Govea-Dissertationdoctoral

2015

The Dissertation Committee for Rosann Mae Govea Certifies that this is the approved version of the following dissertation:

Group III mGluR8 negatively modulates TRP channels

Committee:

Susan M Carlton, Ph.D., Mentor

Darren Boehning, Ph.D.

Hongzhen Hu, Ph.D.

Volker Neugebauer, M.D., Ph.D.

Shao-Jun Tang, Ph.D.

______

Dean, Graduate School of Biomedical Sciences Group III mGluR8 negatively modulates TRP Channels

Rosann Mae Govea, B.S.

Dissertation Presented to the Faculty of the Graduate School of The University of Texas Medical Branch

in Partial Fulfillment of the Requirements for the Degree of

Doctor of Philosophy

The University of Texas Medical Branch March, 2015

Dedication

To my best friend and close confidant Chris Sommerfeld. To my parents Pete and Lucy Govea and my sisters: Roxann, Lucinda, and Mercedes. Thank you for all of your support and encouragement. To my mentor, Dr. Susan Carlton, you are an inspiration. To my very close friend Audrie Medina … our escapes and fantasies of being farmers.

Acknowledgements

Chris, thank you for all of your love and support throughout the years. I appreciated the times you would stay up late to keep me company while I was working. I’d like to thank my family and close friends. First, my parents Pete and Lucy thank you for your encouragement and support. Y’all have always been supportive of all of us. My sisters: Roxann, Lucinda and Mercedes. Thank you for always being there. The months leading up to this point have been rough, and I wouldn’t have been able to come this far without a strong support group. Shengtai, Zhixia, Greg, Junhui, and Dale, thank you for having patience with me as I learned techniques. I have gained a lot of knowledge from all of you. I felt as if I was part of the lab family as soon as I joined the lab. Alyssa, my labmate, I was so glad when you joined the lab. I enjoyed all of our discussions, I’ll miss them. Thanks for being a listening ear when I needed it.

Dr. Carlton, first, thank you for allowing me to join your lab as part of the PREP program. I have come so far from that first day. Thank you for all of our encouragement and support throughout the years. Thank you for your understanding and patience, especially in the months leading up to now. Audrie Medina, I am so fortunate to be your friend. We hit it off from the first time we met. I will miss our coffee break escapes. Thanks for all of your support throughout the years. Dr. Niesel, thank you for your continual support and words of encouragement.

iv Sally, my therapist, you’ve helped me so much. The months leading up to now have been rough to say the least. I would not have made it this far without your help. Lastly, I would like to acknowledge my committee members Dr. Darren Boehning, Dr. Hongzhen Hu, Dr. Volker Neugebauer, and Dr. Shao-Jun Tang. Thank you all for serving on my dissertation committee. I appreciate all of the advice and support.

v Group III mGluR8 negatively modulates TRPA1 activity

Publication No.______

Rosann Mae Govea, Ph.D.

The University of Texas Medical Branch, 2015

Supervisor: Susan M. Carlton, Ph.D.

Several lines of evidence indicate group III metabotropic glutamate receptors

(mGluRs) have systemic anti-hyperalgesic effects. We hypothesized this could occur through modulation of TRP channels on nociceptors. The following studies used a multifaceted approach to examine the interaction between group III mGluRs (mGluR8) and two TRP channels: TRPV1 and TRPA1. In the first study, we examined the interaction between group III mGluRs and TRPV1. Anatomical studies demonstrated that group III mGluR8 is expressed on cutaneous axons and is co-localized with TRPV1 in the dorsal root ganglia (DRG). In behavior studies, peripheral activation of group III mGluRs had no effect on paw withdrawal latency (PWL) to heat in naïve rats. However, local peripheral activation of group III mGluRs significantly attenuated capsaicin (CAP,

TRPV1 agonist)-induced lifting/licking and reduced flinching behavior. Finally peripheral group III mGluR activation reversed forskolin (FSK)-induced heat sensitivity, suggesting that group III could modulate TRPV1 by down-regulating cAMP-PKA activity. In the second part of the study, we examined the interaction between group III mGluR8 and TRPA1. Ca2+ imaging studies demonstrated co-localization and functional

vi coupling of TRPA1 and mGluR8, since DCPG (mGluR8 agonist) significantly reduced the number of mustard oil (MO, TRPA1 agonist) responsive cells. Behavioral studies demonstrated that peripheral DCPG reversed the MO-induced decrease in PWT. At the single fiber level, DCPG significantly attenuated MO-induced nociceptor activity and reversed the MO-induced decrease in mechanical threshold. Furthermore, DCPG significantly reduced the number of MO-induced mechanically sensitive fibers. Inhibition of PKA using RpCAMPS significantly reduced MO-induced calcium mobilization and there was a trend to reduce the number of MO-responsive cells. Taken together, these results show that group III mGluRs can negatively modulate TRPV1 and TRPA1 activity.

Furthermore, it is likely that this modulation occurs at the level of the cAMP/PKA pathway. Additionally, these studies demonstrate that group III agonists may be effective in treatment of mechanical allodynia which can develop as a result of inflammation, nerve injury, chemotherapy or other disease states.

vii TABLE OF CONTENTS

List of Tables ...... xii

List of Figures ...... xiii

List of Abbreviations ...... xiv

CHAPTER 1: EXPANDED INTRODUCTION ...... 17

Pain Pathways ...... 18

Spinothalamic tract ...... 18

Transient Receptor Potential (TRP) ion Channels and Pain ...... 19

TRP channel discovery ...... 19 TRPV1 ...... 20 TRPV1: more than a Pain Receptor ...... 21 TRPA1 ...... 22 TRPA1: more than a pain receptor ...... 23

Metabotropic Glutamate Receptors (mGluRs) ...... 24

Groups I, II, III ...... 24 mGluR8 localization ...... 25 Group III mGluRs (mGluR8 and pain) ...... 25 mGluR8: more than a pain modulator ...... 26

