroying neurons
within the NRM were sufficient to block analgesia produced by glutamate stimulation of
neurons within the periaqueductal gray (PAG) or systemic administration of morphine
[104, 105]. PAG inhibition of spinal neurons was also blocked by lidocaine injected
directly into the NRM, suggesting PAG signaling to the spinal cord is dependent on the
RVM neural activity [106]. Ultrastructural anatomical evidence also demonstrated that
serotonergic neurons originating from the NRM project to laminae I and II of the spinal
cord, regions established as important for pain signaling [107]. Put together, the
functional and anatomical data paint a picture where the RVM receives inputs from the
anterior cingulate cortex, PAG and other rostral pain modulatory nuclei. The RVM in
turn sends serotonergic and non-serotonergic projections to the spinal cord where it can
inhibit pain signaling, thusly influencing pain perception. While this underscores the
importance of the RVM in pain modulation, it is worth pointing out that not all
supraspinal to spinal projections proceed centrally through the RVM, as there are some
inhibitory projections from the PAG that course laterally through the brainstem as well
[108].
RVM as a major player in modulation of pain: Facilitation
More recently appreciated is the ability of RVM neurons to facilitate, or enhance,
noxious input. This phenomenon has been illustrated empirically through experiments
showing that low intensity electrical stimulation or low dose glutamate activation of
neurons within the RVM of anesthetized rats resulted in facilitation of stimulus response
characteristics of spinal cord neurons in response to noxious heat applied to the hind paw
or to the tail [109, 110]. The RVM also plays a significant role in secondary hyperalgesia,
14
as ablation of RVM neurons using ibotenic acid blocked knee hyperalgesia in response to
intra-plantar carrageenan injection [111]. This secondary hyperalgesia appeared to be
mediated by excitatory amino acids within the RVM, as AP5 (an N-methyl-D-aspartate
(NMDA) receptor antagonist) injected directly into the RVM was able to block
facilitation of tail flick latency produced by topical plantar application of mustard oil
[112]. The RVM was not the only supraspinal brain area involved in facilitation of
noxious inputs, as electrical stimulation of the anterior cingulate cortex (ACC) also
produced a reduction in tail flick latency. Consistent with the idea of the RVM being a
central relay for supraspinal pain modulation, blockade of neural activity within the RVM
with lidocaine, or selective antagonism of 2-amino-3-(3-hydroxy-5-methyl-isoxazol-4yl)propanoic acid (AMPA) /kainate receptors in the RVM was able to block ACC
stimulation dependent facilitation [113]. This suggested that ACC signaling to the spinal
cord level proceeded through the RVM.
Substance P in the RVM
It is now very accepted that the RVM can play a major role in the overall pain
state of the organism, as it can serve as a source of modulatory input to the spinal cord as
well as serve as a central relay conveying spinally directed inputs from supra-spinal pain
modulatory sites. Accordingly, considerable effort has been focused on understanding the
molecular determinants of pain modulation within the RVM. Several prominent
candidates have been identified including opioids, excitatory amino acids, GABA,
serotonin, and more recently substance P along with their respective receptor signaling
systems. While most of these systems have been widely studied within the RVM, the
15
body of knowledge addressing the roles of substance P within the RVM is comparatively
small, especially within the context of chronic pain.
Recent work by various research groups has improved our understanding of how
substance P functions within the RVM in experimental models of pain. Inflammatory
injury produced by intraplantar injection of capsaicin or CFA increased the spontaneous
discharge of ON cells within the RVM, a subpopulation of neurons that is postulated to
serve a pain facilitatory role. The responsiveness of the ON cells to mechanical and
thermal stimuli is also increased; this increase was blocked by intra-RVM administration
of NK1 antagonists [114-116]. This increase in ON cell response could also be
recapitulated by direct activation of NK1 receptors with agonist SM-SP [115]. Similarly,
CFA injection in the hindpaw facilitated the excitatory glutamatergic inputs to a subset of
spinally projecting neurons within the RVM, a facilitation that was dependent on the
activity of NK1 receptors within the RVM [56]. This facilitation was dependent on
activation of PKC, but not calcium / calmodulin dependent kinase II (CaMKII),
suggesting selective second messenger recruitment. The activation of PKC following
NK1 receptor activation was consistent with previous reports [117-120].
In the absence of injury, substance P signaling did not appear to play a role in
responsiveness to mechanical or thermal stimuli in the intact animal because
microinjection of an NK1 antagonist did not alter nociceptive threshold [121, 122].
However, following injection of intra-plantar capsaicin, antagonism of NK1 receptors
within the RVM was sufficient to reverse hypersensitivity to noxious mechanical and
thermal stimuli [121, 122]. Following inflammatory injury with CFA, acute and chronic
antagonism of NK1 receptors within the RVM was sufficient to reverse hypersensitivity
16
to thermal but not mechanical stimuli [121, 123]. Interestingly, acute or chronic
application of substance P directly into the RVM of naïve rats resulted in a transient
antinociception followed by a more prolonged hypersensitivity to thermal stimuli [121,
123]. The transient antinociception produced by exogenous substance P was blocked by
NK1 antagonists [121]. This suggests that even in the absence of injury, the signaling
components were present within the RVM, just not activated by substance P. These data
suggest that substance P does not have a critical role in modulating nociceptive threshold
in the absence of injury, but adopts a pronociceptive role following peripheral
inflammatory injury.
Interplay between substance P and NMDA receptors
As with many other signaling systems within the CNS, substance P signaling does
not occur in isolation. One system that has been widely studied in the context of
substance P signaling is the excitatory amino acids and their receptors, specifically the
NMDA receptor (reviewed by [124-126]). For example, coadministration of NMDA and
substance P enhanced the responses of spinothalamic neurons to NMDA alone [127].
This enhancement lasted for hours following substance P application, suggesting a
strengthening of the excitatory signaling system following substance P application.
Coadministration of substance P and NMDA also enhanced behavioral responses to intraplantar formalin, behaviors that have been shown to be NMDA receptor dependent [128].
Interestingly, neither NMDA nor substance P enhanced responses to intra-plantar
formalin when administered alone. Another study showed that inward currents in primary
sensory neurons or dorsal horn neurons evoked by NMDA could be enhanced during and
after substance P administration, again suggesting lasting effects of substance P on
17
NMDA signaling [129, 130]. The relationship between substance P and the NMDA
receptor was further highlighted by studies showing the involvement of substance P in
central sensitization, where it functioned in concert with NMDA receptors to mediate
plasticity ultimately resulting in strengthening of neuronal pathways involved in
nociception [131, 132].
Additional evidence for the interplay between substance P and NMDA receptors
is provided by studies of NK1 internalization. For example, intrathecal administration of
NMDA or application of NMDA to spinal cord slices was sufficient to induce
internalization of NK1 receptors, most likely subsequent to substance P release [133-135].
This NMDA evoked NK1 internalization was similar to NK1 internalization observed
after high frequency stimulation of the dorsal roots that produced action potentials at the
dorsal horn. Both electrical stimulation of primary afferents and NMDA evoked
internalization of NK1 receptors was blocked by general NMDA receptor antagonists and
more specifically by GluN2B antagonists [133, 135, 136]. NMDA-evoked NK1
internalization was not blocked by lidocaine or ω-conotoxin, indicating that NMDAevoked release of substance P occurred independent of action potential generation and
activation of voltage-gated Ca2+ channels [133]. These data suggest that NMDA receptors
are situated on the presynaptic terminal of substance P-containing neurons where they
cause release of substance P by allowing Ca2+ entry into the terminal. In experiments
where substance P concentrations were directly measured from releasates, NMDA
application increased the release of substance P from lumbosacral spinal cord slices and
capsaicin-evoked release of substance P was blocked by NMDA receptor antagonists
[136, 137]. The localization of NMDA receptors to the axon terminals of substance P
18
containing afferents in the spinal cord has been demonstrated by electron microscopy
[138]. Despite the preceding evidence, there was at least one group that concluded
presynaptic NMDA receptors have little to no role in substance P release from primary
afferent terminals based on their studies, as they found that NMDA did not further
enhance behaviors associated with the formalin test nor did NMDA receptor antagonists
greatly reduce NK1 internalization in the formalin test [139].
There is also evidence for an NMDA receptor and substance P relationship in the
RVM. For example, the vast majority of RVM neurons positive for NK1 are also positive
for GluN1 [115]. Increased spontaneous discharge of RVM neurons resulting from
NMDA application was blocked by NK1 receptor antagonists, suggesting a role for
substance P in causing heightened excitability of RVM neurons following NMDA
application [115]. Additionally, facilitation of excitatory glutamatergic inputs to a subset
of RVM neurons following CFA injury was blocked by NK1 antagonists as well as
NMDA receptor antagonists [56]. However, it is worth noting that NMDA receptor
antagonists blocked facilitation only when applied to the perfusate but not when in the
internal solution, which confines its activity to post-synaptic receptors. This suggests a
role for presynaptic NMDA receptors in mediating the observed facilitation. Furthermore,
when the facilitation was mimicked by the application of exogenous substance P, it could
not be blocked by NMDA receptor antagonists in the perfusates [56]. These data suggest
that in this recording paradigm, NMDA receptors exert their action upstream of the
postsynaptic NK1 receptor, i.e. on the axon terminal.
19
Hypothesis and study design
In aggregate, the behavioral and electrophysiological findings discussed above
suggest that substance P plays a critical pronociceptive role in the RVM under conditions
of peripheral inflammatory injury. The mechanisms by which it assumes such a role are
unclear. One possibility is that peripheral inflammatory injury increases the levels and/or
release of substance P in the RVM. This increase could be transcriptional or translational
in nature. Alternatively, it could come about as a result of the activation of presynaptic
NMDA receptors in the RVM. Indeed, persistent inflammatory injury is known to
increase levels of NMDA receptors in the RVM [140]. This thesis explored the
intriguing hypothesis that peripheral inflammatory injury results in a time-dependent
increase in the levels or release of substance P in the RVM as a consequence of the
activation of presynaptic NMDA receptors in the RVM.
Chapter 2 tested the hypothesis that substance P levels in the RVM is increased under
conditions of persistent inflammatory nociception. These experiments used enzyme
immunoassays (EIAs) to determine levels of substance P in the RVM of rats four hours,
four days or two weeks after intra-plantar injection of CFA or saline in one hind paw.
The RVM was divided into ipsilateral and contralateral sides for this analysis. In
addition, for comparison to the literature, levels of substance P were also determined in
the ipsilateral and contralateral dorsal horn and DRG of the same rats.
Chapter 3 tested the hypothesis that substance P-containing afferents in the RVM express
NMDA receptors. These experiments used immunohistochemistry in combination with
20
electron microscopy to obtain definitive evidence for the existence of presynaptic NMDA
receptors on substance P containing afferents in the RVM. This study was not intended
to be quantitative in nature, but rather to demonstrate the existence of presynaptic NMDA
receptors on substance P afferents, a position from which they can modulate substance P
release. These experiments were conducted in rats four days after intra-plantar injection
of saline or CFA in one hind paw.
Chapter 4 tested two hypotheses. First, it used EIAs to determine if the basal and evoked
release of substance P from prisms of RVM tissue was greater in CFA- than salinetreated rats. Second, it determined whether activation of NMDA receptors in these
prisms facilitated the basal or evoked release of substance P in the RVM, and whether
this effect was enhanced in tissue from CFA-treated rats. These studies complemented
those of Chapter 3 by providing data on the functionality of the NMDA receptor.
Data discussed in the following chapters advance our understanding of the
presynaptic mechanisms that may sustain the activity of brainstem pain facilitatory
pathways that contribute to the maintenance of persistent pain states. They also shed
light on a less appreciated concept concerning the existence and function of presynaptic
NMDA receptors within the RVM. More specifically, these data elucidate how an
inflammatory injury alters substance P levels, distribution and release within the RVM.
The data also increase our understanding of how NMDA receptors modulate substance P
release within the RVM.
21
Figure 1. Hypothesis Schematic 1. Spinally projecting neurons in the RVM receive
substance P inputs from various more rostral supraspinal nuclei, including the cuneiform
nucleus and the periaqueductal gray, as well as from substance P producing neurons
within the RVM. Substance P acting on these neurons can indirectly modulate pain
processing neurons at the spinal cord level.
22
Figure 2. Hypothesis Schematic 2. Substance P containing terminal forming a synaptic
contact with a spinal cord projecting neuron. GluN1 is located on the same presynaptic
terminal as substance P, where GluN1 can directly regulate substance P release by
allowing calcium influx into the terminal.
23
CHAPTER II
INVESTIGATING THE EFFECT OF UNILATERAL CFA-INDUCED
INFLAMMATORY INJURY ON SUBSTANCE P LEVELS OF THE RVM, DRG AND
SPINAL CORD OF THE RAT DORSAL HORN
Abstract
Substantial strides have been made in understanding the role of substance P in
peripheral and spinal mechanisms of chronic pain. However, the role that substance P in
the rostral ventromedial medulla (RVM) plays in initiating and sustaining chronic pain
states is far less understood. Recent behavioral and electrophysiological reports document
an important role for substance P in the RVM in the development and the maintenance of
heat hyperalgesia after peripheral inflammatory injury. However, the mechanisms are not
yet clear. The experiments discussed in this chapter were designed to investigate the
hypothesis that tissue levels of substance P in the RVM are increased after peripheral
inflammatory injury. Rats received a single intraplantar injection of complete Freund’s
adjuvant (CFA) or saline in one hind paw and were euthanized 4 hours, 4 days or 2 weeks
later. Tissue punches were obtained from the RVM ipsilateral and contralateral to the
hind paw injection, the contralateral and ipsilateral L4 and L5 dorsal root ganglia (DRG)
and the ipsilateral and contralateral L4-L5 dorsal horn. Substance P levels in these
various tissues were determined by enzyme immunoassay. The data suggest that
substance P levels in the RVM or dorsal horn spinal cord of CFA-treated rats did not
differ from those of saline-treated rats at any of the time points tested. There was a
statistically significant reduction in substance P level in the ipsilateral DRG of CFAtreated rats at the 4 hour and 4 day time points secondary to an increase in substance P
levels of the corresponding DRG in saline treated rats. There were no differences
24
observed in the contralateral DRG. These data indicate that changes in substance P levels
within the RVM are not responsible for the observed role of substance P in initiating and
maintaining inflammatory pain in the CFA model. However, these findings do not rule
out changes in substance P levels in other brain stem areas that project to the RVM, nor
do they preclude an increased mobilization or release of substance P from terminals. The
findings likewise do not preclude an upregulation of the neurokinin-1 receptor number or
function as the mechanism by which substance P acts in the RVM to initiate or maintain
heat hyperalgesia after peripheral inflammatory injury. These data provide a context for
data obtained from substance P release experiments discussed in a later chapter of this
thesis.
Introduction
The role of substance P in peripheral and spinal nociception is very well
appreciated (see Ref: [102, 141] for review). What remains relatively unclear is the role
of substance P in supraspinal modulation of nociception, despite its high expression in
various supraspinal sites with established roles in pain perception such as the RVM,
periaqueductal gray (PAG) and thalamus [45, 142-144].
Recent work has advanced our understanding of the role of substance P in
supraspinal modulation of pain within the RVM. For example, NK1 receptor antagonism
in the RVM was sufficient to prevent and reverse mechanical hypersensitivity that
developed following capsaicin induced inflammation [121, 122]. It was also sufficient
to prevent and reverse f thermal hypersensitivity in both the capsaicin and CFA models of
inflammatory injury [121-123]. Further, NK1 expression was upregulated from 2 hours
up to 4 days following peripheral inflammatory injury, demonstrated by both western
25
blotting and immunohistochemistry [47, 123]. These data provide strong support for a
critical role of substance P in the RVM in the initiation and maintenance of persistent
pain after peripheral inflammatory injury. Complementary data from electrophysiology
recordings show that peripheral inflammatory injury resulted in facilitation of excitatory
inputs to a subset of spinally projecting neurons. This facilitation was blocked by NK1
receptor antagonists [56].