CHAPTER 2: GROUP III MGLURS NEGATIVELY MODULATE TRPV1 ACTIVITY .....28

Introduction ...... 28

Methods...... 30

Animal Use and Care ...... 30 Immunostaining of the digital nerve at the electron microscopic level ...... 30 Immunostaining the dorsal root ganglia at the light microscopic level ...... 31 Tissue Collection ...... 31 Immunohistochemistry ...... 32 Cresyl Violet ...... 32

viii Analysis...... 33 Behavior ...... 33 Drug Preparations ...... 33 Drug Injections...... 34 Habituation for behavioral testing ...... 34 Testing for thermal and CAP-induced behaviors ...... 35 Data Analysis ...... 35

Results ...... 36

mGluR8-labeled digital axons ...... 36 mGluR8 and TRPV1 are co-localized on rat DRG cells ...... 37 Group III mGluR agonist L-AP-4 attenuates CAP-induced nociceptive behaviors ...... 40 Group III mGluR activation modulates the cyclic AMP (cAMP)/Protein Kinase A (PKA) pathway ...... 44

Discussion ...... 45

Conclusion ...... 50

Acknowledgments...... 50

CHAPTER 3: GROUP III MGLUR8 NEGATIVELY MODULATES TRPA1 ACTIVITY ...51

Introduction ...... 51

Methods...... 53

Animal use and care ...... 53 Drug preparations ...... 53 Drugs: Topical Application and Injection ...... 53 Habituation for behavioral testing ...... 54 Testing for Mustard oil-induced behaviors and mechanical sensitivity ...... 54 Electrophysiological recordings ...... 55 In vitro skin-nerve preparation ...... 55 Thermal and chemical testing procedures ...... 55 Thermal stimulation...... 55 Mechanical stimulation...... 56

ix Chemical stimulation...... 56 Conduction velocity...... 56 Spike Sorting ...... 57 Calcium Imaging...... 57 Cell Culture ...... 57 Cytosolic Ca2+ imaging ...... 58 Data analysis ...... 58

Results ...... 59

DCPG reduces the number of MO-responsive cells ...... 59 DCPG attenuates MO-induced mechanical sensitivity ...... 60 DCPG attenuates MO-induced nociceptor activity and mechanical sensitivity ...... 62 PKA inhibition attenuates MO-induced Ca2+ mobilization ...... 66

Discussion ...... 68

mGluR8 and TRPA1 are expressed in the rat DRG ...... 68 mGluR8 and TRPA1 are co-localized in the rat DRG...... 69 mGluR8 activation attenuates TRPA1-induced mechanical sensitivity ...... 70 mGluR8 modulates TRPA1 activity through the cAMP/PKA pathway...... 71

Conclusion ...... 73

CHAPTER 4: EXPANDED DISCUSSION ...... 74

TRPV1/TRPA1 interactions ...... 74

TRP channel modulation...... 76

TRPV1 Sensitization...... 76 TRPV1 Desensitization...... 77 TRPA1 Sensitization...... 78 TRPA1 Desensitization...... 78 TRPA1 and cold sensitivity ...... 79

x mGluR Signaling ...... 83

Concluding Remarks ...... 84

REFERENCES ...... 85

VITA ……………………………………………………………………………….116

xi List of Tables

Table 2.1: Percent of mGluR8-labeled axons in hindpaw digital nerves ...... 37

Table 2.2: Percent mGluR8- and TRPV1-labeled cells in the DRG ...... 39

xii List of Figures

Figure 2.1: Localization of mGluR8 on axons in the digital nerves ...... 36

Figure 2.2: Co-localization of mGluR8 and TRPV1 in rat DRG ...... 38

Figure 2.3: Diameters of single- and double-labeled DRG neurons ...... 40

Figure 2.4: L-AP-4, a group III agonist, has no effect in the naïve state ...... 42

Figure 2.5: L-AP-4 produces an anti-hyperalgesic effect on CAP-induced nociception

...... 43

Figure 2.6: L-AP-4 inhibits FSK-induced heat sensitization ...... 44

Figure 3.1: DCPG Dose-Response Relationship ...... 60

Figure 3.2: DCPG inhibits MO-induced mechanical sensitivity in vivo ...... 62

Figure 3.3: DCPG attenuates MO-induced nociceptor activity in vitro ...... 64

Figure 3.4: DCPG attenuates the MO-induced decrease in mechanical threshold in

vitro ...... 65

Figure 3.5: DCPG reduces the number of mechanically sensitive fibers in vitro...... 66

Figure 3.6: RpCAMPS attenuates MO-induced Ca2+ mobilization ...... 67

Figure 4.1: MO decrease cold response in primary afferents ...... 81

Figure 4.2: DCPG reduced the number of cold responsive fibers ...... 82

xiii

List of Abbreviations

4HNE 4-hydroxynoneal

AC adenyl cyclase

AMPA alpha-amino-3-hydroxy-5-methylisoxazolone-4-propionic acid

BK bradykinin cAMP cyclic AMP

CaM calmodulin

CaMKII calmodulin-dependent kinase II

CAP capsaicin

CFA complete freund’s adjuvant

CGRP calcitonin gene related peptide

CM c-mechano

CMH c-mechanoheat

DCPG (S)-3,4-Dicarboxyphenylglycine

DRG dorsal root ganglia

EPSC excitatory postsynaptic current

ES extracellular solution

FSK forskolin iGluR ionotropic glutamate receptor

IL interleukin

KO kockout

L-AP-4 L-(+)-2-Amino-4-phosphonobutyric acid

xiv mGluR metabotropic glutamate receptor

NDS normal donkey serum

NGF nerve growth factor

NGS normal goat serum

NMDA N-Methyl-D-aspartate

NTS nucleus tractus solitarius

MO mustard oil

PAG periaqueductal grey

PB phosphate buffer

PBS phosphate buffered saline

PGE2 prostaglandin E2

PGI2 prostaglandin I2

PKA protein kinase A

PKC protein kinase C

PLC phospholipase C

PWL paw withdrawal latency

PWT paw withdrawal threshold

RpcAMPS Rp-cyclic 3′,5′-hydrogen phosphorothioate adenosine

triethylammonium salt

RT room temperature

SGC satellite glial cells

SIF synthetic interstitial fluid

TG trigeminal ganglion

xv THC D9-tetrahydrocannabinol

TRPV1 transient receptor potential vanilloid 1

TSA tyramide signal amplification

TrkA tyrosine kinase A

UBP α-methyl-3-methyl-4-phosphonophenylglycine (UBP1112)