In the absence of injury, a single injection of substance P directly into the RVM
resulted in hypersensitivity of noxious heat stimuli to the hind paw [121, 122]. In the
absence of injury, continuous infusion of substance P into the RVM also resulted in
hypersensitivity to thermal stimuli lasting 24 hours [123]. Exogenous substance P
facilitated excitatory inputs to RVM neurons, mimicking the effect of peripheral
inflammatory injury [56]. Interestingly, in the absence of injury, NK1 receptor
antagonists injected directly into the RVM did not result in altered responsiveness to
thermal or mechanical stimuli [121]. These data suggest that in the uninjured state,
endogenous substance P does not play a role in responsiveness to noxious stimuli.
However, administration of exogenous substance P to the uninjured RVM recapitulates
various aspects of peripheral inflammatory injury.
These data showed that following peripheral inflammatory injury, substance P
acts in the RVM to initiate and maintain persistent pain states. The mechanism by which
substance P assumes this role following peripheral inflammatory injury remains unclear.
The experiments detailed in this chapter tested whether substance P levels within the
RVM are increased following peripheral inflammatory injury. Such an increase could
underlie the pro-nociceptive role for substance P following peripheral inflammatory
26
injury. To test this hypothesis, RVM punches were obtained from rats injected with either
CFA or saline into one hind paw for 4 hours, 4 days or 2 weeks. RVM punches were
obtained ipsilateral and contralateral to the hind paw injection. The substance P levels of
these punches were measured using an enzyme immunoassay kit (Cayman Chemicals).
Substance P levels were also measured in selected DRG and dorsal horn segments. As
substance P levels have already been reported for these regions, data from these tissues
provided a measure of validation of the method used to measure substance P levels in the
present study. The results obtained from this analysis have provided a comprehensive
picture of substance P levels within the RVM, DRG and dorsal horn in response to
peripheral inflammatory injury over time. They also provide context to data obtained
from substance P release experiments detailed later in this thesis.
Materials and Methods
Animals
The use of animals as described in this chapter was approved by the University of
Iowa Animal Care and Use Committee. All animal testing was performed based on
guidelines set forth by the National Institutes of Health and the International Association
for the Study of Pain [145]. Adult male Sprague Dawley Rats weighing 225 – 250 g
obtained from Charles River (Raleigh, NC) were used for this study. All rats were
allowed at least 48 hours following arrival to acclimate to the novel environment of the
University of Iowa Animal Care facility, where they were housed in a temperature and
humidity controlled room on a 12 hour light / dark cycle with free access to food and
water. Rats were housed 2 per cage, until intraplantar injection of CFA or saline after
which they were caged individually.
27
Model of hind paw inflammatory pain
The method of inducing hind paw inflammatory pain was comparable to that
previously reported by this lab [121, 146, 147]. All rats were lightly anesthetized with
isoflurane before receiving a single intraplantar injection of 0.15 ml CFA (150 µg of
Mycobacterium butyricum, 85% Marcol 52, and 15% Aracel A mannide monoemulsifier;
Calbiochem, San Diego, CA) or saline (pH 7.4 and passed through a 0.22 micron filter)
into one hind paw, and were allowed to fully recover from anesthesia before being
returned to the housing facility.
Assessment of localized inflammatory hypersensitivity
Paw withdrawal latencies (PWLs) and paw thickness were determined before
(baseline) and after the intraplantar injection of CFA or saline. PWLs to noxious heat
were obtained as previously described [146]. Briefly, the rats were placed on a glass
surface maintained at 25°C and allowed 15 minutes to acclimate to the novel
environment. A radiant heat source was used to apply a noxious heat stimulus to the
plantar aspect of each hind paw. A timer automatically detected when the rat removed its
hind paw to terminate the stimulus. To prevent tissue damage, the test was terminated at
20 seconds in the absence of a withdrawal response. Rats that did not respond within 20
seconds were assigned that value. PWLs were assessed for both the injected and
uninjected hind paw. Paw thickness was determined using digital calipers.
Tissue Collection for Substance P Level analysis
At the designated time point 4 hours, 4 days or 2 weeks after the intraplantar
injection of CFA or saline, PWL and paw diameters were reassessed. The rats were then
28
euthanized with carbon dioxide (CO2), exsanguinated and experimenters worked to
simultaneously remove the brain and expose the spinal cord regions. To obtain the RVM,
the cerebellum was removed to expose the brain stem. A 2 mm thick coronal section of
brainstem beginning 3 mm and ending 5 mm rostral to the obex was quickly dissected
and placed on dry ice. This brainstem section contains the RVM [148]. Two tissue
punches (0.75 mm diameter) were obtained from the RVM, one ipsilateral and the other
contralateral to the injected hind-paw. The L4 and L5 segments of the spinal cord were
harvested as one unit after following dorsal roots to the corresponding section of the
spinal cord. The ipsilateral and contralateral sides were separated and the dorsal horn of
each side was obtained. Additionally, L4 and L5 DRG were also collected and pooled for
each side in each rat. All tissue samples were collected into Eppendorf tubes on dry ice
and stored at -80 ºC until analysis. A schematic of this process is presented in figure 2.1.
Measurement of substance P Levels
Substance P was extracted from the collected tissue using a variation of the method
described by Savard and colleagues [149]. Tissue sections were weighed and then
mechanically homogenized in 0.5 ml of 2 M acetic acid. The homogenates were
centrifuged at 10,100g for 10 minutes using an Eppendorf 5415r tabletop centrifuge and
then the supernatant carefully removed. The supernatants were lyophilized using a Savant
Speed Vac SC110 and then reconstituted in enzyme immunoassay buffer before
determination of Substance P levels using a kit from Cayman Chemicals. Substance P
concentrations were determined based on a standard curve with a detection range of 3.9 –
500 pg of substance P per ml. The linear part of the standard curve was between 20 –
80 % bound / maximal binding. All samples were diluted to yield substance P
29
concentrations within this range; i.e. by 15-fold for DRG and RVM and by 150-fold for
dorsal horn. The kit was used as directed by the manufacturer. All substance P
concentrations obtained from the standard curves were further standardized to wet tissue
weight. For the remainder of this document, this value (substance P level measured by
EIA) will be referred to simply as substance P level.
Immunohistochemistry
The immunohistochemistry experiments were conducted in cohorts. A cohort was
comprised of one rat in each of four treatment groups: intraplantar injection of CFA or
saline four days or two weeks earlier. The timing of the injections was such that all rats in
a cohort were euthanized and perfused on the same day. The tissue from the four rats
was then processed concurrently. This study consisted of a total of three cohorts.
Four days or two weeks after intraplantar injection of CFA or saline, rats were
deeply anesthetized using sodium pentobarbital. When unresponsive to a hard pinch to
the tail or hind paw, the chest cavity was opened and 0.1 ml heparin (1000 USP units per
0.1 ml) was administered into the heart. All rats were transcardially perfused using a
peristaltic pump (Masterflex L/S, Model 7518-10) and tubing (Masterflex 6424-25)
tipped with a feeding needle inserted into the ascending aorta. The perfusion sequence
consisted of first flushing the circulatory system using 0.9% saline until the perfusate ran
clear, then perfusing with ice cold 4% paraformaldehyde solution (pH 7.4) for
consecutive three minute periods at 40 and 30 ml / minute, respectively. Following
perfusion, rat brains were removed and post fixed in 4% paraformaldehyde for 30
minutes, before transfer to 30% sucrose in 0.1 M phosphate buffer solution for cryoprotection.
30
The brainstem was blocked, frozen in optimal cutting temperature (OCT) compound
media (Sakura Finetek, Torrance, CA), and transverse sections of 50 µm thickness were
collected into 0.1 M phosphate buffered saline (PBS) using a Leica CM3050 S cryostat.
Immuno-labeling experiments were performed on free floating sections. Selected serial
sections were treated with 1.67% hydrogen peroxide in methanol for 20 minutes, washed
several times with 0.1 M PBS and then incubated in 1% sodium borohydride for 30
minutes. Following several more 0.1 M PBS washes, sections were then blocked with 0.1
M phosphate buffer containing 2% normal donkey serum and 0.3% Triton X for 2 hours.
This solution also served as the diluent for primary and secondary antibodies used later in
these experiments. Following blocking, sections were incubated with guinea pig antisubstance P antibody diluted to 1:160,000 (Neuromics, GP14103 lot number 401159) for
40 hrs at 4ºC. After several washes, the sections were incubated in Biotin SP donkey antiguinea pig (1:500, Jackson Immunoresearch, 706-485-148) for 1 hour. The sections were
further processed using an ABC Elite kit and ImmPACT DAB (Vector Laboratories).
The ABC solution was prepared as specified by the manufacturer. The RVM sections
were incubated in this solution for 5 minutes, followed by several washes with 0.1 M
PBS. ImmPACT DAB solution was prepared at half the manufacturer’s recommended
concentration and each RVM section was incubated in this solution for 90 seconds before
transfer to double distilled water for 5 minutes to terminate the DAB deposition reaction.
This was followed by several rinses in 0.1 M PBS. Sections were mounted out of water
onto glass slides, dried and then cleared in xylenes (Research Products International, Mt.
Prospect, IL) before coverslipping with DPX mounting medium (VWR International,
Poole, England)
31
Quantification of RVM substance P immunoreactivity
All quantification was done by an investigator blinded to the treatment condition.
Brainstem sections containing the RVM were identified using landmarks as noted in The
Rat Brain Atlas [148]. Each RVM was further divided into two aspects: ipsilateral or
contralateral to the injected hind paw. Stereo Investigator software (MBF Bioscience)
was used to randomly sample three regions of interest on each side from which images
were obtained. Six sections spanning the rostrocaudal length of the RVM were analyzed
in each rat. Thus, substance P immunoreactivity was determined from 18 regions of
interest within the ipsilateral, as well as the contralateral RVM. All images were acquired
using a Nikon Eclipse E800 microscope equipped with an RT Slider SPOT (Diagnostic
Instruments) digital camera. The same camera settings were used for all images acquired
for a cohort.
Metamorph software (Molecular Devices) was used to establish an immunoreactive
threshold for each image. The immunoreactive threshold was determined by first
measuring the pixel intensity (possible values being 0 -255 on a standard grayscale) of six
distinct substance P immunoreactive structures in the region of interest, averaging these
six values and computing a range defined as three standard deviations above and below
that average. All immunoreactive structures whose intensity fell within that range were
then thresholded to generate a binary image of labeled and unlabeled structures. Figure
2.2 provides an example of an image and the corresponding binary image. Metamorph
was further used to calculate total number of pixels that were immunoreactive as defined
by the thresholding function in each region of interest. To reiterate, three regions were
sampled in each of six sections of both the ipsilateral and contralateral RVM. These 18
32
values were then averaged to yield a single value for that side of the RVM in that rat.
The values for each rat in a treatment group were then averaged to generate a mean and
S.E.M. for that treatment condition.
Statistical Analysis
All data are expressed as mean ± standard error. A two-way ANOVA was used to
compare PWL and paw thickness between saline and CFA-treated rats. One factor was
treatment (saline vs CFA) and the other factor was time (before vs after). Levels of
substance P were compared between CFA and corresponding saline-treated rats using a
two-way ANOVA in which one factor was treatment and the other factor was time (4 hr,
4 days or 2 weeks). For analysis of substance P immunoreactivity, all values in a cohort
were normalized to the value in the ipsilateral side of the saline-treated rat. Values in the
saline-treated rats were normalized to the average value for the group. A two-way
ANOVA was used to compare values in saline and CFA treated rats at 4 days or 2 weeks
after injection. A P < 0.05 was considered significant for all analyses.
Results
Characterization of CFA induced inflammatory injury
Intraplantar injection of CFA produced significant heat hyperalgesia in the
ipsilateral hindpaw that was evident within 4 hours and persisted through 4 days and 2
weeks as compared to saline-treated rats at the corresponding time points (P < 0.01 for all
groups, see figure 2.3). PWL of the contralateral hind paw of CFA and saline treated rats
did not differ at any time point. Intraplantar injection of CFA also produced inflammation
as evidenced by an increase in the thickness of the ipsilateral hind paw of CFA-treated
rats compared to the corresponding hindpaw of saline treated rats at all time points (P <
33
0.01 for all groups, see figure 2.4). The thickness of the contralateral hindpaw of CFA
treated rats did not differ from that of the contralateral hindpaw of corresponding saline
treated rats at any time point (See figure 2.4).
Substance P Levels
All substance P measurements were made using EIA kits that yielded values in
picograms per ml (pg / ml). All values were subsequently corrected for the wet weight of
the source tissue in milligrams to yield a final measurement of substance P in picograms
per milliliter per milligram protein (pg / ml per mg protein). Levels of substance P in
either the ipsilateral or the contralateral RVM of CFA treated rats did not differ from
those in corresponding saline treated rats at any time point (figure 2.5). In saline treated
rats, Substance P levels varied as a function of time with higher levels apparent at four
days. Levels of substance P in the ipsilateral L4 and L5 DRG were significantly less
than those of saline-treated rats 4 hours and 4 days after CFA, but did not differ at two
weeks. Whether this is a biologically relevant decrease is unclear given that levels of
substance P in the DRG of CFA treated rats were comparable to those of the contralateral
DRG of both saline and CFA treated rats. Thus, the apparent decrease may be secondary
to an increase in levels in the saline treated rats. Substance P levels in the contralateral
L4 and L5 DRG did not differ in saline or CFA treated rats (figure 2.6). Finally,
substance P levels in the ipsilateral and contralateral dorsal horn of CFA treated rats were
comparable at all time points to that in saline treated rat. Interestingly, substance P levels
in saline treated rats in the dorsal horn appeared to be lower at four days than at 4 hours
or two week time points.
34
Quantification of RVM substance P Immunoreactivity
4 days and 2 weeks after peripheral injury
Figures 2.8 and 2.9 are representative photomicrographs of substance P
immunoreactivity within the RVM four days and two weeks, respectively, after
intraplantar injection of CFA or saline. Immunoreactivity was confined to puncta and
processes in the neuropil; immunoreactive somata were not observed. Figure 2.10 depicts
the result of quantitative densitometry. Four days after CFA-treatment, substance P
immunoreactivity was significantly increased by 2.5 fold in the ipsilateral RVM
compared to the ipsilateral RVM of saline treated rats, and also marginally higher
compared to the contralateral side of CFA. Two weeks after CFA, substance P
immunoreactivity in the ipsilateral RVM did not differ from that of saline treated rats.
There was a modest increase in substance P immunoreactivity in the contralateral RVM
of the CFA treatment group compared to the contralateral RVM of the saline treatment
group. These data suggest that while the gross substance P levels within the RVM do not
change at any of the tested time points following inflammatory injury, there appear to be
differences in how substance P is mobilized to terminals following the CFA treatment
compared to the saline controls.
Discussion
Previous reports from this lab and others have demonstrated that antagonism of
NK1 in the RVM reversed heat hyperalgesia at 4 hours, 4 days and 2 weeks following
intraplantar CFA injection [121-123]. However, microinjection of NK1 antagonists
directly into the RVM of uninjured rats did not alter PWL [121]. Further, substance P
applied directly to the RVM was able to facilitate excitatory transmission of RVM
35
neurons following peripheral inflammatory injury [56]. Taken together, these data
suggest that while the actions of substance P in the RVM do not influence responsiveness
to thermal stimuli in the uninjured rat, they are important to the maintenance of thermal
hyperalgesia in the acute and persistent components of CFA induced inflammatory pain.
This observation prompted us to ask whether the expression of a pro-nociceptive role of
substance P in the RVM of injured rats is the result of increased levels of substance P
within the RVM.