WT wild type

xvi

CHAPTER 1: EXPANDED INTRODUCTION

Over 1.5 billion people across the globe suffer from chronic pain (AAPM). For many, treatments include taking opioids and/or NSAIDs. Unfortunately the consumption of these drugs can lead to detrimental side effects. Patients on opioid therapy often experience various adverse effects, including, but not limited to: addiction, nausea, constipation, vomiting, and dizziness. In the case of NSAIDs, prolonged use may lead to gastrointestinal ulcers, including bleeding ulcers and kidney failure. Thus, development of non-opioid and non-NSAID drugs to relieve both acute and chronic pain would improve the quality of life for millions of people.

Pain is “an unpleasant sensory and emotional experience associated with actual or potential tissue damage, or described in terms of such damage,” as defined by the

International Association for the Study of Pain (IASP). Primary afferent nociceptors

(Levine et al., 1993) are sensory neurons that respond to stimuli that have the potential for causing damage (Willis, 2007). Nociceptors are pseudounipolar with cell bodies contained within the dorsal root ganglia (DRG) or trigeminal ganglion (TG) with both a peripheral process and a central process (Willis, 2007). Early studies classified afferents 17

as either thermoreceptors, mechanoreceptors or polymodal nociceptors (Perl, 1968;

Bessou and Perl, 1969). Several studies indicated that nociceptors were polymodal, responding to noxious heat, irritant chemicals, and moderate mechanical stimuli with weak activity in response to low temperatures (Bessou and Perl, 1969; Torebjörk, 1974).

Nociceptors are described as either unmyelinated C fibers or lightly myelinated Aδ fibers with each group having different conduction velocities (Burgess and Perl, 1967; Bessou

and Perl, 1969; Torebjörk, 1974; Harper and Lawson, 1985a, b; Traub and Mendell,

1988). The various classifications of nociceptors include ADM (Aδ-mechano), ADMH

(Aδ-mechanoheat), ADMC (Aδ-mechanocold), CMH (C-mechanoheat), CM (c- mechano), CMC (c-mechanocold), and CMHC (c-mechanoheatcold) (Leem et al., 1993).

Using an in vivo preparation, Leem et al. (1993) reported that on average Aδ nociceptors had a mean CV of 14.17±5.86 m/s while C nociceptors had a mean CV of 0.77±0.14 m/s.

Using our in vitro skin-nerve preparation, Du et al. (2001) reported C fibers as having

CVs of <1.6 m/s and Aδ fibers as having CVs in the range of 1.6 – 12 m/s. Beginning with the nociceptor, painful information can be sent to the brain through various pathways.

Pain Pathways

There are at least five pathways by which information regarding tissue damage is transmitted to the brain. These pathways include the spinothalamic, spinoreticular, spinomesencephalic, cervicothalamic, and spinohypothalamic tracts. The most prominent and well-studied pathway is the spinothalamic tract since it brings pain, temperature and crude touch information to conscious levels.

SPINOTHALAMIC TRACT

When a noxious stimulus activates nociceptors, signals are sent from the peripheral processes through the central processes and enter the spinal cord through

Lissauers tract. At the spinal cord, the axons may ascend or descend 1-2 spinal segments before making the first synapse in either laminae I (marginal zone) or II (substantia gelatinosa). It should be noted that there are some projections from laminae II to laminae

V, which is part of the nucleus proprius. From the synapse at either laminae I, II or V, the

2nd order neuron crosses to the anterolateral quadrant of the contralateral side of the spinal cord via the anterior commissure and ascends through the brainstem and synapses at the ventral posterior medial or lateral nucleus of the thalamus. From the thalamus the

3rd order neuron carries the signal through the internal capsule to the post central gyrus, the primary somatic sensory cortex (Purves et al., 2001). It is likely that information from

TRP channels utilize the spinothalamic tract, since nociceptors express TRP channels.

Transient Receptor Potential (TRP) ion Channels and Pain

TRP CHANNEL DISCOVERY

The TRP channels were first described on photoreceptors in Drosophila. The trp gene mutation was first described by Cosens and Manning (1969), and later termed the transient receptor potential mutant (trp) by Minke et al. (1975). Flies with the trp mutation, which was found to occur spontaneously, were indistinguishable from WT in appearance (Cosens and Manning, 1969). In the presence of a continuous light stimulus, the WT photoreceptor maintained a sustained potential during stimulation. However, the trp mutation resulted in a transient receptor potential in the presence of continuous light during which the potential decayed to baseline (Minke et al., 1975). Since these first studies, over 30 TRP isoforms have been identified, making up 7 subfamilies: dTRP,

TRPC, TRPV, TRPM, TRPML, TRPP, TRPA and TRPN, the latter is only found in

Xenopus and zebrafish (Hardie, 2007). Of these TRP isoforms, TRPV1 and TRPA1 have been shown to play an important role in pain signaling (Patapoutian et al., 2009;

Brederson et al., 2013; Julius, 2013).