Findings for the RVM
This study provided the first quantitative determination of substance P levels in
the RVM. Previous qualitative studies using immunohistochemical approaches reported
the RVM contained moderately high levels of substance P. The present results support
those prior reports and indicate that the RVM contains significant amounts of substance P,
with levels approximately twice those of the DRG and about one-tenth that of the dorsal
horn on a wet-weight basis. For the present study, the RVM was divided ipsilateral and
contralateral to injury. The basis for this division is since published observations that
following CFA injury, the number of neurons expressing NK1 in the RVM was further
increased contralateral to the hind paw injury [47]. Other groups have also noted lateral
effects of injury on neurons within the RVM [150].
Contrary to our hypothesis, substance P levels were not increased in the RVM at
any time point after intraplantar injection of CFA compared to saline. Substance P levels
in the RVM of saline treated rats was higher at both four day and two week time points,
which may be a result of adaptive changes to a new housing environment (food, bedding,
handlers). Given that there was so little variance in the PWLs, no attempt was made to
36
determine whether there was a relationship between substance P levels and the magnitude
of heat hyperalgesia.
A shortcoming of this approach was the lack of cellular resolution. Substance P
levels were measured in homogenates of tissue using EIA. This approach would obscure
increases in substance P that occurred in a subpopulation of neurons or only in
presynaptic terminals, as it also measures substance P within other structures like cell
bodies. To address this limitation, immunohistochemistry was used to visualize and label
substance P in serial sections of the RVM from CFA and saline treated rats that were
processed as cohorts. The immunohistochemical data indicated that 4 days after injection
of CFA, substance P immunoreactivity was increased by 2.5 fold in the ipsilateral RVM
and by about 1.5 fold in the contralateral RVM compared to the corresponding side in
saline treated rats. Two weeks after CFA, the density of substance P immunoreactivity
was comparable in both CFA and saline treated rats. When the immunohistochemical
data are considered along with the EIA data, they suggests that whereas the levels of
substance P may not change, the distribution of substance P is altered shortly after CFA
injection. The increased labeling of processes and puncta suggest that there may be
increased synthesis and trafficking of substance P to the nerve terminals. Indeed,
trafficking of substance P to the nerve terminal appears to be a very active given that
substance P immunoreactive cell bodies are not visible without the use of colchicine to
inhibit transport [31]. Although the immunohistochemical conditions do not permit us to
determine if there is an increase in substance P immunoreactive cells bodies in the RVM,
use of colchicine makes animals extremely ill and would have introduced an unwanted
confound in this study.
37
These findings do not preclude other mechanisms by which substance P may
assume a pro-nociceptive role after injury. For example, conditions of inflammatory pain
may alter the release of substance P, allowing for more substance P to be released into the
synaptic cleft following presynaptic depolarization. This scenario is not unlike previous
reports demonstrating that electrically stimulated substance P release is increased in
spinal cords slices obtained from rats injected with intraplantar CFA [79]. It is also
possible that changes may occur post-synaptically to alter the number or affinity of NK1
receptors, or its ability to mobilize its second messenger systems. Recent work has
demonstrated increased protein levels of NK1 receptors within the RVM between 2 hours
and 3 days following intraplantar CFA injection [123], a finding that has been confirmed
in this lab using immunohistochemistry and stereological analysis [47], lending credence
to post-synaptic changes in the RVM in response to intraplantar CFA. While there are
substance P producing neurons within the RVM, the RVM also receives substance P
inputs from various supraspinal locations implicated in nociceptive processing [40, 151].
It is possible that intraplantar CFA may alter tissue levels in any of these other areas
containing substance P, affecting the properties of its projections to the RVM.
Findings for the DRG
This study also examined effects of intraplantar CFA on substance P levels of the
L4 and L5 DRG. The data indicate that intraplantar CFA decreases substance P levels in
the ipsilateral DRG compared to the saline controls at the 4 hour and 4 day time points,
but not at the 2 week time point. There was no effect of intraplantar CFA on substance P
levels in the contralateral DRG at any time point. Reports from other groups have
concluded that intraplantar CFA increased substance P levels in the ipsilateral DRG 2, 5
38
and 15 days following injection [152-154]. The disparity between the conclusions of the
previous studies and this one may lie in the difference in experimental design. SafiehGarabedian et al., Smith et al. and Donnerer et al. compared ipsilateral DRG levels to the
contralateral side of the same rat to reach their conclusions, while the current study
compared ipsilateral DRGs of CFA treated rats to ipsilateral DRG of saline treated rats. If
the same comparison is made with the present data, a marginal increase in substance P
levels is observed at the 2 week time point as reported by Smith et al. In another study,
Calza and colleagues found slightly reduced substance P immunoreactivity in the DRG at
5 days and 13 days after CFA injection, but significantly increased substance P
immunoreactivity 21 days after injection. It is worth noting that in another study of CFAinduced polyarthritis, Lembeck and colleagues found increased levels of substance P in
the T12-S3 DRG but not in the spinal cord dorsal horn 20 days following CFA injection
[155]. Finally, it should be appreciated that the decrease in substance P observed at 4
hours and 4 days may actually be secondary to increased levels of substance P in the
ipsilateral DRG of saline treated rats, which were also greater than levels of the
contralateral DRG. The basis for this increase is unknown.
Findings for the dorsal horn
Substance P levels of the dorsal horn determined in this study were in good
agreement with several prior reports [36, 38, 64]. These results increase confidence in
data obtained for the RVM. Weihe and colleagues reported slight increases in staining
intensity in the ipsilateral spinal cord compared to the contralateral spinal cord 7 days
following injection of CFA into the hind paw [78]. They also similarly report
significantly reduced substance P immunoreactivity in the spinal cord at 5 days into their
39
polyarthritis model, and significantly increased levels after 21 days [156]. The data from
this study indicated that following CFA-induced inflammatory injury, there was no
change in substance P levels in the ipsilateral or contralateral spinal cord dorsal horn at
any of the time points tested. This finding is in line with reports from other groups that
have reported seeing no changes in the spinal cord dorsal horn substance P levels at 5
days following intraplantar CFA [152]. However, another study from Galeazza et al.
reported decreases in substance P levels of the L4-L6 spinal cord following intraplantar
CFA [157]. Again, the disparity in the conclusions of these studies may be explained by
differences in experimental design, as the study from Galeazza and colleagues compared
substance P levels in the CFA treated rats to untreated controls, while the current study
compared substance P levels in CFA treated rats to controls injected with intraplantar
saline. Differences in the concentration of CFA administered in the different studies may
also play a role.
Summary
The results of this study indicate that CFA injection does not increase levels of
substance P in the RVM and are contrary to our hypothesis. However, the
complementary immunohistochemical results suggest that persistent inflammatory injury
increases trafficking of substance P to the nerve terminal. These observations, coupled
with the results of an earlier electrophysiological study that demonstrated a facilitated
release of substance P in CFA-treated rats, led us to modify our hypothesis. The
modified hypothesis was that CFA induced an increased release of substance P in the
RVM and that presynaptic NMDA receptors played a role.
40
Figure 3. Tissue Analysis Schematic. Illustration of the experimental design and
approach to quantification of levels of substance P in the RVM, dorsal horn and DRG
of CFA- and saline-treated rats.
41
Figure 4. Representative image of substance P in the RVM and
corresponding binary image. A representative example of the use of a
thresholding program to generate a binary image of substance P
immunoreactivity from a photomicrograph of a region of interest in the RVM.
The number of pixels was then counted to generate a single value for the region
of interest. See the text for details.
42
Figure 5. Characterization of CFA-induced heat hyperalgesia 4 hours, 4 days and
2 weeks after intraplantar injection. Panels A, B and C depict PWL before and 4
hours, 4 days or 2 weeks after injection of saline or CFA in one hindpaw. Data are the
mean ± S.E.M. of determinations in 6 – 14 rats in each treatment group. * P < 0.05,
** P < 0.01 compared to preinjection values. ┼ P < 0.05, ╪ P < 0.01 compared to
corresponding saline group.
43
Figure 6. Characterization of CFA-induced inflammation 4 hours, 4 days and 2
weeks after intraplantar injection. Panels A, B and C depict paw diameter before
and 4 hours, 4 days or 2 weeks after injection of saline or CFA in one hindpaw. Data
are the mean ± S.E.M. of determinations in 6 – 14 rats in each treatment group. * P <
0.05, ** P < 0.01 compared to preinjection values. ╪ P < 0.01 compared to
corresponding saline group.
44
Figure 7. Time course of substance P levels in the RVM ipsilateral (A) and
contralateral (B) to peripheral inflammatory injury. Substance P levels measured
in tissue punches from the RVM at various time points following intra-plantar
injection of CFA (closed circles) or saline (open circles). Data expressed as picograms
substance P per mg wet tissue weight. Each value is mean ± SEM of 8-15 rats per
group.
45
Figure 8. Time course of substance P levels in the DRG ipsilateral (A) and
contralateral (B) to peripheral inflammatory injury. Substance P levels measured
in the L4 and L5 DRG at various time points following intra-plantar injection of
CFA (closed circles) or saline (open circles). Data expressed as picograms substance
P per mg wet tissue weight. Each value is mean ± SEM of 8-15 rats per group. * P <
0.05, ** P < 0.01 compared to corresponding saline group.
46
Figure 9. Time course of substance P levels in the dorsal horn ipsilateral (A) and
contralateral (B) to peripheral inflammatory injury. Substance P levels measured in
the L4 and L5 dorsal horn at various time points following intra-plantar injection of
CFA (closed circles) or saline (open circles). Data expressed as picograms substance P
per mg wet tissue weight. Each value is mean ± SEM of 8-15 rats per group.
47
Figure 10. Representative photomicrographs of substance P immunoreactivity in
the RVM 4 days after intra-plantar saline (A) or CFA (B). Note that substance P
immunoreactivity is largely confined to processes within the neuropil in both images.
48
Figure 11. Representative images of substance P immunoreactivity in the RVM 2
weeks after intra-plantar saline (A) or CFA (B). Note that substance P
immunoreactivity is largely confined to processes within the neuropil in both images.
49
Figure 12 Quantification of substance P Immunoreactivity in the RVM 4 days (A)
and 2 weeks (B) following intra-plantar CFA or saline. Data at both time points
have been normalized to relative immunoreactivity of the respective ipsilateral saline
group. All data expressed as relative immunoreactivity ± SEM, n = 3 for all groups.
50
CHAPTER III
ULTRASTRUCTURAL IDENTIFICATION OF AXON TERMINALS CONTAINING
BOTH NMDA RECEPTORS AND SUBSTANCE P
Abstract
A growing body of work has highlighted the interactions between substance P and
the NMDA receptor signaling systems. For example, in the spinal cord, co-application of
substance P and NMDA results in synergistic increases in the excitability of dorsal horn
neurons. Further, application of NMDA results in release of substance P in the RVM,
documented by NK1receptor internalization. More recent work has demonstrated that
NMDA receptors are located on substance P containing axon terminals in the dorsal horn
and thus situated to directly affect the probability of release. Recent findings have shown
that the interactions between substance P and the NMDA receptor extend to the RVM as
spontaneous firing of RVM neurons was increased in an NMDA receptor dependent
manner. Additionally, peripheral inflammatory injury results in facilitation of
glutamatergic inputs to a subset of spinally projecting RVM neurons. This facilitation
was dependent on the activity of NK1 receptors, and while it could be blocked by bath
application of NMDA receptor antagonists, it was not reversed by blocking only postsynaptic NMDA receptors, indicating a role for presynaptic NMDA receptors. The
mechanism by which this facilitation occurs is unclear. The hypothesis of this current
work is that NMDA receptors are situated on substance P-containing axon terminals in
the RVM. This localization would allow NMDA receptors to directly affect the release
and or extent of substance P release by participating (or not participating) in events
preceding neurotransmitter release. Additionally, morphological changes in presynaptic
51
terminal properties may underlie the changes that occur in substance P release following
peripheral inflammatory injury. To test this hypothesis, RVM sections were obtained
from rats injected four days earlier with CFA or saline into a hind paw. These sections
were dual labeled for substance P and GluN1, the obligate NMDA receptor subunit, and
then examined using electron microscopy. The substance P antibody used in this study
was validated by immunohistochemistry experiments using tissues obtained from Tac1
(the gene coding substance P) null mice to demonstrate a loss of substance P staining.
The GluN1 antibody was validated by western blotting experiments showing it detected a
band at the correct molecular weight, and detection could be blocked by preabsorption
with a neutralizing peptide. Additionally, knock down of GluN1 using miRNA resulted in
diminished labeling of GluN1. At the ultrastructural level, substance P and GluN1 were
colocalized to axons terminals in the RVM of both CFA and saline treated rats. While no
overt differences were observed in the two treatment conditions, a quantitative
component would have to be incorporated into the present study to make any conclusive
statements. This finding identifies a means by which NMDA receptors may regulate
substance P release in the RVM, a topic that is explored in more depth in chapter IV of
this thesis.
Introduction
The relationship between the substance P and glutamate receptor signaling system,
especially the NMDA receptor, has been the subject of intense investigation. Some of the
first evidence suggesting a relationship was data showing that combined microiontophoretic application of substance P and NMDA resulted in sensitization of
spinothalamic neurons long after substance P was removed [127]. Responses to
52
intraplantar formalin are also enhanced in response to combined intrathecal
administration of NMDA and substance P. No enhancement is observed when either
substance P or NMDA is administered alone [128]. Additionally, incubation of spinal
cord slices with NMDA results in substance P release, evidenced by internalization of the
NK1 receptor, likely subsequent to substance P release [134-136]. This NK1
internalization was blocked by NMDA receptor antagonists, suggesting NMDA receptors
are involved in this release of substance P. Capsaicin evoked substance P release is also
blocked by NMDA receptor antagonists [137], indicating the involvement of NMDA
receptors in substance P release evoked by various stimuli. More recently, ultrastructural
data from the spinal cord has shown NMDA receptors are expressed on the same
presynaptic terminals containing substance P [138]. This finding provides anatomical
evidence for a mechanism by which NMDA receptors can regulate substance P release.
More recently, studies have demonstrated that the relationship between the
glutamate receptor and substance P signaling systems extend to the RVM. For example,
increases in spontaneous firing of RVM neurons following NMDA application is blocked
by NK1 receptor antagonists, suggesting the NMDA application within the RVM results
in substance P release [115]. Also, following peripheral inflammatory injury, excitatory
inputs to a subset of spinally projecting RVM neurons are facilitated [56]. This
facilitation is blocked by NK1 receptor antagonist, bath applied NMDA receptor
antagonists, but not by postsynaptic NMDA receptor antagonist, implicating presynaptic
NMDA receptors in mediating this facilitation. Further, this facilitation can be
recapitulated in RVM neurons from uninjured rats by application of exogenous substance
P. While all these data point to presynaptic NMDA receptors in the RVM mediating this
53
facilitation after injury, conclusive anatomical evidence does not exist as in the spinal
cord. This led to the experiments detailed in this chapter, which explored whether
substance P and NMDA receptors exist on the same presynaptic terminals within the
RVM. Such a location would allow for NMDA receptors to directly modulate substance
P release. This was tested by obtaining RVM sections from rats injected with either CFA
or saline into one hind paw for four days. These sections were then dually
immunolabeled and examined by electron microscopy to determine colocalization of both
markers in presynaptic terminals. These data provide a plausible mechanism by which
NMDA receptors can directly regulate substance P release in the RVM. They also serve
as an anatomical basis of functional experiments detailed in chapter IV of this thesis,
exploring NMDA receptor facilitation of substance P release within the RVM following
injury.
Materials and Methods
Animals
The use of animals as described in this chapter was approved by the University of
Iowa Animal Care and Use Committee. All animal testing was performed based on
recommended guidelines set forth by the National Institutes of Health and the
International Association for the Study of Pain [145]. Adult Sprague Dawley Rats
weighing 225 – 250g obtained from Charles River (Raleigh, NC) were used for this study.
All rats were allowed at least 48 hours following travel for acclimation to the novel
housing environments at the University of Iowa Animal Care facility, where they were
housed in a temperature and humidity controlled room on a 12 hour light / dark cycles
54
with free access to food and water. Rats were housed 2 per cage, until intraplantar
injection of CFA or saline after which they were caged individually.
Model of hind paw inflammatory pain
The method of inducing hind paw inflammatory pain was comparable to that
previously reported by this lab [121, 146, 147]. All rats were lightly anesthetized using
isoflurane before receiving a single intraplantar injection of 0.15 ml CFA (150 µg of
Mycobacterium butyricum, 85% Marcol 52, and 15% Aracel A mannide monoemulsifier;
Calbiochem, San Diego, CA) or saline (pH 7.4 and passed through a 0.22 micron filter)
into one hind paw, allowed to fully recover from the anesthesia effects before being
returned to the housing facility.
Tissue Preparation
Light Microscopy: Four days following intraplantar CFA or saline injection, rats were
deeply anesthetized using sodium pentobarbital. When unresponsive to a hard pinch to
the tail or hind paw, the chest cavity was opened and 0.1 ml heparin (1000 USP units per
0.1 ml) was administered into the heart. All rats were transcardially perfused using a
peristaltic pump (Masterflex L/S, Model 7518-10) and tubing (Masterflex 6424-25)
tipped with a feeding needle inserted into the ascending aorta. The perfusion sequence
consisted of first flushing the circulatory system using 0.9% saline until the perfusate ran
clear, then perfusing with ice cold 4% paraformaldehyde solution (pH 7.4) for
consecutive three minute periods at 40 and 30 ml / minute, respectively. Following
perfusion, rat brains were removed and post fixed in 4% paraformaldehyde for 30
minutes, before transfer to 30% sucrose in 0.1 M phosphate buffer solution for cryoprotection. The brainstem was blocked, frozen in optimal cutting temperature (OCT)
55
compound media (Sakura Finetek, Torrance, CA), and transverse sections of 50 µm
thickness were collected into 0.1 M phosphate buffered saline (PBS) using a Leica
CM3050 S cryostat. Immuno-labeling experiments were performed on free floating
sections. Selected serial sections were treated with 1.67% hydrogen peroxide in methanol
for 20 minutes, washed several times with 0.1 M PBS and then incubated in 1% sodium
borohydride for 30 minutes. Following several more washes with 0.1 M PBS, sections
were then blocked with 0.1 M phosphate buffer containing 2% normal donkey serum and
0.3% Triton X for 2 hours. This solution also served as the diluent for primary and
secondary antibodies used later in these experiments.
Bright field Immunohistochemistry: Following blocking, sections were incubated with
goat anti-GluN1 (1:500; Santa Cruz Biotech, sc-1467) for 40 hrs at 4ºC. After several
washes, the sections were incubated in Biotin SP donkey anti-goat (1:500, Jackson
Immunoresearch, 705-065-003). The sections were further processed using an ABC Elite
kit and ImmPACT DAB (Vector Laboratories). ABC Elite kit was prepared as specified
by the manufacturer. The RVM sections were incubated in this solution for 5 minutes,
followed by several washes with 0.1 M PBS. ImmPACT DAB solution was prepared at
half the manufacturer’s recommended concentration and each RVM section was
incubated in this solution for 90 seconds before transfer to double distilled water for 5
minutes to terminate the DAB deposition reaction. This was followed by several rinses in
0.1 M PBS. Section were mounted on glass slides out of water, dried and then cleared in
xylenes (Research Products International, Mt. Prospect, IL) before coverslipping with
DPX mounting medium (VWR International, Poole, England)
56
Fluorescent Immunohistochemistry: Following blocking, sections were incubated with
goat anti-GluN1 (1:500; Santa Cruz Biotech, sc-1467) alone or with rabbit anti-GFP
(1:2000; Abcam, ab290) for 40 hrs at 4ºC. After several washes in 0.1 M PBS, the
sections were incubated in Dylight 649 donkey anti-goat (1:800, Jackson
Immunoresearch, 705-495-147) and Cy3 donkey anti-rabbit (1:400, Jackson
Immunoresearch, 711-165-152). After several washes in 0.1 M PBS, the sections were
mounted on glass slides out of water, dried and then cleared in xylenes (Research
Products International, Mt. Prospect, IL) before coverslipping with DPX mounting
medium (VWR International, Poole, England). Images were acquired using a confocal
microscope (Zeiss LSM 510). All images are the projection of 2-3 scans (z = 0.86
microns per scan).
Electron Microscopy tissue preparation: Rats were deeply anesthetized by intraperitoneal
pentobarbital overdose followed by intracardiac injection of 0.1 ml heparin (1000 USP
units per 0.1 ml). When unresponsive to hard pinch to the tail or paw, the rats were
transcardially perfused using the Perfusion One (www.myneurolab.com) apparatus as
directed by the manufacturer. Rats were first perfused with a 10% sucrose solution in
water at 300 mm Hg, followed by fixative (4% paraformaldehyde, 0.1% glutaraldehyde
in 0.1 M phosphate buffer) also at 300 mm Hg. The brains remained in-situ for 4 hours
before being dissected out and stored in 0.1M phosphate buffer until sectioning less than
18 hours later.
Electron Microscopy: To facilitate sectioning, brains were embedded in 4% agarose.
Transverse sections 150 microns thick were collected into 0.1 M PBS using a vibratome
(Vibratome 1500, Intracel, UK). Selected sections were treated with 1% NaBH4 for 30
57
minutes, washed several times with 0.1 M PBS, then blocked with 0.1 M phosphate
buffer containing 2% normal donkey serum for 2 hours. The sections were then incubated
overnight with guinea pig anti-substance P antibody (1:5000; Neuromics, GP14103) and
goat anti-GluN1 (1:500; Santa Cruz Biotech, sc-1467). Following a series of washes with
0.1 M PBS, sections were subsequently incubated with biotin SP donkey anti-guinea pig
(1:500, Jackson Immunoresearch, 706-485-148) and donkey anti-goat ultrasmall gold
secondary (1:50, Electron Microscopy Sciences, Cat # 25800), donkey anti-goat 6 nm
gold secondary (1:50, Electron Microscopy Sciences, Cat # 25802) or donkey anti-goat
15 nm gold secondary (1:50, Electron Microscopy Sciences, 25806). All sections were
further processed using ABC Elite kit and ImmPACT DAB (Vector Laboratories). ABC
Elite kit was prepared as specified by the manufacturer. The RVM sections were
incubated in this solution for 5 minutes, followed by several washes with 0.1 M PBS.
ImmPACT DAB solution was prepared at half manufacturer’s recommended
concentration and each RVM section was incubated in this solution for 90 seconds before
transfer to double distilled water for 5 minutes to terminate the DAB deposition reaction.
This was followed by several rinses in 0.1 M PBS. Sections immuno-labeled with
ultrasmall or 6 nm gold particles were silver enhanced using Aurion SE-EM Gent kit (Cat
# 500.033, Aurion Immuno Gold Reagents & Accessories, Wageningen, The Netherlands)
for 50 minutes. Tissue punches containing the RVM (Harris Uni-core, 1.5 mm diameter,
Ted Pella, Redding, CA) were obtained from selected immuno-labeled sections. These
were incubated in 1% OsO4 and 1.5% potassium ferrocyanide in 0.1% phosphate buffer
for 1 hour. Following several washes (3 changes in 1 hour) with 1X phosphate buffer, the
sections were treated with 2.5% uranyl acetate for 20 minutes. The sections were then
58
dehydrated by incubating in increasing concentrations of ethanol, with each incubation
lasting 30 minutes, except for the final incubation in 100 % ethanol that lasted for 1 hour.
This was followed by embedding the sections in Eponite resin (Ted Pella, Redding, CA)
and curing at 60 °C overnight. Thin sections were obtained using an ultramicrotome
(Leica EM UC6, Leica Microsystems, Wetzler, Germany) and placed on nickel formvar
grids. Grids were examined using a JEOL 1230 TEM equipped with a Gatan Ultrascan
1000 2k x 2k CCD camera for image acquisition.
Western Blotting
Rats naïve to any experimental treatment were euthanized by carbon dioxide (CO2)
asphyxiation. Working quickly on ice, the whole brain was removed into ice cold lysis
buffer (1% Triton X-100, 50 mM Tris pH 7.4, and 150 mM NaCl in water) supplemented
with a protease inhibitor cocktail tablet (Roche complete mini, EDTA-free, Roche, IN).
The brains were then homogenized in a 15 ml Potter-Elvehjem Teflon-glass tissue
grinder coupled to a motorized overhead stirrer (both from Wheaton Science Products,
Millville, NJ) to yield a homogenous solution. This solution was then homogenized at
70000 rpm (2626400 g) using a Thermo-Sorvall Discovery M150 Ultracentrifuge for 15
minutes at 4°C. The protein concentration of the supernatant was determined using a
BCA protein assay (Pierce, Rockford, IL) and appropriate working concentrations were
achieved by dilutions in ice cold lysis buffer.
A loading mixture was obtained by mixing appropriate amounts of protein sample
with 0.1 M dithiothreitol (DTT) and 1X lithium dodecyl sulfate (LDS) buffer (Pierce,
Rockford, IL) then heated at 70°C for 10 minutes. A loading mixture volume of 40 µl,
along with visible and chemiluminescent molecular weight ladder (Bio-Rad, Hercules,
59
CA) was loaded onto a NuPage 4-12 % Bis-Tris polyacrylamide gel (Life Technologies,
Carlsbad, CA), run for 2 hours then transferred onto a PVDF membrane. The membrane
was then blocked for 1 hour with a solution of 5% non-fat dry milk (Genesee Scientific,
San Diego, CA) in 1X TBS-Tween (TBST) buffer followed by incubation with a 1:2000
dilution of goat-anti-GluN1 antibody (sc-1467, Santa Cruz Biotech, Santa Cruz, CA) in
5% bovine serum albumin in TBST buffer overnight. For pre-absorption control
experiments, the primary antibody was incubated with a 3-fold molar excess of the preabsorption peptide (sc-1467-p, Santa Cruz Biotech, Santa Cruz, CA) for 3 hours before
dilution to 1:2000 in 5% bovine serum albumin buffer overnight. Following washes with
1X TBS-Tween buffer, the membrane was subsequently incubated for 2 hours in 1:2000
donkey anti-goat HRP (Santa Cruz Biotech, Santa Cruz, CA), and 1:5000 of streptavidin
(Bio-Rad, Hercules, CA), both diluted in 5% milk in TBST buffer. The membrane was
incubated with an enhanced chemiluminescence (ECL Plus) substrate (GE Healthcare
Life Sciences, Piscataway, NJ) for 3 minutes before image acquisition using an EC3
imaging system (UVP, Upland, CA) equipped with a CCD camera.
Knockdown of GluN1 in the RVM
Knock down of GluN1 within the RVM was achieved by direct microinjection of
the FIV coding miRNA to GluN1 and GFP (or control expressing only GFP) into the
RVM. Both miRNA coding viruses were characterized by DaSilva and colleagues [158].
All injections were carried out using aseptic and sterile tip methods. Rats were deeply
anesthetized with a mixture of ketamine (70 mg/kg) and xylazine (13 mg/kg),
administered intraperitoneally. When unresponsive to a hard pinch, rats were positioned
on a stereotaxic instrument based on coordinates corresponding to the RVM in adult rats
60
[148]. These coordinates have been used successfully in this lab to target the RVM in the
past [121, 147]. A midline incision was made to expose the skull and a drill was used to
make a small hole along the midline. A Hamilton 10 µl syringe coupled to a syringe
pump was used to inject 0.5 µl of FIV-miRNA or control. The needle was left in place for
60 seconds following injection to reduce diffusion of virus up the needle tract. Upon
completion of the injection, staples were used to close the midline incision and rats were
allowed to fully recover from the anesthesia before being returned to the housing facility.
Rats were housed individually thereafter. All rats used in this portion of the study were
euthanized and perfused 2 weeks after the injection.
Antibody Characterization
The substance P antibody used in this study was GP14103 from Neuromics
(Edina, MN), lot 401159. To verify the specificity of the antibody, sections of
diencephalon from wild type and Tac1 knockout mice (B6.CG-Tac1tm1Bbm/J, The Jackson
Laboratory, Farmington, CT) were processed concurrently to immunolabel for substance
P. The pattern of substance P labeling was compared in tissues from the wildtype and
Tac1 knockout mice. The GluN1 antibody used in the study was SC-1467 from Santa
Cruz Biotech (Santa Cruz, CA); two different lots, J2010 and J3012, were used in this
study. Immunolabeling by each lot was indistinguishable. The specificity of both lots of
this antibody was verified using western blotting (correct molecular weight, protein
dependent staining intensity and loss of binding following pre-incubation with
neutralizing peptide) and showing reduced immune-fluorescent labeling intensity
following viral knockdown of GluN1.
61
Results
Characterization of Substance P Antibody
The substance P labeling in the RVM by this antibody has been discussed at
length in chapter II. To characterize this antibody using tissue from wild type and Tac1
knockout mice, the striatum was chosen because of the especially dense immunolabeling
for substance P in this region. Labeling with this antibody resulted in dense labeling of
processes in the medial preoptic nucleus adjacent to the third ventricle and the basal
nucleus of the striatum as expected (Figure 3.1A,B). In contrast, there was a complete
absence of labeling in the same regions of the Tac1 knockout mouse (Figure 3.1C).
Characterization of the GluN1 primary antibody
Immunoreactivity for GluN1 was evident in the cytoplasm of cell bodies and
proximal dendrites (see Figure 3.2). This pattern is consistent with other reported
instances of neuronal GluN1 immuno-labeling [159, 160]. At higher magnification,
various puncta along fine processes are labeled by this antibody, likely representing
terminals containing GluN1 (see Figure 3.2).
Mice that are null mutants for GluN1 are neonatally lethal, and thus not available
to validate the specificity of the antibody. Two alternative and complementary
approaches were therefore used. Western blotting for GluN1 in whole brain lysates
revealed a broad band with a molecular weight of approximately 115 kDa, the expected
molecular weight of GluN1 (see Figure 3.3 A and C). Loading less protein resulted in a
less intense band, indicating that the binding of this antibody was dependent on ligand
concentration. Additionally, this band was absent when the primary antibody was
preabsorbed with a three-fold molar excess of the antigen peptide (see Figure 3.3 B and
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D). Of note, this antibody also stained a large number of bands of lesser and greater
molecular weight, which were not appreciably reduced by preabsorption of the antibody.
The second approach entailed microinjection in the RVM of a recombinant FIV
expressing GluN1 targeted miRNA and green fluorescent protein (GFP) in order to knock
down GluN1 protein in the RVM. This virus has been previously characterized and used
to knock down GluN1 in the RVM [158]. Representative photomicrographs of the
finding are presented in figure 3.4.
In sections obtained from the rat treated with the miRNA coding virus, many
neurons close to the injection site expressed GFP. Neurons that were immunoreactive for
GluN1 but not GFP were also identified. Neurons that expressed GFP exhibited less
intense labeling for GluN1 as compared to neurons in the same focal plane that were
immunoreactive for GluN1, but did not express GFP (See 3.4A-D). In sections of the
RVM obtained from the rat treated with the FIV-control virus, many neurons close to the
injection site expressed GFP. Of these, many were immunoreactive for GluN1. Also
present were neurons that were immunoreactive for GluN1 but not GFP. There was no
appreciable difference in the intensity of GluN1 labeling in neurons that did or did not
express GFP in the same focal plane (see 3.4 E and F). This result, along with the western
blotting results, supports the idea that this antibody identifies GluN1 protein.
Ultrastructural Morphology of the RVM
To obtain samples of RVM sections for examination by electron microscopy,
tissue samples were obtained as close as possible to the tissue / resin interface, as these
sections were most likely to be immuno-labeled as a result of limited antibody
penetration into the tissue. Micrographs of the RVM were photographed based on the
63
presence of labeling for either substance P or GluN1, or the presence of other structures
of interest.