TRPV1

Caterina et al. (1997) first cloned TRPV1, which appeared to resemble TRP-like ion channels, and termed it Vanilloid Receptor subtype 1 due to its site of action

(Szallasi, 1994). Expression has been shown in keratinocytes, smooth muscle, neurons, astrocytes, microglia, oligodendrocytes, and ependymal cells (line ventricular system of brain and spinal cord), the hypothalamus (Cavanaugh et al., 2011), GABA cells of the dorsal horn (Kim et al., 2012), and in sensory neurons of the dorsal root ganglia (DRG) and trigeminal ganglia (TG) (Caterina et al., 1997; Fernandes et al., 2012; Zhang et al.,

2014b). Further analysis identified it as the capsaicin (CAP) receptor (Caterina et al.,

1997). CAP is an ingredient found in hot chili peppers. In addition to CAP, TRPV1 is activated by acidic pH, spider toxins, noxious heat (> 40 ºC), and endocanabinoids

(Caterina et al., 1997). In recent years, TRPV1 has gained much attention due to evidence which implicates it as an important transducer of pain information or noxious stimulation.

Several lines of evidence indicate that TRPV1 plays an important role in the development of hyperalgesia, neuropathic and inflammatory pain (Caterina et al., 2000; Davis et al.,

2000; Stucky et al., 2009; Carlton et al., 2011; McDougall, 2011; Govea et al., 2012;

Nakamura et al., 2012; Barabas and Stucky, 2013). For example, studies from knockout

(KO) mice indicate that these mice maintain normal responses to noxious stimuli, while displaying an attenuated development of hyperalgesia in the inflammatory state (Caterina et al., 2000; Davis et al., 2000; Bölcskei et al., 2005). Compared to WT, TRPV1 KO mice display an attenuated development of carrageenan-induced mechanical hyperalgesia

(Watanabe et al., 2015). Interestingly, in contrast to WT mice, the KO mice did not develop contralateral mechanical hypersensitivity, suggesting that TRPV1 plays a role in

development of contralateral secondary hypersensitivity following inflammation. In an incision model, TRPV1 KO mice display an attenuated development of thermal hyperalgesia compared to WT (Barabas and Stucky, 2013). Several studies indicate that

TRPV1 activity can be enhanced by various modulators or inflammatory mediators (De

Petrocellis et al., 2001; Bhave et al., 2002; Rathee et al., 2002; Huang et al., 2006; Jeske et al., 2008; Chaudhury et al., 2011; Malek et al., 2015), including PKA, which potentially increases surface membrane expression (Of note, a study with Xenopus oocytes and Aplysia demonstrates conflicting reports of the cAMP/PKA pathway in sensitizing TRPV1 currents, see Lee et al. [2000]). Repeated application of CAP induces desensitization of the TRPV1 receptor (Ruparel et al., 2008). This TRPV1 desensitization is attenuated by PKA activation, resulting in an enhanced 2nd response to CAP compared to control (Bhave et al., 2002). Taken together, these findings suggest that TRPV1 plays an important role in both thermal hyperalgesia and mechanical hypersensitivity and its activity can be modulated by PKA.

TRPV1: MORE THAN A PAIN RECEPTOR

Studies suggest that TRPV1 plays a role in body temperature regulation (Iida et al., 2005), since administration of TRPV1 antagonists induces hyperthermia (Swanson et al., 2005b; Gavva et al., 2007b; Gavva et al., 2007a; Honore et al., 2009; Wong and

Gavva, 2009). Inhibition of TPRV1 appears to attenuate anxiety-like behaviors, fear conditioning behaviors and stress sensitization (Zhang and Liao, 2014). Interestingly, activation of TRPV1 appears to induce hypothermia which has been shown to be neuroprotective in a transient middle cerebral artery occlusion model (Cao et al., 2014).

Furthermore, studies indicate that TRPV1 is an attractive target in other hypoxic/ischemic

events (Khatibi et al., 2011; Xu et al., 2011) . Additionally, TRPV1 function has been demonstrated in dendritic cell maturation, vascular dilation, adipocyte differentiation, pro-inflammatory responses of CD4+T cells, and neurogenesis (Basu and Srivastava,

2005; Hwang et al., 2005; Hsu and Yen, 2007; Fernandes et al., 2012; Bertin et al., 2014;

Stock et al., 2014). In keratinocytes, TRPV1 activation induces the release of several inflammatory mediators, including prostaglandin E2 (PGE2) and interleukin (IL)-8

(referred to as an autocrine inflammatory mediator [Atta-ur-Rahman et al., 1999])

(Southall et al., 2003; Fernandes et al., 2012). These latter findings suggest that TRPV1 indirectly plays a role in potentiating inflammation and likely inflammation-associated pain.

TRPA1

TRPA1 is the first receptor identified in the TRPA subfamily (Jaquemar et al.,

1999). This receptor is known as the mustard oil (MO) receptor (Jordt et al., 2004). In addition to MO, TRPA1 is activated by various stimuli, including cinnamaldehyde, noxious mechanical and noxious cold (<17 °C) (Story et al., 2003; Jordt et al., 2004;

Kwan et al., 2006; Barabas et al., 2012). Similar to TRPV1, several reports have highlighted the importance of TRPA1 in pain transmission (Lapointe and Altier, 2011;

Bautista et al., 2013; Premkumar and Abooj, 2013). For example, studies indicate that

TRPA1 is key to the development of mechanical sensitivity following inflammation or nerve injury (Obata et al., 2005; Ji et al., 2008; da Costa et al., 2010; Lennertz et al.,

2012). Administration of a TRPA1 antagonist significantly attenuates complete Fruends

Adjuvant (CFA)-induced mechanical hypersensitivity (da Costa et al., 2010). In line with these findings, studies at the single fiber level demonstrate that administration of a

TRPA1 antagonist prevents CFA-induced mechanical sensitization in C fibers (Lennertz et al., 2012).

In addition to mechanical hypersensitivity, cold hypersensitivity has been shown to develop following inflammation or nerve injury (Jasmin et al., 1998; Allchorne et al.,

2005; Scherer et al., 2010). While TRPA1 as a cold receptor is controversial, several lines of evidence indicate that TRPA1 is important in the development of this pain related cold hypersensitivity (Obata et al., 2005; Ji et al., 2008; da Costa et al., 2010). Obata et al.

(2005) found that CFA induces an increase in TRPA1 expression in the DRG.

Furthermore, knockdown of TRPA1 using an antisense oligodeoxynucleotide prevents and reverse both CFA- and nerve injury-induced cold hypersensitivity.

Similar to TRPV1, TRPA1 appears to be modulated by various mediators (Wang et al., 2008; Schmidt et al., 2009), including PKA. Wang et al. (2008) showed that forskolin (FSK), an adenylyl cyclase (AC) activator, potentiated MO-induced TRPA1 currents. Interestingly, Schmidt et al. (2009) found that FSK can induce translocation of

TRPA1 to the surface membrane. Moreover, agonist activation of TRPA1 can induce translocation. Taken together, these studies suggest that TRPA1 is important in the development of both mechanical and cold hypersensitivity in various pain states.

TRPA1: MORE THAN A PAIN RECEPTOR

Expression of TRPA1 has been detected in the gastrointestinal tract and in the lung (Lapointe and Altier, 2011), smooth muscle (bladder) (Du et al., 2008b), skeletal muscle (Lapointe and Altier, 2011), keratinocytes (Atoyan et al., 2009), inner hair cells

(Corey et al., 2004), cerebellar neurons (Garrison and Stucky, 2011), and in sensory ganglia expressing TRPV1 (Story et al., 2003; Nagata et al., 2005). Additionally, TRPA1

function has been demonstrated in immune cells (monocytes and macrophages) (Lee et al., 2005; Chao et al., 2008) as well as in the vasculature (Earley et al., 2009).

Findings from Corey et al. (2004) initially proposed TRPA1 as a hair cell transduction channel. However, subsequent studies demonstrated that TRPA1 KO mice display a normal startle response, sense of balance, auditory brainstem response and transduction of currents in vestibular hair cells (Kwan et al., 2006; Prober et al., 2008).

Interestingly, in macrophages and monocytes, agonist activation of TRPA1 appears to play an anti-inflammatory role, inhibiting either gram negative bacteria-induced nitric oxide release (Lee et al., 2005), or IL-1 and TNF release (Chao et al., 2008), respectively. Additionally, TRPA1 has been shown to play a role in keratinocyte proliferation, differentiation and cell cycle regulation, as well as regulating the release of pro-inflammatory ILs: IL-1α and IL-1β (Atoyan et al., 2009). Similar to TRPV1, these latter findings suggest that TRPA1 is indirectly involved in the inflammatory response and likely to potentiate inflammatory pain.

Metabotropic Glutamate Receptors (mGluRs)

GROUPS I, II, III

The metabotropic glutamate receptors are divided into 3 groups based on sequence-homology and pharmacology (Conn and Pin, 1997; Schoepp and Conn, 2002;

Niswender and Conn, 2010). Group I mGluRs, consisting of mGluR1 and mGluR5, are positively coupled to phospholipase C (PLC) and are associated with neuronal excitation.

Group II mGluRs include mGluR2 and mGluR3. Group III mGluRs include mGluR4, and mGluR6-8. Group II and III mGluRs are negatively coupled to AC and the

cAMP/PKA pathway and are thus associated with neuronal inhibition (Goudet et al.,

2009). Several lines of evidence, detailed below, suggest that group III mGluRs are attractive targets for modulating nociceptor activity.

MGLUR8 LOCALIZATION

Prominent mGluR8 expression has been found in numerous brain regions, including the thalamus (Saugstad et al., 1997; Turner and Salt, 1999), amygdala (Palazzo et al., 2008; Ren et al., 2011), periaqueductal grey (PAG) (Marabese et al., 2007a;

Marabese et al., 2007b), NTS (Young et al., 2008; Liu et al., 2012), enteric nervous system (Tong and Kirchgessner, 2003), spinal cord (Thomas et al., 2001), sensory neurons (Carlton and Hargett, 2007; Govea et al., 2012; Boye Larsen et al., 2014) as well as in both myelinated and unmyelinated digital axons (Govea et al., 2012).

GROUP III MGLURS (MGLUR8 AND PAIN)

Drugs targeting group III mGluRs are currently being investigated for treatment of Parkinson’s disease, anxiety, schizophrenia as well as epilepsy(Conn et al., 2005;

Swanson et al., 2005a; Patil et al., 2007; Folbergrová et al., 2008; Conn et al., 2009;

Hopkins et al., 2009). Drugs (e.g. gabapentin, pregabalin, carbamazepine, valproate) that successfully reduce epileptic events, associated with neuronal hyperexcitability, often become part of the arsenal for treating chronic pain patients, where neuronal hyperexcitability is contributing to and/or driving the pain. The group III mGluRs have been shown to be effective in attenuating pain behaviors (Neugebauer et al., 2000;

Marabese et al., 2007a; Goudet et al., 2009; Govea et al., 2012). Group III mGluRs are often localized on the presynaptic terminals, potentially outside of the active zone

(Schoepp, 2001), inhibiting neurotransmitter release (Niswender and Conn, 2010).

Several lines of evidence indicate mGluR8 is important in modulating pain centrally

(Thomas et al., 2001).

Activation of mGluR8 in the PAG using DCPG, a potent agonist with 100-fold selectivity over mGluR4 (Thomas et al., 2001; Zhai et al., 2002), attenuated both formalin and carrageenan-induced pain behaviors (Marabese et al., 2007a). Furthermore, administration of DCPG into the central nucleus of the amygdala of the amygdala attenuated spinal nocifensive reflexes in arthritic animals, and inhibited both audible and ultrasonic vocalizations in arthritic animals compared to control (Palazzo et al., 2008). To date no studies examining the effects of peripheral administration of DCPG have been reported.