When the RVM is examined by electron microscopy, several structures associated
with the central nervous system are readily visible as identified by Peters et al. [161].
Many of these structures of relevance to neurotransmission are depicted in figure 3.8 A.
These include several axon terminals (T) forming synapses (arrowheads) with a single
large dendrite (D). Axon terminals are identified based on the presence of vesicles.
Additionally, several mitochondria (m) are readily visible within axon terminals,
consistent with the energy intensive process of neurotransmitter release. While majority
of the vesicles within the axon terminals are of the small clear variety, several large dense
core vesicles are also present (arrows). The small clear vesicles are thought to contain
monoamine and amino acid neurotransmitters, while the large dense core vesicles are
thought to contain peptide neurotransmitters. Other structures identified include
myelinated axons, cell bodies and their various organelles, endothelial cells of blood
vessels and various neuroglial cells (see Fig. 3.5). In the examined tissue, myelin and
other lipid containing structures appear dark (electron dense) as a result of treating the
tissue samples with osmium tetroxide, which embeds heavily in lipid rich structures like
cell membranes. There are two general classifications of synapses, types 1 and 2. Type 1
synapses, characterized by asymmetric synapse density distribution, are generally
associated with rounder and generally larger vesicles (see Figure 3.6 A and B). These
synapses are thought to be excitatory. Type 2 synapses, characterized by symmetrical
synapses, generally contain flattened or elongated vesicles (see Figure 3.6 C and D).
These synapses are thought to be inhibitory. Both types of synapse were readily
64
identifiable within the sections examined, consistent with literature regarding the types of
neurotransmission within the RVM.
Ultrastructural GluN1 labeling
At the ultra-structural level, gold particles representing GluN1 labeling were
present in axon terminals (see Fig 3.7). Within terminals, particles were either seemingly
loosely localized within the terminal, or attached to the membranes of small clear vesicles
(see Figures 3.7 B and C). Gold particles were also found post-synaptically, along the
synaptic density immediately adjacent (see Figures 3.8 A and B). When the synapse was
readily visible, terminals labeled with GluN1 generally formed asymmetrical synapses
(see figure 3.8 B), consistent with its established role in excitatory glutamatergic
neurotransmission. In addition to synaptic elements already described, GluN1 was also
found on other structures, likely axons or dendrites, not visibly associated with synapses
(see Figure 3.8 C). Interestingly, there were several instances of GluN1 labeling of
mitochondria (see Fig 3.9). While not pertinent to the current study, this observation
supports a recent report identifying functional NMDA receptors on nervous system
mitochondria [162].
Ultrastructural Substance P labeling
At the ultra-structural level, the vast majority of observed DAB deposits,
representing substance P labeling, were in axon terminals (see Figure 3.10). DAB
deposits were sometimes localized to immunolabeled vesicles, although there were
several other instances where large portions of terminals contained DAB. When the
synapse was readily visible, substance P, like GluN1, was found in terminals forming
asymmetrical synapses. While these synapses generally contained round small clear
65
vesicles, substance P was associated with larger dense core (granular) vesicles. Also,
readily observable were other large dense core vesicles unlabeled for substance P,
sometimes in the same presynaptic terminals. This finding is consistent with the presence
of other peptide neurotransmitters within the RVM.
GluN1 and substance P colocalization in both CFA and
saline-treated rats
There were several instances in which substance P and GluN1 labeling were
observed within the same axon terminal in the RVM of CFA (see Figure 3.11) and saline
(see Figure 3.12) treated rats. No differences in the morphology or distribution of
substance P and or GluN1 labeling in the terminals of saline and CFA treated rats were
immediately apparent. Many of these micrographs showed DAB deposits in large
portions of the terminals, mainly associated with large dense core vesicles. Additionally,
discrete gold particles representing GluN1 were also present in these terminals, mainly
associated with the membranes of small clear vesicles. In some of these micrographs,
electron dense labeling (likely gold particles) is also is found along the presynaptic aspect
of the synapse, consistent with the synaptic localization of the NMDA receptor. When the
synapses of these dual labeled terminals are readily visible, they appear to be
asymmetrical in nature, consistent with the excitatory role of GluN1 in neurotransmission.
Discussion
The findings of this study confirm our hypothesis that substance P containing
terminals in the RVM express NMDA receptors, and provide definitive evidence of an
anatomical basis for the ability of NMDA to promote the release of substance P in the
RVM. The findings are strengthened by careful studies that first established the
66
specificity of the antibodies for the GluN1 subunit of the NMDA receptor and for
substance P. Total loss of substance P labeling was observed in the Tac1 knockout mice.
Western blot analysis showed GluN1 labeling at the correct molecular weight in a protein
concentration dependent manner, and labeling could be blocked by pre-incubation with a
neutralizing peptide. Additionally, the reduction in GluN1 labeling following viral
knockdown is in good agreement with the result of another study [158]. The results
detailed here also provide a basis for performance of release studies to determine whether
the receptors are functional, which are described in chapter IV.
There is precedent for the existence of NMDA receptors on substance P
immunoreactive terminals in the spinal cord [138]. This anatomical evidence of substance
P and GluN1 colocalization in presynaptic terminals in the spinal cord was preceded by
functional studies showing that activation of NMDA receptors resulted in NK1 receptor
internalization in dorsal horn neurons, shown at both the light and ultrastructural levels
[134, 135]. A more recent study has shown this NMDA receptor regulation of substance
P release is mediated by the GluN2B subunit, and requires NMDA receptor
phosphorylation by Src Kinases [133]. The presence of NMDA receptors on the same
terminals as substance P provides a plausible mechanism by which this regulation may
occur.
Technical Considerations
A natural extension of the current study would be to be to incorporate a
quantitative component, likely through stereology. Similar analyses have been performed
at the ultrastructural level [163, 164]. By adding such a component to the current study,
several intriguing questions can be addressed. For example, following peripheral
67
inflammatory injury, is there an increase the number of substance P immunoreactive
terminals? Does injury result in increased number of substance P immunoreactive
vesicles per terminal? Does the proportion of substance P terminals expressing NMDA
receptors change after a peripheral inflammatory? Are any particular NMDA receptor
subunits upregulated following injury and by how much? Additionally, by slightly
altering the experiment design (adding a tracer to label spinally projecting neurons), one
could address whether substance P inputs to spinally projecting neurons are changed
following peripheral inflammatory injury.
However, before such questions are tackled, several technical considerations must
be addressed. A means must be devised to consistently sample similarly labeled sections
within and between treatment groups. This difficulty is compounded by limitations of
antibody penetration and the requirement that tissue sections have to be reliably obtained
at the same distance from the tissue / resin interface to prevent potential bias.
Additionally, given the apparent small percentage of substance P immunoreactive
terminals that also colocalize GluN1, random sampling of a sufficient area within the
RVM in order to reach any meaningful conclusion would not be a trivial task.
The combination of DAB and gold conjugated secondary antibodies has been used in
other ultrastructural studies [138, 163, 164]. However, there is a major technical
consideration when using this technique, namely the balance between getting adequate
antibody penetration and using a gold particle large enough to be easily distinguishable
from the DAB deposits. Ultrasmall gold particles will provide the best tissue penetration,
but are hardest to distinguish from DAB, especially profuse DAB within the same
compartment. At the other end of the spectrum, larger gold particles, like the 15 nm used
68
in the present study, are more readily distinguishable from DAB but do not penetrate far
into the tissue, resulting in labeling only at the surface portions of the section. The best
recourse is to use a larger gold particle with the understanding that sections have to be
collected very close to the tissue / resin interface or risk completely missing gold labeled
portions of the tissue and limit the DAB reaction to the shortest length of time possible to
still get deposits. Thus, it is possible that this study has underestimated the proportion of
substance P terminals that express the NMDA receptor.
Summary
To conclude, the data presented in this chapter of the thesis validated the
antibodies used in this study. They also provided ultrastructural evidence that substance P
and the GluN1 colocalize on the same presynaptic terminals within the RVM. This
provides an anatomical basis by which the NMDA receptor can regulate substance P
release within the RVM, which is explored further in experiments detailed in chapter 4 of
this thesis. This colocalization was readily observed in RVM sections from both CFA and
saline treated rats.
69
Figure 13. Representative photomicrographs of substance P immunolabeling in
the diencephalon of (A,B) a wildtype mouse and (C) tac1 null mouse. Note the
intense labeling of processes in the medial preoptic nucleus of the hypothalamus and
the bed nucleus of the striatum in the wildtype mouse and complete absence of
labeling in the tac1 null mouse. These tissues were processed concurrently. Panel B
is a higher magnification of the bed nucleus of the striatum. Scale bars are 200
microns in panels A and C, and 100 microns in panel B.
70
Figure 14. Representative photomicrographs of GluN1 immunoreactivity in the
(A,C,D) RVM and (B) facial motor nucleus. Strong labeling is present in the cell
bodies (arrowheads) as well as along neuronal processes (arrows). Comparable
labeling patterns were observed with either fluorescent secondary (A and B) or a
peroxidase labeling system (C and D). Scale bars for panels A and B are 50 microns for
panels A and B, 40 microns for panel C and 100 microns for panel D.
71
Figure 15. Western blots labeled with the GluN1 antibodies. These blots were
conducted using the two lots of GluN1 antibody used in this study. Blots in panels A and
B were labeled using lot J2010, and those in panels C and D were labeled using lot
J3012. In A and C, a broad band was evident at approximately 115 kDa, the expected
molecular weight of GluN1. Loading half the amount of protein resulted in a less intense
fainter band. Neither lot of antibody labeled bands in the absence of sample. Incubation
of the antibody with a three molar excess of the antigen peptide (see B and D) resulted in
a loss of the expected band at 115 kDa. However, it did not diminish the intensity of
other bands suggesting that the antibody has non-specific or off-target binding.
72
Figure 16. Representative high magnification photomicrographs of GluN1
immunoreactivity in the RVM of rats microinjected with FIV-miRNA to knock
down GluN1 or empty vector virus. Neurons that have been successfully transfected
with the viral vector are identified by expression of GFP (A,C,E green). GluN1
immunolabeling appears as red (B,D,F). Panels A-D are from a rat in which FIVmiRNA-GRN1 was microinjected in the RVM two weeks earlier. Panels E and F are
from a rat in which the empty vector was microinjected in the RVM two weeks earlier.
All tissues were processed concurrently. Panels B and D demonstrate that the intensity
of GluN1 labeling in GFP expressing neurons (corresponding neurons in panels A and C)
is less than GluN1 labeling in neurons that do not express GFP. Panels E and F
demonstrate that the intensity of GluN1 labeling in neurons that express GFP is
equivalent to those that do not express GFP, indicating that the empty vector is without
affect. Scale bar is 20 microns.
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Figure 17. General morphology of the RVM at the ultrastructural level. Panels A
and B are representative electron micrograph that illustrates the appearance of myelinated
axons (Ax), dendrites (D) and axon terminals filled with vesicles (T). The very fine
structures along the surface of the leftmost axon in panel B are likely microtubules. Panel
C illustrates a myelinated axon as it traverses the tissue Ax(t). Mitochondria (m), a
necessary component of the energy intensive process of neurotransmitter release, are also
present. Panel D is of the cytoplasm within a neuron. The Golgi apparatus (G) and
endoplasmic reticulum (ER) are both visible. Punctate structures around the ER likely
represent free ribosomes (r).
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Figure 18. Electron micrographs of symmetrical and asymmetrical synapses within
the RVM. Panel A illustrates an asymmetrical synapse (see arrowheads) between an
axon terminal and a dendrite. Panel B is a higher magnification image of the area
demarcated by the blue rectangle (dashed lines) in panel A. Note the difference in
electron dense structures between the post-synaptic density and the apposed presynaptic
region, as well as the relatively round vesicles adjacent to the synapse. Based on these
characteristics, this synapse is likely excitatory. Panel C illustrates a symmetrical synapse
(arrowheads), while Panel D is a higher magnification image of the region demarcated by
the blue rectangle (dashed lines) in panel C. Notice the relatively even distribution of
electron dense structures on either side of the synapse, as well as the ellipsoid shape of
the vesicles in the axon terminal. This synapse is likely inhibitory.
75
Figure 19. Examples of GluN1 labeling with silver enhanced 6 nm gold particles
in axon terminals within the RVM. The photomicrograph in Panel A shows an area
of the RVM containing several axon terminals. Although the majority are unlabeled,
two terminals (top left and bottom right) contain gold particles, indicating the presence
of GluN1. Panels B and C are higher magnifications of those labeled terminals,
demarcated by the blue rectangle (dashed lines) in panel A.
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Figure 20. Examples of post-synaptic GluN1 labeling with gold particles within
the RVM. Panel A is an electron micrograph illustrating a dendrite that receives input
from a presynaptic terminal. Panel B is a magnification of the peri-synaptic area
demarcated by a blue rectangle (dashed lines), illustrating electron dense material,
likely silver enhanced 6 nm gold particles labeling GluN1, clustered at the postsynaptic density as well as immediately around the postsynaptic density. This pattern
is consistent with post synaptic localization of the NMDA receptor. Panel C is an
example of silver enhanced ultra-small gold particle labeling, likely GluN1, within
various dendrites. None of these dendrites appear to receive synaptic contacts in this
focal plane.
77
Figure 21. GluN1 labeling on or around mitochondria within the RVM. Panel A
shows an example of an axon terminal containing silver enhanced 6 nm gold particles.
Higher magnification of this terminal (B) as demarcated by the blue rectangle (dashed
lines) in panel A shows gold particle accumulation around the outer membrane of the
mitochondria within the terminal. Other instances (C and D), show discrete gold particles
on cristae within the mitochondria.
78
Figure 22. Substance P labeling within the RVM. Peptidergic terminals are
characterized by the presence of dense core vesicles. These vesicles are generally larger
and usually outnumbered by the small round clear vesicles found within the same
terminal. In these electron micrographs from sections stained for substance P, electron
dense deposits of DAB overlay the dense core vesicles.
79
Figure 23. GluN1 and substance P immunoreactivity colocalize in axon terminals
in the RVM of CFA treated rats. The electron micrograph in Panel A illustrates an
axo-dendrite synapse. Panel B is a higher magnification of the region demarcated by
the blue rectangle (dashed lines) in panel A, illustrating discrete silver enhanced 6 nm
gold particles (GluN1) on various small clear vesicles within the terminal, as well as
DAB deposits (substance P) atop a larger structure, likely a dense core vesicle. Panel
C is a profile from the RVM showing various structures, including multiple unlabeled
terminals and a single dual labeled terminal. Panel D is a higher magnification of the
region demarcated by the blue rectangle (dashed lines) in panel C, again showing
DAB deposits (substance P) as well as multiple small clear vesicles labeled with silver
enhanced 6 nm gold particles (GluN1).
80
Figure 24. GluN1 and substance P immunolabeling colocalize to axon terminals
in the RVM of SALINE treated rats. Electron micrographs in Panels A and C show
profiles from the RVM of axo-dendritic synapses. Higher magnification of the regions
demarcated by blue rectangles (dashed lines) in panels A and C are shown in panels B
and D, respectively. These higher magnification images show DAB deposits
(substance P) as well as discrete 15 nm gold particles (GluN1) around small clear
vesicles. Also note the accumulation of electron dense material presynaptic (B and D)
and post synaptic (D) in these images, likely gold particles but possibly DAB.