MGLUR8: MORE THAN A PAIN MODULATOR

Several lines of evidence indicate that mGluR8 plays a role in epilepsy

(Folbergrová et al., 2008), stress and anxiety (Linden et al., 2002; Pałucha et al., 2004;

Duvoisin et al., 2005; Swanson et al., 2005a; Palazzo et al., 2008; Fendt et al., 2010), schizophrenia (Robbins et al., 2007), alcohol-seeking behavior (Bäckström and Hyytiä,

2005), as well as in gastrointestinal motility and insulin secretion (Tong et al., 2002;

Tong and Kirchgessner, 2003). Higher levels of anxiety are seen in mGluR8 KO mice compared to WT littermates using the elevated plus maze and the open field test (Linden et al., 2002; Duvoisin et al., 2005). In the pancreas, group III mGluR8 activation on α– cells reduces glucagon release (Tong et al., 2002). In the gastrointestinal tract, agonist activation of mGluR8 increases both the rate of propulsion and stimulation-induced contraction of longitudinal muscle of ileal segments compared to control (Tong and

Kirchgessner, 2003). Interestingly, DCPG dose-dependently reduces ethanol self- administration and attenuates reinstatement behavior (Bäckström and Hyytiä, 2005). It should be noted that in this study, all doses of DCPG also reduce ambulatory and vertical locomotor activity potentially altering behavioral responses. Folbergrová et al. (2008) demonstrate that administration of DCPG has a pronounced anticonvulsant effect on DL-

HCA-induced seizures. Furthermore, results from this study also indicate that pretreatment with DCPG has a substantial neuroprotective effect.

The following studies in this dissertation use a multifaceted approach to examine the interaction between group III mGluRs (mGluR8) and two TRP channels: TRPV1 and

TRPA1. In the first study, we demonstrate for the first time that mGluR8 is localized on digital axons using electron microscopy. Immunohistochemistry in DRG neurons demonstrate that TRPV1 and mGluR8 are co-localized. Behavioral assays demonstrate the functional relevance of this co-localization as activation of group III mGluRs significantly attenuate TRPV1-induced nocifensive responses. Furthermore, a behavioral assay demonstrated that group III mGluRs negatively modulate TRPV1 through the cAMP/PKA pathway. In the second study, Ca2+ imaging is used to show co-localization of TRPA1 and mGluR8. Functional relevance of this co-localization is addressed in a behavioral assay where activation of mGluR8 significantly attenuates TRPA1-induced mechanical hypersensitivity. Using the skin-nerve preparation, TRPA1-induced mechanical hypersensitivity of Aδ and C fibers is attenuated by activation of mGluR8.

Using Ca2+ imaging, modulation of TRPA1 by mGluR8 was found to be partially mediated through the cAMP/PKA pathway.

CHAPTER 2: GROUP III MGLURS NEGATIVELY MODULATE TRPV1

ACTIVITY

Introduction

Glutamate, a major excitatory neurotransmitter in the nervous system, is important in peripheral pain transmission and acts on both ionotropic and metabotropic receptors (Carlton, 2001; Goudet et al., 2009). The eight cloned metabotropic glutamate receptors (mGluRs) are G-protein coupled receptors and are divided into three groups depending on sequence-homology and pharmacological properties. Group I (mGluRs 1 and 5) are excitatory and coupled to Gq, thus associated with the stimulation of phospholipase C (PLC) and intracellular calcium signals. Group II (mGluRs 2 and 3) and

III (mGluRs 4, 6, 7, and 8) are inhibitory and coupled to Gi/o, thus negatively coupled to adenylyl cyclase (AC) (Goudet et al., 2009). The inhibitory functions of group II and III make these receptors attractive targets for peripheral pain modulation. Several lines of evidence have implicated group II receptor agonists in the attenuation of peripheral pain

(Anjaneyulu et al., 2008; Du et al., 2008a). Data indicating that group III mGluR agonists are also effective in treatment of pain are accumulating. For example, administration of the group III agonist, L-(+)-2-Amino-4-phosphonobutyric acid (L-AP-4), in the primate dorsal horn, inhibits capsaicin (CAP)-induced central sensitization (Neugebauer et al.,

2000; Goudet et al., 2009). Additionally, application of L-AP-4 to spinal lamina II in nerve injured rats results in greater inhibition of evoked excitatory postsynaptic currents

(EPSCs) compared to control rats (Zhang et al., 2009). Finally, systemic administration of (S)-3,4-dicarboxyphenylglycine (DCPG), a selective mGluR8 agonist, attenuates

formalin and carrageenan-induced hyperalgesia (Marabese et al., 2007a). Currently, studies investigating the effects of group III mGluR activation in the periphery are limited (Walker et al., 2001; Jin et al., 2009; Chen et al., 2010). Furthermore, no studies have examined the relationship between peripheral group III mGluR agonists and the transient receptor potential ion channels.

Transient receptor potential vanilloid 1 (TRPV1) is most widely known as the

CAP receptor. CAP is an ingredient found in hot chili peppers and activates the channel

(Caterina et al., 1997). In addition to CAP, TRPV1 can be activated by a variety of noxious stimuli, including heat (above 40°C) (Caterina et al., 1997), acidic pH

(Tominaga et al., 1998; Jordt et al., 2000), cannabinoid anandamide (Zygmunt et al.,

1999; Smart et al., 2000), HETE (Hwang et al., 2000), and spider toxins (Costa et al.,

1997; Siemens et al., 2006). Thus, TRPV1 is a polymodal receptor. Several studies have demonstrated that TRPV1 knockout mice maintain responses to acute noxious stimuli while showing attenuated development of hyperalgesia in the inflammatory state

(Caterina et al., 1997; Davis et al., 2000; Bölcskei et al., 2005). These studies highlight the role TRPV1 plays in development of hyperalgesia and the importance of targeting

TRPV1 in the treatment of pain.

Pain modulation at the source is an attractive strategy, especially in aiming to reduce side-effects common to most widely available analgesics (Patapoutian et al., 2009;

McDougall, 2011). Since the TRP family makes up a large group of temperature sensing ion channels (Patapoutian et al., 2009), it is of great interest to examine the modulation of

TRP channels, TRPV1 in particular, in the periphery. In the present study, we examined

the effects of group III mGluR activation by L-AP-4 on CAP-induced nociception in the periphery.

Methods

ANIMAL USE AND CARE

All experiments were approved by the University Animal Care and Use committee and followed the guidelines for the ethical care and use of laboratory animals

(Zimmermann, 1983). Steps were taken to minimize both the number of animals used and their discomfort.

IMMUNOSTAINING OF THE DIGITAL NERVE AT THE ELECTRON MICROSCOPIC LEVEL

Naïve male Sprague-Dawley rats (n = 3, 250-300 g) were deeply anesthetized with pentobarbital (100 mg/kg) and perfused transcardially with heparinized saline followed by a mixture of 2.5 % glutaraldehyde, 1.0 % paraformaldehyde and 0.1 % picric acid in 0.1 M phosphate buffer (PB) at 4° C. The digital nerves were dissected and cut into 2.0-2.5 mm segments. Prior to immunostaining, all tissue was placed for 1 hr in 1 % sodium borohydride to remove excess glutaraldehydes and rinsed in graded alcohols to increase antibody penetration. The tissue was placed in 10 % normal goat serum (NGS) for 1 hr and incubated in antiserum made in guinea pig directed against mGluR8 (1:2000,

Chemicon, Temecula, CA) for 72 hr at 4° C. Following incubation, the tissues were rinsed in phosphate buffered saline (PBS), followed by 3 % NGS for 30 min, incubated in biotinylated goat anti-guinea pig IgG in 1 % NGS for 1 hr, rinsed in 1 % NGS followed by 3 % NGS for 30 min each and incubated in the ABC reagent (Vector Laboratories,

Burlingame, CA) for 2 hr. The tissue was incubated in a solution of diaminobenzidine

(0.05 %) containing 0.01 % hydrogen peroxide for approximately 4-6 min. After immunostaining, tissue was placed in 1 % phosphate buffered osmium tetroxide for 2 hr, dehydrated and embedded in plastic. For all tissue, ultrathin sections of the digital nerves were cut at right angles to the long axis of the fibers. The sections were mounted on formvar coated slot grids and viewed on a JEOL 100CX electron microscope. A digital nerve cut in cross section from each animal was photographed and montaged, and all labeled and unlabeled axons counted in order to obtain a percentage of mGluR8-labeled axons. For some tissues, the primary antiserum was omitted and the tissue was otherwise treated as described above. Absorption controls were also run with the peptide obtained from Chemicon. Tissue was incubated in a solution containing antibody (1:2000) that was preabsorbed with 100 µg/ml of peptide. Specific immunoreaction product was absent in both of these controls.

IMMUNOSTAINING THE DORSAL ROOT GANGLIA AT THE LIGHT MICROSCOPIC LEVEL

Tissue Collection

Naïve male Sprague-Dawley rats (n = 3, 250-300 g) were deeply anesthetized with pentobarbital (100 mg/kg) and perfused transcardially with 4 % paraformaldehyde and 0.1 % picric acid in 0.1 M PB at 4º C. The L4 dorsal root ganglia (DRG) were dissected out. The ganglia were fast frozen using liquid nitrogen in a cryoembedding compound. The tissue was sectioned (8 µm thick) on a cryostat (Microm International,

GmbH), along the short axis and placed on gelatin-dipped 3-well, Teflon-printed slides

(Electron Microscopy Sciences, Fort Washington, PA). Serial section sets (3 – 5 sets per ganglia) were collected with a defined section separation so that cell counts could be

determined by stereological analysis. Tissue sections were allowed to dry overnight at room temperature (RT).

Immunohistochemistry

Slides containing tissue were first incubated in PB+0.2 % TritonX for 10 min.

Sections were blocked in 10 % normal donkey serum (NDS) for 1 hr. Sections were incubated in guinea pig polyclonal anti-mGluR8 (1:200,000, Chemicon) for 48 hr at RTº and rinsed with PBS. Next, sections were incubated in biotinylated goat anti-guinea pig

IgG (1:200, Vector Laboratories) for 1 hr. After rinsing in PBS, sections were incubated in Vectastain Elite ABC peroxidase reagent (avidin-biotin complex, Vector Laboratories) for 1 hr and rinsed in PBS. Sections were incubated in Cyanine 3-labeled tyramide (1:75,

Perkin-Elmer Life Science, Waltham, MA) for 7 min, using the TSA (Tyramide Signal

Amplification) protocol, rinsed in PBS, and incubated in 0.03 % H2O2 for 20 min to inactivate residual peroxidase, followed by a rinse in PBS. Next, sections were incubated in goat anti-TRPV1 (1:500 Santa Cruz, Santa Cruz, CA) for 24 hr. Sections were rinsed in PBS and incubated in a Cyanine 2-conjugated mouse anti-goat IgG (1:300 Jackson

Laboratories, West Grove, PA) for 1 hr and were rinsed in PBS, then dH2O. Slides were cover slipped using VectaShield mounting media (Vector Laboratories). In separate sets of tissue used for controls, the primary antiserum was omitted and the tissue was otherwise treated as described above. Absorption controls for TRPV1 (Carlton et al.,

2009) and mGluR8 (Carlton and Hargett, 2007) have been reported previously from our lab.

Cresyl Violet

To obtain total cell counts, one serial section set representing each DRG was stained with cresyl violet for 2 min.

Analysis

Pairs of serial sections (disector pairs) were photographed and analyzed using the physical disector method (Coggeshall, 1992; Coggeshall and Lekan, 1996; West, 1999).

Total cell counts were determined for each DRG by multiplying the number of Tops

(cells only present in 1 of the 2 disector sections) by section separation, and dividing by 2

(the number of disector sections). Tissue labeled with mGluR8 and TRPV1 were used to determine percentages of single- and double-labeled cells. Percentages were calculated by dividing the number of single- and double-labeled Tops by the total cell count

(obtained from the cresyl violet stained sections) and multiplying by 100. Cell diameters were determined by taking the sum of the length and width of neurons containing a visible nucleus and dividing by 2.