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CHAPTER IV
RELEASE OF SUBSTANCE P IN THE RAT RVM: MODULATION BY
PERIPHERAL INFLAMMATORY INJURY AND PRESYNAPTIC NMDA
RECEPTORS
Abstract
It is evident that substance P functions in the RVM to facilitate pain through
bulbospinal pathways. For example, antagonism of NK1 receptors reverses
hypersensitivity to noxious thermal stimuli in the capsaicin and CFA models of
inflammatory injury. Additionally, following inflammatory injury, excitatory
glutamatergic inputs to a subset of spinally projecting non-serotonergic neurons in the
RVM are facilitated in a manner dependent on NK1 receptor activation, likely subsequent
to substance P release. This facilitation can be recapitulated in uninjured rat RVMs by
application of exogenous substance P. This facilitation is blocked by bath applied NMDA
receptor antagonists, but not NMDA receptor antagonists in the internal solution, which
only act on post-synaptic NMDA receptors. Interestingly, in the absence of injury, NK1
receptor antagonists microinjected into the RVM are of no consequence to
responsiveness to noxious thermal stimuli. These data suggest that following a peripheral
inflammatory injury, substance P assumes its pro-nociceptive role where it acts to
maintain hypersensitivity to noxious thermal stimuli in a manner dependent on presynaptic NMDA receptors. Further, as substance P does not modulate responses to
noxious heat in the absence of injury, the data suggest changes in substance P release in
the RVM following CFA injury. This study therefore investigated whether release of
substance P is increased in the RVM following peripheral inflammatory injury, whether
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this release can be is modulated by NMDA receptors and whether this modulation is
altered following peripheral inflammatory injury. Cohorts of male rats were injected in
one hind paw with either saline or CFA. Four days after the injections, the rats were
euthanized and tissue prisms were prepared from the RVM. Prisms were incubated with
Krebs Ringer buffer (or buffers inducing depolarization conditions) for four periods at
37 °C for, each period lasting 15 minutes. Depolarization was induced with potassium or
veratridine, with or without NMDA. Releasates were collected after each period and
analyzed for substance P using enzyme immunoassay. Measurements were made of basal
release, release evoked by potassium (K+) or veratridine, as well as in combination with
by NMDA. Basal release of SP did not differ between CFA and saline groups (0.020 ±
0.001 pg substance P /µg protein for both groups). Incubation with potassium and
veratridine yielded release of substance P in RVM prisms from both the CFA and saline
groups. The magnitude of this release was not different between groups. The combination
of high concentration (50 mM) of potassium with NMDA yielded facilitation of
substance P release in both CFA and saline groups. A more modest concentration of
potassium (25 mM) combined with NMDA yielded facilitation of substance P release in
the saline, but not the CFA group. Both concentrations of veratridine tested (50 and 25
µM) yielded facilitation of substance P release when combined with NMDA in the saline
group, but not in the CFA group. These data demonstrate that basal and evoked release of
substance P from RVM prisms are not increased following peripheral inflammatory
injury. Additionally, NMDA receptor facilitation of substance P release is also not
increased by peripheral inflammatory injury. These data suggest post synaptic
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mechanisms underlie previously noted changes in substance P activity within the RVM
following peripheral inflammatory injury.
Introduction
The majority of the studies looking at substance P release in the context of pain
have been conducted in the spinal cord. For example, substance P was released in the
spinal cord in response to acute noxious mechanical or thermal stimuli [60, 62]. It was
also released in experimental models of inflammatory pain [83, 159]. More recently
appreciated was the role of NMDARs in regulating this release of substance P. For
example, NMDA-dependent excitatory activity of dorsal horn neurons was enhanced by
the addition of substance P [127, 129]. Capsaicin evoked release of substance P was
blocked by NMDA receptor antagonists [137]. Additionally, spinal application of NMDA
caused internalization of NK1 receptors, suggesting that the release of substance P was
dependent on pre-synaptically located NMDA receptors [134, 135]. This internalization
was mimicked by high frequency stimulation of dorsal roots and blocked by NMDAR
and NK1 antagonists, providing further evidence that this release of substance P involved
activation of NMDA receptors [135]. More recent work demonstrated that NMDA
evoked release of substance P was mediated by NMDA receptors containing the GluN2B
subunit, as well as phosphorylation of the receptor by Src family kinases [133]. Despite
the preceding evidence, at least one study found little relationship between NMDA
receptor activation and substance P release [139].
The data concerning release of substance P in the RVM, especially within the
context of inflammatory injury, is relatively limited. A recent publication from this lab
examined the effect of peripheral inflammatory injury on substance P inputs to spinally
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projecting non-serotonergic neurons in the RVM using electrophysiology [56]. Following
injury, a subset of these neurons showed facilitated responsiveness to a range of
excitatory inputs to the RVM i.e. these neurons from CFA-treated rats showed a greater
response to the excitatory inputs compared to the saline treated controls. This facilitation
was reversed by NK1 receptor antagonists, indicating a role for NK1 receptors, likely
subsequent to substance P release. Additionally, this facilitation was blocked by bath
application of NMDA receptor antagonist, but not by NMDA receptor antagonist in the
internal solution of the patch pipette. This finding was significant as this meant blocking
all NMDA receptors reversed the facilitation but blocking only the post synaptic
receptors did not block facilitation, implicating presynaptic NMDA receptors. When
exogenous substance P was applied with tetrodotoxin (blocks presynaptic transmission)
an inward current was still observed. This inward current was blocked by an NK1
antagonist, indicating that substance P’s effect is mediated by postsynaptic NK1 receptors.
Put together, the data paint a picture of presynaptic NMDA receptors mediating increased
substance P release following inflammatory injury in a subset of spinally projecting
neurons. However, this hypothesis has not been directly tested.
Other studies have examined the functional significance of substance P release in
the RVM. For example, in the capsaicin model of inflammatory pain, substance P release
in the RVM was necessary to maintain hypersensitivity of noxious mechanical and
thermal stimuli [121, 122]. In the CFA model of inflammatory pain, substance P release
was similarly necessary to maintain hypersensitivity to noxious thermal [121, 123].
Interestingly, in the absence of injury, antagonism of NK1 receptors in the RVM does not
affect responsiveness to noxious mechanical or thermal injury, suggesting that any
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ongoing release of substance P does not play a role in responsiveness to these stimuli in
the absence of injury.
Put together, the data suggest that before injury, substance P was not involved in
responsiveness to noxious stimuli. After peripheral inflammatory injury, substance P
release becomes important for the maintenance of hypersensitivity to noxious stimuli.
Additionally, excitatory inputs to a subset of neurons within the RVM were facilitated in
a manner dependent on presynaptic NMDA receptors and post-synaptic NK1 receptors
following CFA injury.
Data discussed in chapter II of this thesis show that total amount of substance P in
the RVM does not change following inflammatory injury. However, substance P
mobilization to terminals within the RVM appeared to be increased 4 days following
inflammatory injury. Additionally, electron microscopy data show NMDA receptors are
expressed on the same presynaptic terminals positive for substance P. The experiments
detailed in this chapter tested whether peripheral inflammatory injury increases substance
P release (basal and evoked) in the RVM. They also tested whether NMDA receptors
facilitate substance P release in the RVM, and whether this facilitation is altered after
inflammatory injury. Such changes could be the mechanism by which substance P
assumes its pro-nociceptive role following inflammatory injury.
Materials and Methods
Animals
The use of animals as described in this chapter was approved by the University of
Iowa Animal Care and Use Committee. All animal testing was performed based on
recommended guidelines set forth by the National Institutes of Health and the
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International Association for the Study of Pain [145]. Adult Sprague Dawley Rats
weighing 225 – 250g obtained from Charles River (Raleigh, NC) were used for this study.
All rats were allowed at least 48 hours following travel for acclimation to novel housing
environments at the University of Iowa Animal Care facility, where they were housed in
a temperature and humidity controlled room on a 12 hour light / dark cycles with free
access to food and water. Rats were housed 2 per cage, until intraplantar injection (see
below) after which they were caged individually.
Model of hind paw inflammatory pain
The method of inducing hind paw inflammatory pain was comparable to that
previously reported by this lab [121, 146, 147]. All rats were lightly anesthetized with
isoflurane before receiving a single intraplantar injection of 0.15 ml CFA (150 µg of
Mycobacterium butyricum, 85% Marcol 52, and 15% Aracel A mannide monoemulsifier;
Calbiochem, San Diego, CA) or saline (pH 7.4 and passed through a 0.22 micron filter)
into one hind paw, and were allowed to fully recover from anesthesia before being
returned to the housing facility.
Substance P Release from RVM Tissue Prisms
Four days following intraplantar injection of either CFA or saline, the rats were
euthanized by carbon dioxide asphyxiation. The rats were then weighed and hind paw
thicknesses were measured to verify the development of a localized inflammatory state.
Brains were quickly removed onto ice and the RVM isolated as previously described
[147]. To isolate the RVM, the cerebellum was removed to expose the brain stem. A 2
mm thick coronal section of brainstem beginning 3 mm and ending 5 mm rostral to the
obex was quickly dissected and placed on ice as this region contained the RVM [148]. A
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triangular section containing the RVM was further dissected, with the top corner of the
triangle on the midline and immediately ventral to the ventricle and the remaining corners
of the triangle on lateral edge of each pyramid. The RVM triangle was immediately
placed on a chilled aluminum disc, and then cross-chopped three times (60° angles, each
cut 300 microns) to yield tissue prisms [160]. The tissue prisms were immediately
collected into chilled Eppendorf tubes containing 0.5 ml of ice cold standard KrebsRinger buffer (125 mM NaCl, 2.5 mM KCl, 1.25 mM NaH2PO4, 2 mM CaCl, 1 mM
MgCl, 25 mM NaHCO3 and 25 mM glucose). Tissue prisms were subsequently aliquoted
into 96-well filter plates (1.2 microns, Millipore Multiscreen, Millipore Corporation,
Bedford, MA) and the ice cold buffer removed and discarded using a vacuum manifold
(Millipore Corporation, Bedford, MA).
Prisms were incubated in 200 µl of buffer at 37 °C for four consecutive 15 minute
periods, with filtrate collected by the vacuum manifold into 96 well plates after each
incubation period. Filtrates were stored on ice until analyzed for substance P
concentration on the same day. Freezing the filtrates for as little as overnight resulted in
a complete loss of substance P. At the completion of the release experiment, filter plates
containing tissue prisms were frozen until assessment of the amount of protein in the
prism . Substance P levels in the filtrate were assessed in duplicate (50-µl aliquots) using
a substance P enzyme immuno-assay kit (Cayman Chemical, Ann Arbor, MI) as directed
by the manufacturer.
Drugs and Depolarization Buffers
Potassium (K+) depolarization buffers were obtained by equimolar substitution of
sodium (Na+) for potassium in the standard Krebs-Ringer buffer detailed above. For the
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50 mM K+ depolarization buffer, the Krebs Ringer buffer was prepared as follows: 77.5
mM NaCl, 50 mM KCl, 1.25 mM NaH2PO4, 2 mM CaCl, 1 mM MgCl, 25 mM NaHCO3
and 25 mM glucose. The 25 mM K+ buffer was prepared as follows: 102.5 mM NaCl, 25
mM KCl, 1.25 mM NaH2PO4, 2 mM CaCl, 1 mM MgCl, 25 mM NaHCO3 and 25 mM
glucose. NMDA, D-Serine and veratridine were obtained from Sigma Aldrich (St. Louis,
MO). All drugs were stored frozen as single use aliquots. To use, the frozen aliquots were
thawed on ice and then diluted to the specified concentration in standard Krebs Ringer
buffer or the two depolarization buffers described above. Veratridine, a sodium channel
activator, was dissolved in ethanol and then diluted to the appropriate concentration with
Krebs Ringer buffer such that the final concentration of ethanol was 0.5%.
Prism Protein Quantification
To measure prism protein , the frozen filter plates were thawed on ice. To each
well was added 200 µl of Solvable™ (PerkinElmer Life and Analytical Sciences, Boston,
MA) and the plate was incubated at 37 °C for 2 hours. The plates were then centrifuged at
787 G-force using a Beckman Coulter Allegra 6 centrifuge equipped with a Beckman
PTS 2000 rotor to collect filtrates into 96 well collection plates. All protein quantification
was performed in duplicate in 96-well plates using a BCA protocol (ThermoScientific,
Rockford, IL). Stock purified protein (2 mg / ml) was provided in the BCA kit.
Subsequent dilutions for the standard curve were made as directed by the manufacturer’s
protocol. The diluent for the standard curve was a 1:1 solution of Solvable™ and double
distilled water. Protein filtrates were also diluted 1:1 with double distilled water to yield
concentrations on the linear range of the standard curve. Protein sample concentrations
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were assessed by measuring optical density (560 nm) using a Spectramax 384 plus plate
reader.
Experimental Design
Experiments were designed such that each prism sample within the treatment
groups (CFA or saline) was obtained from different rats i.e. the sample size indicated in
the figure legend also represents the number of rats used in each treatment group. In
experiments with multiple sub-treatment groups (e.g. CFA + veratridine and CFA +
veratridine + NMDA), RVM prisms prepared from a single rat of a treatment group were
used for all matching sub-treatment groups, e.g. in the example above, aliquots of RVM
prisms from each CFA treated rat were used in both the ‘CFA + veratridine’ and ‘CFA +
veratridine + NMDA’ sub-treatment groups. All substance P release experiments were
similarly designed. RVM prisms were incubated in buffer for four 15-minute periods.
Preliminary experiments indicated that basal release of substance P stabilized by the
second period and remained constant through periods three and four. Thus, period 2 was
considered to reflect basal release. Unless otherwise stated, all pharmacological
manipulations occurred during period 3. Data were expressed both as mean ± S.E.M. pg
substance P / µg protein and as a percent of basal release using the value determined for
that sample.
Statistical Analysis
All data are expressed as mean ± S.E.M pg substance P/µg protein. Student’s ttest was used to compare basal release from prisms of saline and CFA treated rats. Twoway analysis of variance for repeated measures was used to compare release between
different treatment conditions. For this analysis, treatment was one factor and period was
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the second, repeated factor. Posthoc comparisons among group mean values were made
using Newman-Keul’s test. Substance P release was also expressed as percent basal
release, in which release during period 2 was designated as the basal value and equated to
100%.
Results
Evoked, but not Basal Release is Calcium-Dependent
The first set of experiments confirmed the calcium-dependence of substance P
release from RVM prisms (Figure 4.1). RVM prisms prepared from naïve rats were
incubated in either standard Krebs buffer or in calcium free buffer for all four periods.
Release of substance P during the first period was relatively high and of comparable
values whether or not the buffer contained calcium (P > 0.6). Basal release of substance
P was significantly decreased by period 2, and again the levels of release were unaffected
by the absence of calcium (P > 0.9). Upon depolarization with Krebs buffer containing
50 mM K+ in the presence of calcium, substance P release increased by three-fold. In
contrast, this same stimulus failed to increase substance P release in calcium free buffer
(Figure 4.1). Upon removal of the depolarization stimulus in the fourth period, substance
P release returned to (or remained at) basal levels. These data indicate that basal release
of substance P from prisms is not calcium dependent, whereas release induced by
depolarization requires calcium. For the purpose of clarity and because period 1 is a
washout period, the data for period 1 are excluded from all subsequent analyses and
graphs.
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Basal Release of Substance P is Similar in Saline and
CFA-treated Rats
To establish whether basal release of substance P from RVM prisms of CFA
treated rats is greater than that of saline treated rats, data for period 2 from all the
experiments were pooled to yield a single mean and S.E.M. for prisms from saline treated
rats and CFA treated rats. Basal release in prisms from saline-treated rats was 0.020 ±
0.001 pg/µg protein (N = 104) and 0.020 ± 0.001 pg/µg protein (N = 105) from prisms of
CFA treated rats (P > 0.6). Thus, peripheral inflammatory injury does not increase basal
release of substance P.
Potassium Evoked Release of Substance P is Similar in
RVM Prisms of Saline and CFA-treated Rats
A depolarizing stimulus of 50 mM K+ was chosen for these experiments based on
robust release obtained in pilot experiments. Incubation of prisms with 50 mM K+ buffer
increased substance P release to a similar extent in saline- and CFA-treated rats (P > 0.4)
(figure 4.2). The increased variance in the saline treated group is attributed to one rat
whose basal release was lower than other samples, but did not qualify as an outlier.
NMDA Alone Does Not Evoke Substance P Release in the Absence of Depolarization
Incubation in either 10 or 100 µM NMDA (Figures 4.3 and 4.4) did not evoke any
appreciable release of substance P from prisms of either saline or CFA-treated rats (P >
0.1 both concentrations). To exclude the possibility that this finding was a result of
insufficient levels of the obligate NMDA receptor co-agonist at the glycine binding site,
the experiments were repeated in the presence of 10 µM D-Serine using prisms from
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naive rats (Figure 4.5). Neither concentration of NMDA increased the release of
substance P even in the presence of 10 µM D-Serine (P > 0.3 both concentrations).
NMDA Facilitates Evoked Release of Substance P from
Prisms of Saline- and CFA-treated Rats.
To test whether NMDA receptor activation facilitates substance P release in RVM
prisms, and whether this facilitation is enhanced following peripheral injury, 10 µM
NMDA was added to the 50 mM K+ depolarizing buffer (Figure 4.6). As expected 50
mM K+ depolarizing buffer significantly increased substance P release from prisms of
both CFA and saline treated rats. Addition of 10 µM NMDA resulted in an even greater
increase in substance P release in the CFA and saline groups compared to release
obtained with the 50 mM K+ depolarizing buffer alone (P < 0.01) (Figure 4.6). Upon
removal of the 50 mM K+ depolarization buffer (and 10 µM NMDA) during the fourth
period, substance P release for all four groups returned to basal levels. This result
demonstrates that while the 50 mM K+ depolarization buffer causes similarly robust
release of substance P from both the CFA and saline prisms, addition of NMDA results in
facilitation of substance P release in both groups. Contrary to the hypothesis, NMDA did
not produce a significantly greater facilitation in CFA-treated rats (P > 0.6).
To explore the possibility that a differential facilitation was not observed because
50 mM K+ is a supramaximal stimulus, the experiments were repeated using 25 mM K+
depolarizing buffer (Fig 4.7). Approximations based on the Goldman-Hodgkin-Katz
equation indicate that 25 mM K+ should raise the resting membrane potential to -47 mV,
which would make this a marginal depolarization stimulus. This expectation was
confirmed when the subsequent data were analyzed. A very small, but statistically
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significant increase occurred in saline treated rats, but not in CFA treated rats; however,
the effect was not different between the groups (P > 0.6). Further examination of the data
revealed that addition of 25 mM K+ depolarizing buffer caused a 50% or greater increase
in substance P release in only 4 of 8 samples from saline-treated rats and 2 of 8 samples
from CFA treated rats. The proportion of responsive samples was not significantly
different (χ2 test, P > 0.5).
Figure 4.8 depicts the results obtained when prisms were exposed to 25 mM K+
depolarizing buffer in the presence of 10 µM NMDA. The addition of 10 µM NMDA
significantly enhanced substance P release only in prisms from saline treated rats (P <
0.01), and did not significantly increase release from prisms of CFA treated rats (P > 0.4).
To determine whether NMDA facilitation of substance P release in CFA and saline
groups was dependent on NMDA concentration, two other concentrations of NMDA (100
and 1 µM NMDA) were paired with the 25 mM K+ depolarization buffer. The addition of
100 µM NMDA to the 25 mM K+ depolarization buffer again significantly increased
substance P release from prisms of saline-treated rats, and did not produce a statistically
significant increase in prisms from CFA treated rats (Fig. 4.9). Addition of 1 µM NMDA
did not increase the release of substance P from prisms of either saline or CFA treated
rats (data not shown).
Figure 4.10 depicts the results obtained when RVM prisms from naive rats were
exposed to 25 mM K+ depolarizing buffer and NMDA with or without 10 µM D-Serine.
The addition of D-Serine did not further enhance the release produced by NMDA (P >
0.7). These data indicate that the obligatory co-agonist was not a limiting factor in the
findings.
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Veratridine Evoked substance P Release
The results obtained using K+ as a means to evoked release were contrary to our
hypothesis. To determine whether this finding was stimulus-dependent, veratridine was
used to evoke release in the next experiments. Veratridine functions by binding to voltage
gated sodium channels in a manner that causes them to remain open, ultimately leading to
increases in intracellular calcium and causing neurotransmitter release. This is in contrast
to depolarization with high extracellular potassium concentrations that reverses the
membrane potential, causing the opening of voltage gated ion channels ultimately leading
to neurotransmitter release. As these two mechanisms are sufficiently dissimilar, using
veratridine to evoke substance P release from CFA and saline prisms is an appropriate
means to ensure the results already detailed in this chapter regarding evoked release of
substance P are not dependent on the means of depolarization.
RVM prisms from CFA and saline treated rats were incubated with 50 µM
veratridine in the presence or absence of 10 µM NMDA (Figure 4.11). The experimental
design was adjusted so that aliquots of the same tissue were exposed to veratridine with
and without NMDA so that a paired t-test could be used to assess the significance of any
facilitation produced by NMDA. Prisms from saline and CFA treated rats responded to
50 µM veratridine with robust substance P release of comparable magnitude. Addition of
10 µM NMDA to the veratridine significantly increased release of substance P from the
prisms of saline treated rats (P < 0.05), but did not significantly (P = 0.06) increase
release from prisms of CFA treated rats. The interaction of 10 µM NMDA and a lower
concentration of veratridine, 25 µM, was also investigated (Figure 4.12). Again, the
magnitude of the increase did not differ between prisms from saline and CFA treated rats.
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Addition of 10 µM NMDA significantly increased the release of substance P from prisms
of saline treated rats, but failed to significantly (P = 0.077) increase release in CFA
treated rats. In contrast to 50 µM K+, substance P levels did not immediately return to
basal values follo0wing removal of the 25 µM veratridine during the fourth period.
Discussion
The current study investigated two major questions regarding substance P release
in the RVM. First, does a peripheral inflammatory injury alter substance P release (basal
and evoked) in the RVM? Secondly, does NMDA receptor activation facilitate substance
P release in RVM, and is this effect enhanced after peripheral inflammatory injury? The
findings do not support either hypothesis, and suggest that different factors are
responsible.
Basal Release
Basal release of substance P was not enhanced in prisms from CFA treated rats. In
retrospect, this finding may not be surprising. Basal release was not calcium-dependent.
Moreover, no difference was found in substance P levels in the RVM of CFA and saline
treated rats. Finally, recent data indicate that internalization of NK1 receptors is only
observed after delivery of a noxious stimulus [47], i.e. release of substance P is evoked.
Evoked Release
Contrary to expectations, potassium evoked release was not enhanced in prisms
from CFA treated rats. This finding was surprising because in vivo studies demonstrated
that a noxious heat stimulus significantly increased the number of RVM neurons that
internalized the NK1 receptor in CFA treated rats compared to saline treated rats [47].
Moreover, whole cell patch clamp recordings of spinally projecting neurons
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demonstrated that excitatory inputs were facilitated, a facilitation that could be blocked
by an NK1 antagonist and recapitulated in the RVM in naïve rats by application of
exogenous substance P [56]. Additionally, immunohistochemical data described in
chapter II of this thesis showed four days after peripheral inflammatory injury, more
substance P appeared mobilized to terminals, a finding suggestive of an increase in the
releasable pool of substance P.
Use of another depolarizing agent confirmed that evoked release of substance P is
not enhanced in the RVM of CFA treated rats. When depolarized with high
concentrations of potassium, the membrane potential is reversed leading to the opening of
several voltage gated ions channel opening and ultimately calcium channel opening
which will lead to neurotransmitter release. While this leads to robust release of
neurotransmitter, the membrane remains depolarized as long as the high extracellular
potassium is present. This does not allow the voltage gated ion channels to transition
from their inactivated state to the closed state, when they can be activated again. As the
voltage gated ion channel population is finite, a state where there are insufficient
channels that can be activated is eventually reached, meaning no more neurotransmitter
released. This is in contrast to veratridine, which functions by activating voltage gated
sodium channels. While this ultimately results in calcium channel activation and
neurotransmitter release, binding kinetics allow for the activated channel to cycle through
to the inactivated then closed state, where it is available to be activated again. This cycle
can theoretically occur repeatedly for the duration of the veratridine incubation, yielding
neurotransmitter release each time. If this duration is long enough, the veratridine will
eventually lead to more neurotransmitter release. Both 25 and 50 µM veratridine
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produced a 6- to 7-fold increase in substance P release suggesting that any findings with
the K+ depolarizing stimulus were not limited by a ceiling effect.
There may be several explanations for this unexpected finding. Using
electrophysiology, it is possible to identify individual spinally projecting neurons of
specific subtypes, a level of selectivity not possible with the method used in this study.
Preparation of prisms may have interfered with the functioning of substance P afferents
to the RVM. Release of substance P from the terminals of substance P neurons in the
RVM could also have masked increased release from a subpopulation of afferents to the
RVM. Finally, it is also possible that the disparity between the current results and the
hypothesis is a result of well documented developmental changes that occur in the RVM
[161, 163] during the transition from preadolescence pup to the adult rat. These changes
are relevant here as the study hypothesis was partly based on data from rat pups, while
the current experiments were completed using adult rats. This possibility can be
addressed by performing these substance P release experiments RVM prisms from rat
pups. These experiments are intriguing as they potentially bridge the current results and
the hypothesis, and shed more light on documented developmental changes occurring in
the RVM.
NMDA Mediated Facilitation of Substance P release
The data show that NMDA is able to facilitate substance P release from RVM
prisms when coupled with a depolarization stimulus. This finding shows that the
presynaptically located GluN1 channels on substance P positive terminals identified
earlier in this thesis are functional and their activation results in substance P release from
the axon terminal. This finding is in line with other work in the spinal cord showing that
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NMDA receptor activation, most likely presynaptic, results in substance P release [134136]. It also provides more evidence for the growing body of work linking the NMDA
receptor and substance P / NK1 signaling systems as discussed in chapter I of this thesis.
Interestingly, the current data show that NMDA application in the absence of any
depolarization does not result in substance P release, in contrast to the spinal cord
experiments where NMDA alone is able to induce NK1 internalization [133-135]. An
explanation for this difference might be that the studies in the spinal cord used slices,
which would have retained more of the native circuitry compared to tissue prisms. It is
also possible that spinal cord slices are able to retain more of the post-translational
modifications to the NMDA receptor that have been demonstrated as important for
substance P release [133]. It is also possible that differences might be inherent to the
different tissues sampled.
There was no clear difference in NMDA facilitation of substance P release
following peripheral inflammatory injury. This lack of a difference in NMDA facilitated
substance P release following inflammatory injury was unexpected. There are many
potential reasons for this result, some of which have already been discussed earlier in this
section (e.g. lack of selectivity of the method, developmental changes).
It is also worth noting that a third population of spinally projecting non-serotonergic
neurons exist that do not show facilitation of excitatory inputs following peripheral
inflammatory injury. Interestingly, application of an NK1 receptor antagonist decreased
the spontaneous activity of these neurons and significantly reduced the slope of the inputoutput curve of type 3 neurons in CFA-treated rats. This suggests that this population of
spinal projecting neurons may receive tonic inputs from substance P afferents that are not
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changed by inflammatory injury [56]. While all three types of RVM neurons are equally
represented within the RVM [164], differences that may occur in substance P inputs to
types 1 and 2 spinally projecting non-serotonergic neurons might be masked by the lack
of difference in the type 3 neurons along with other substance P inputs to non-spinally
projecting neurons. Finally, although admittedly not quantitative, the electron
microscopic findings did not reveal any obvious differences in the incidence of GluN1
labeling of substance P containing afferents in the RVM.
Sufficient Obligate Co-Agonist
The data showed that in the RVM prisms, D-Serine was without effect when
coupled with NMDA in the presence and absence of depolarization. This is in contrast to
spinal cord experiments where it is a standard component of experiments examining the
role of NMDA receptors in neurotransmitter release [133, 139]. D-Serine, along with
glycine, functions as a co-agonist of the NMDA receptor, where it is needed, along with
glutamate or NMDA and membrane depolarization for NMDA receptor activation [125].
As it was without effect in these experiments, this suggests that there is sufficient
endogenous co-agonist in the RVM prism preparation.
Conclusions
In conclusion, the data discussed in this chapter indicate that substance P release
in the RVM, both basal and evoked, is not different after a peripheral inflammatory injury.
NMDA receptor activation facilitates substance P release, but this facilitation is not
enhanced after peripheral inflammatory injury. Lastly, NMDA evoked release of
substance P from RVM prisms does not occur in the absence of depolarization. The
finding that peripheral inflammatory injury does not alter substance P release or NMDA
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receptor dependent facilitation of substance P release is contrary to the study hypothesis.
While there are a few confounding factors, these data point to a post-synaptic basis for
documented changes in substance P / NK1 receptor signaling following peripheral
inflammatory injury. These include different recruitment of second messenger systems
like PKC following peripheral inflammatory injury [56]. They also include increases in
the number of neurons expressing NK1 following peripheral inflammatory injury as
recently reported by this lab [47]. Other postsynaptic explanations include changes in
binding properties of the receptor to substance P and or changes to surface expressed
receptors following injury. These options have not been directly tested.
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Figure 25. Evoked, but not basal release of substance P is calcium dependent.
RVM prisms were obtained from naïve rats. Panel A shows substance P measured
from releasates incubated with calcium containing buffers (filled circles) or calcium
free buffers (open circles). Incubations during periods 1, 2 and 4 were with Krebs
Ringer buffer while period 3 was with 50 mM K+ Krebs Ringer buffer. Panel B
depicts the same data expressed as a percent of values in period 2 for that sample,
which was set to 100%. Data are mean ± S.E.M. pg substance P/µg protein. N = 4
per group. ** P < 0.01 compared to period 2; ╪ P < 0.01 compared to calcium free
buffer at the corresponding period.
102
Figure 26. Substance P release in response to 50 mM K+ Krebs Ringer is similar
in prisms from saline and CFA treated rats. RVM prisms obtained from CFA (open
symbols) and saline (filled symbols) treated rats. Panel A shows substance P measured
from releasates incubated with Krebs Ringer buffer during periods 1, 2 and 4 and with
50 mM K+ Krebs Ringer buffer during period 3. Panel B depicts the same data
expressed as a percent of values in period 2 for that sample, which was set to 100%.
Data are mean ± S.E.M. pg substance P /µg protein. N = 6-8 samples per group. ** P
< 0.01 compared to period 2.
103
Figure 27. 10 µM NMDA alone does not increase release of Substance P from
RVM prisms of saline or CFA treated rats. RVM prisms obtained from CFA (filled
symbols) and saline (open symbols) treated rats. Panel A shows substance P measured
from releasates incubated with Krebs Ringer buffer during periods 1, 2 and 4 and with
10 µM NMDA in Krebs Ringer buffer during period 3. Panel B depicts the same data
expressed as a percent of values in period 2 for that sample, which was set to 100%.
Data are mean ± S.E.M. pg substance P/µg protein. N = 6 per group.
104
Figure 28. 100 µM NMDA alone does not increase release of Substance P from
RVM prisms of saline or CFA treated rats. RVM prisms obtained from CFA (filled
symbols) and saline (open symbols) treated rats. Panel A shows substance P measured
from releasates incubated with Krebs Ringer buffer during periods 1, 2 and 4 and with
10 µM NMDA in Krebs Ringer buffer during period 3. Panel B depicts the same data
expressed as a percent of values in period 2 for that sample, which was set to 100%.
Data are mean ± S.E.M. pg substance P/µg protein. N = 4-6 per group.
105
Figure 29. Addition of D-Serine does not enable either 10 or 100 µM NMDA to
release Substance P from RVM prisms of naive rats. Panel A shows substance P
measured from releasates incubated with Krebs Ringer buffer during periods 1, 2 and
4 and with 10 µM or 100 µM NMDA in Krebs Ringer buffer containing 10 µM DSerine during period 3. Panel B depicts the same data expressed as a percent of values
in period 2 for that sample, which was set to 100%. Data are mean ± S.E.M. pg
substance P/µg protein. N = 4 per group.
106
Figure 30. Addition of 10 µM NMDA increases substance P release evoked by 50
mM K+ Krebs Ringer from RVM prisms from saline and CFA treated rats. RVM
prisms obtained from CFA (filled symbols) and saline (open symbols) treated rats.
Panel A shows substance P measured from releasates incubated with Krebs Ringer
buffer during periods 1, 2 and 4. Incubations during period 3 were with 50 mM K+
Krebs Ringer buffer (circles) or with 50 mM K+ Krebs Ringer buffer and 10 µM
NMDA (squares). Panel B depicts the same data expressed as a percent of values in
period 2 for that sample, which was set to 100%. Data are mean ± S.E.M. pg
substance P/µg protein. N = 6 per group. ** P < 0.01 compared to period 2; P <
0.05, ╪ P < 0.01 compared to 50 mM K+ Krebs Ringer buffer alone at the
corresponding period.
107
Figure 31. Substance P release in response to 25 mM K+ Krebs Ringer is
different in prisms from saline and CFA treated rats. RVM prisms obtained from
CFA (filled symbols) and saline (open symbols) treated rats. Panel A shows substance
P measured from releasates incubated with Krebs Ringer buffer during periods 1, 2
and 4 and with 25 mM K+ Krebs Ringer buffer during period 3. Panel B depicts the
same data expressed as a percent of values in period 2 for that sample, which was set
to 100%. Data are mean ± S.E.M. pg substance P/µg protein. N = 8 samples per
group. * P < 0.05 compared to period 2.
108
Figure 32. Addition of 10 µM NMDA increases substance P release evoked by 25
mM K+ Krebs Ringer from RVM prisms from saline and CFA treated rats. RVM
prisms obtained from CFA (open symbols) and saline (filled symbols) treated rats.
Panel A shows substance P measured from releasates incubated with Krebs Ringer
buffer during periods 1, 2 and 4. Incubations during period 3 were with 25 mM K+
Krebs Ringer buffer and 10 µM NMDA. Panel B depicts the same data expressed as a
percent of values in period 2 for that sample, which was set to 100%. Data are mean ±
S.E.M. pg substance P/µg protein. N = 7-8 per group. ** P < 0.01 compared to
period 2.
109
Figure 33. Addition of 100 µM NMDA increases substance P release evoked by
25 mM K+ Krebs Ringer from RVM prisms from saline, but not CFA treated
rats. RVM prisms obtained from CFA (filled symbols) and saline (open symbols)
treated rats. Panel A shows substance P measured from releasates incubated with
Krebs Ringer buffer during periods 1, 2 and 4. Incubations during period 3 were with
25 mM K+ Krebs Ringer buffer and 100 µM NMDA. Panel B depicts the same data
expressed as a percent of values in period 2 for that sample, which was set to 100%.
Data are mean ± S.E.M. pg substance P/µg protein. N = 8-10 per group. * P < 0.05,
** P < 0.01 compared to period 2.
110
Figure 34. Substance P measured from RVM prism releasates depolarized with
25 mM K+ Krebs Ringer, 10 µM D-Serine and 10 µM NMDA during period 3.
RVM prisms obtained from naïve rats. Panel A shows substance P measured from
releasates incubated with Krebs Ringer buffer during periods 1, 2 and 4 and with 25
mM K+ Krebs Ringer, 10 µM NMDA and 10 µM D-Serine in Krebs Ringer buffer
(filled circles) or 25 mM K+ Krebs Ringer and 10 µM NMDA (open circle) during
period 3. Data are mean ± S.E.M. pg substance P/µg protein. N = 8 per group. Panel
B depicts the same data expressed as a percent of values in period 2 for that sample,
which was set to 100%. * P < 0.05, ** P < 0.01 compared to period 2, two-way
repeated measures ANOVA.
111
Figure 35. 10 µM NMDA facilitates substance P release induced by 50 µM
veratridine in RVM prisms from saline, but not CFA treated rats. RVM prisms
obtained from CFA (filled symbols) and saline (open symbols) treated rats. Panel A
depicts substance P measured from releasates incubated with Krebs Ringer buffer
during periods 1, 2 and 4. Incubations during period 3 were with 50µM veratridine in
Krebs Ringer buffer (circles) or with 50 µM veratridine and 10 µM NMDA in Krebs
Ringer buffer (squares). Data are mean ± S.E.M. pg substance P/µg protein. N = 8 per
group. Panel B depicts the same data expressed as a percent of values in period 2 for
that sample, which was set to 100%. ** P < 0.01 compared to period 2, two-way
repeated measures ANOVA; P < 0.05 paired t-test compared to corresponding
control in period 3.
112
Figure 36. 10 µM NMDA facilitates substance P release induced by 25 µM
veratridine in RVM prisms from saline, but not CFA treated rats. RVM prisms
obtained from CFA (filled symbols) and saline (open symbols) treated rats. Panel A
depicts substance P measured from releasates incubated with Krebs Ringer buffer
during periods 1, 2 and 4. Incubations during period 3 were with 25 µM veratridine in
Krebs Ringer buffer (circles) or with 25 µM veratridine and 10 µM NMDA in Krebs
Ringer buffer (squares). Data are mean ± S.E.M. pg substance P/µg protein. N = 8 per
group. Panel B depicts the same data expressed as a percent of values in period 2 for
that sample, which was set to 100%. * P < 0.05, ** P < 0.01 compared to period 2,
two-way repeated measures ANOVA; P < 0.05 paired t-test compared to
corresponding control in period 3.
113
CHAPTER V
CONCLUSIONS
It is evident that following peripheral inflammatory injury, substance P assumes a
pro-nociceptive role within the RVM, where it is intimately involved in the maintenance
of hypersensitivity to various noxious stimuli [121-123]. The means by which substance
P assumes this role after injury remains unclear. A potential mechanism is that following
peripheral tissue injury and inflammatory injury, substance P levels within the RVM are
increased. Alternatively, the mechanism could be altered pre-synaptic NMDA receptor
regulation of substance P release following injury, especially since the NMDA receptor
expression is known to be upregulated following peripheral inflammatory injury [140].
The experiments detailed in this thesis explored the hypothesis that peripheral
inflammatory injury results in a time dependent increase in levels or release of substance
P, resultant from altered regulation by pre-synaptic NMDA receptors in the RVM.
Substance P Levels within the RVM following peripheral Inflammatory Injury
Substance P levels were measured within the RVM at various time points (4 hours,
4 days and 2 weeks) in rats injected with CFA or in saline controls. Contrary to the
hypothesis, there were no differences in substance P levels within the RVM at any of the
time points tested. However, changes in substance P levels that may involve discrete
regions within the RVM could be masked when assessing levels of tissue homogenates.
Therefore, an immuno-histochemical (IHC) approach in conjunction with computer aided
densitometry was added to assess possible changes in substance P immunolabeling in
serial sections of the RVM from CFA (4 day and 2 week) and saline control rats. Within
the 4 day group, immunolabeled substance P within the RVM was greater in both CFA
groups compared to the saline group. In the 2 week group, while substance P labeling
114
was again increased in the CFA groups, these increases were not as robust as observed in
the 4 day group. This study could be further focused to assess changes of substance P
inputs to RVM neurons that project to the spinal cord, particularly as spinally projecting
neurons are hypothesized to play a role in the facilitation of nociception under conditions
of persistent inflammatory injury. Retrograde tracers could be used to label spinally
projecting neurons [46], which constitute approximately 39 % of all RVM neurons [158].
Immunostaining for substance P would enable us to determine whether substance P
inputs to these specific neurons are altered. This approach would enable us to ask
whether the percentage of spinally projecting neurons receiving substance P inputs is
altered following inflammatory injury, or whether the density of inputs per spinally
projecting neuron is altered following injury. This study could also be paired with
immunolabeling of the NK1 receptor. Nonetheless, these studies all suffer from the
limitation that they are static measurements of tissue levels. Thus, the incorporation of
approaches that permit assessment of dynamic events at the synapse is also required.
Colocalization of NMDA receptors and substance P in the same presynaptic terminals
One approach to assess dynamic events at the synapse involves measurement of
neurotransmitter release either from synaptosomes, tissue slices or prisms or by in vivo
microdialysis. However, such studies are best undertaken when there is evidence of a
neuroanatomical basis for receptor action. Chapter 3 of this thesis sought to document
that NMDA receptors are indeed situated presynaptically on substance P containing axon
terminals in the RVM. Colocalization was observed in RVM sections obtained from both
CFA and saline treated rats. This study was intended to by a qualitative assessment of
whether NMDA receptors are located on the same presynaptic terminals as substance P.
115
This localization is significant as it identifies NMDA receptors in a position where they
can regulate substance P release. What was not assessed was whether the extent of this
colocalization is altered following peripheral inflammatory injury. This question can be
answered by extending this study to include a quantitative component that would allow
for assessment of the effect of peripheral inflammatory injury on the quantity and or
morphology of presynaptic terminals containing both substance P and the NMDA
receptor. This technique has been employed at the ultrastructural level [163, 164].
Additionally, incorporation of a tracer to label spinally projecting neurons can provide
more context to this study, as it would allow for identification of substance P (with and
without the NMDA receptor) inputs to spinally projecting neurons and determination of
whether injury changes the total number and or percentage of these inputs.
This localization of NMDA receptors on the presynaptic terminal is a departure
from its established postsynaptic role [125]. However, it adds to the growing body of
literature recognizing the existence and importance of presynaptic NMDA receptors in
regulating neurotransmitter release [48, 165-167].
Substance P release (basal and evoked) and its regulation by NMDA receptors
Neither basal nor evoked (potassium or veratridine) substance P release were
different between RVM prisms obtained from CFA (4 days) or saline treated rats. This
was a surprising finding in light of background data showing facilitation of excitatory
inputs to a subset of spinally projecting RVM neurons following peripheral inflammatory
injury [56]. This facilitation was dependent on NMDA receptor and NK1 activity.
Additionally, IHC data presented in this thesis was presumptive of increased mobilization
of substance P to terminals following peripheral inflammatory injury.
116
A potential explanation for the disparity between the background data and the
current results is that the data showing facilitation of excitatory inputs to a subset of
spinally projecting neurons was obtained in preadolescent rat pups. In contrast, the
current data were obtained in adult rats. Given this gap in age, developmental changes
may underlie the differences between the background and current data. Developmental
changes have been documented in the RVM [161, 163]. The possible role for
developmental changes can be investigated by repeating some of the release experiments
looking at basal and evoked release of substance P in rat pups. If those data are not
different from the data presented in this thesis, then developmental changes do not
underlie the disparity between the background and current data. Another possibility is
that electrophysiology, as employed in the background experiments, allows for exquisite
targeting of a particular subset of spinally projecting neurons. That level of specificity is
lost when measuring substance P in from RVM prisms. Even if there are increases in
substance P inputs to a subset of neurons following injury, such changes (unless
overwhelming) would be diluted by other substance P within the RVM.
NMDA receptor activation facilitated substance P release in evoked by both
veratridine and potassium. The extent of facilitation did not differ between prisms
obtained from CFA and saline RVM. The finding that NMDA receptor activation
facilitates substance P release is in line with several others showing involvement of
NMDA receptors in substance P release [134-136]. The finding that there was no
difference in substance P release from RVM prisms after injury was surprising. However,
the possible explanations discussed earlier to explain the lack of a difference in basal and
evoked release of substance P apply here.
117
Hints regarding how pre-synaptic NMDA receptors regulate substance P release
are provided by some recent reports. Chen and colleagues showed that NMDA evoked
NK1 internalization in the spinal cord was not blocked by either lidocaine or ω-conotoxin
[133]. Based on this finding, the authors concluded that NMDA evoked substance P
release did not require the firing of action potentials or the opening of voltage gated
calcium channels. Additionally, the authors demonstrated that NMDA receptors involved
in substance P release contained the GluN2B subunit, and that the release process was
regulated by Src family kinases. Kunz and colleagues further show that neurotransmitter
release mediated by NMDA receptors was dependent on extracellular calcium and
sodium, and partially regulated by protein kinase C [166]. Extrapolating from these
results to the present data, it is possible that the presynaptic NMDA identified cause
release of substance P by allowing sufficient sodium and calcium into the presynaptic
terminal to culminate in neurotransmitter release in a manner dependent on Src family
kinases and PKC.
Present Findings relative to NK1 antagonist failures as analgesics
The failure of NK1 receptor antagonists as analgesics in clinical trials, despite the
wealth of preclinical evidence suggesting they would be great analgesics, has been well
chronicled [168-170]. The studies described in this thesis were part of a larger effort in
the laboratory to focus on substance P signaling within the RVM. The majority of the
preclinical data regarding substance P signaling focused on its roles in the periphery as
well as in the spinal cord. Relatively nothing was known about its signaling in the
brainstem, including the RVM. It was therefore possible that uncovering novel actions of
substance P within the brainstem, e.g. a predominantly anti-nociceptive role, would
118
reconcile the disparity between the preclinical studies of substance P in pain and the
failures of NK1 antagonists in clinical trials. As these studies draw to a close,
reconciliation of the previously mentioned disparity is not in the offing. If anything, the
present data and other recent findings support the idea that substance P functions in a
pronociceptive manner following peripheral inflammatory injury, and thus NK1
antagonists should have been successful in clinical trials. The failure of this class of drugs
in clinical trials remains a mystery.
Disclosure Regarding Kits used to Measure Substance P
Direct measurement of substance P was an integral part of data collection for this
thesis. This was accomplished by use of an enzyme immuno-assay (EIA) kit,
commercially available from Cayman Chemical (Ann Arbor, Michigan). In May of this
year, after completion of all the experiments detailed in this thesis, communication from
Cayman Chemical revealed that in the process of developing an EIA kit for hemokinin1(HK-1), they discovered that their substance P kit was unable to distinguish between
HK-1 and substance P. HK-1 is a recently discovered tachykinin that is expressed in both
the periphery as well as the central nervous system, including the brainstem [171, 172].
Additionally, HK-1 and substance P bind to the NK1 receptor with identical affinity [171,
173], and HK-1 is thought to be a full agonist at the receptor as NK1-dependent calcium
influx is identical when either substance P or HK-1 is used as the agonist [173]. As a
result, it is necessary to acknowledge that while the conclusions of the substance P levels
and release studies included in this thesis have focused on substance P, it is likely that
substance P, in some combination with HK-1 was measured in those studies.
119
Concluding Remarks
Substance P remains a relevant topic of research decades since its discovery. This is
especially true within the pain field, where substance P signaling was once as promising
as any other druggable target for the treatment of pain (for review, see [168, 169]). The
failure of drugs targeting substance P signaling underscored the need for greater
understanding of the myriad actions of substance P, especially beyond the periphery and
the spinal cord. This thesis explored the actions of substance P within the RVM in the
context of injury and in relation to its regulation by NMDA receptors. The data show the
RVM contains significant amounts of substance P and that presynaptically located
NMDA receptors are able to regulate its release. However, the data contained herein do
not provide evidence for presynaptic changes in substance P levels or release underlying
reported changes in substance P’s role following injury. While the case can still be made
for presynaptic changes, it is likely that post-synaptic changes in receptor properties and
distribution, as well as second messenger systems, serve as the mechanistic bases for
substance P’s actions following peripheral inflammatory injury. These changes can
include increased number of neurons within the RVM that express the NK1 receptor [47],
providing more sites of action for presynaptically released substance P. They can also
include increased synthesis of AMPA receptors, as well as increased activation of protein
kinase C (PKC) downstream of NK1 activation which can phosphorylate AMPA
receptors and lead to their increased insertion to the membrane [174, 175], leading to
amplification of signaling by presynaptically release glutamate. These types of
postsynaptic changes can result in amplification of excitatory inputs following peripheral
inflammatory injury.
120
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