BEHAVIOR

Drug Preparations

A 10 % CAP stock solution was diluted with the CAP vehicle (7 % Tween-80 in saline) in order to produce a working solution of 0.05 % CAP. The following drugs were dissolved in 1N NaOH and made up as a 100 mM stock solution: L-AP-4 (Tocris

Cookson Ltd., Ellisville, MO, USA), a selective group III mGluR agonist and α-methyl-

3-methyl-4-phosphonophenylglycine (UBP1112 [UBP], Tocris), a potent group III mGluR antagonist. Forskolin (FSK) was dissolved in distilled water to a 1 mM stock

solution. L-AP-4, UBP and FSK stock solutions were diluted to desired concentrations using PBS (pH 7.4) and the pH was adjusted to 7.4.

Drug Injections

Intraplantar drug injections were administered using a 28-gauge needle attached to a 50 µl Hamilton syringe using PE20 tubing. Since spontaneous nociceptive behaviors are most robust during the first 5-10 min following CAP injection, rats were not anesthetized during these drug injections. In contrast, rats receiving injections for the

FSK paradigm were anesthetized with 3% isofluorane (anesthesia could be used since it would not interfere with the evoked heat responses being measured). The needle punctured the plantar skin and was guided through the subcutaneous space to a site just proximal to the pads. All drugs were administered into the same place in the chosen hind paw and all injected volumes were kept constant within an experimental paradigm. Each animal was only used once and the investigator was unaware of the drug combinations injected.

Habituation for behavioral testing

After arrival at the Animal Care Facility, animals were acclimated for at least 3 days before behavioral testing began. Animals were housed in groups of 3 in plastic cages with soft bedding under a reversed light/dark cycle of 12 hr/12 hr. All behavioral testing was performed between 8AM and 5PM, during the animal’s dark cycle. Care was taken to keep the animals in low lighting conditions during transport, habituation and testing. Rats were habituated for thermal testing for 1 hr on each of 5 days by placing them in plexiglass cages (8x8x18 cm) on a glass plate which was ¼ inch thick and

maintained at 23˚ C. During the habituation period, each hind paw was tested twice 10 min apart. A modified Hargreaves et al. (1988) method was used, however, the radiant heat source was replaced by a laser. The laser system was custom-made in house and consisted of a 980 nm (near infrared) continuous wave, solid-state laser (4W) with a spot size of 2 mm. Rats were habituated for CAP-induced behavioral testing by placing them on a wire screen platform in plexiglass cages for 1 hr. Each rat was habituated twice before being placed into an experimental group.

Testing for thermal and CAP-induced behaviors

Paw withdrawal latency (PWL) was assessed by examining changes in the length of time the hind paw remained on the glass, when the laser heat stimulus was applied. For the L-AP-4 dose response relationship, PWL was tested at 10 min intervals, for 60 min post-injection and plotted as the percent change from baseline. For the FSK paradigm,

PWL for each group was tested at 30 and 60 min post-injection. CAP-induced spontaneous behaviors were assessed by counting the number of flinches and the number of seconds an animal spent lifting and/or licking (L/L) the injected paw at 5 min intervals for 60 min. A flinch was defined as a spontaneous, rapid jerk of the hind paw whether it was on the screen or in the air. The time course of the nociceptive behaviors was plotted as the mean number of flinches and amount of time spent L/L in 5-min intervals for 60 min.

Data Analysis

Data are presented a Mean ± SE (unless noted otherwise). Differences between groups were evaluated with a one way ANOVA. Differences between groups over a time

course were evaluated with a two way RM ANOVA (if a normality test was passed). *P

< 0.05 was considered significant.

Results

MGLUR8-LABELED DIGITAL AXONS

Analysis of montages of hind paw digital nerves demonstrated mGluR8-labeled axons. An axon was considered labeled when dense reaction product was observed associated with the axonal membrane or with microtubules in the axoplasm (Figure 1). In naïve rats 21.9 ± 6.2 % of unmyelinated axons and 19.4 ± 6.9 % of myelinated axons were labeled for mGluR8 in the digital nerves (Table 2.1).

Figure 2.1: Localization of mGluR8 on axons in the digital nerves Electron microscopic plate of labeled (arrows) and unlabeled (asterisks) axons. The top panel (A) contains unmyelinated axons while lower panel (B) demonstrates myelinated axons. Scale bar = 10 µm.

Table 2.1: Percent of mGluR8-labeled axons in hindpaw digital nerves

Rat # Unmyelinated axon Percent Myelinated axons Percent

Labeled/total Labeled /total

# 195 53/195 27.2 20/136 14.7

# 196 38/251 15.1 21/129 16.2

# 197 57/245 23.3 29/106 27.4

Mean ± S.D. 21.9 ± 6.2 19.4 ± 6.9

MGLUR8 AND TRPV1 ARE CO-LOCALIZED ON RAT DRG CELLS

Initial observations indicated a large number of mGluR8-labeled cells and a much smaller number of TRPV1-labeled cells in the DRG. Cells labeled for mGluR8 displayed numerous (red) fluorescent puncta in the cytoplasm but the nuclei were unlabeled (Figure

2.2A). Among the DRG cells, the degree of mGluR8 fluorescent label varied from light to heavy. In some cells the puncta were distributed throughout the cytoplasm while in others the puncta were concentrated in one area of the cell. Cells were considered labeled when they contained several fluorescently labeled puncta in the cytoplasm. Control sections contained no puncta. mGluR8 label was also found in satellite glial cells (SGC), the support cells surrounding DRG cells.

Figure 2.2: Co-localization of mGluR8 and TRPV1 in rat DRG Immunostaining for mGluR8 (A) and TRPV1 (B) and merged (C) in the same section. Examples of single- (arrowhead) and double-labeled (arrows) DRG neurons are indicated. Scale bar = 50 µm.

Unlike mGluR8, cells labeled for TRPV1 displayed a homogeneous (non-punctate green) fluorescence that filled the cytoplasm but the nuclei were unlabeled (Figure 2.2B).

Cells were considered labeled when the fluorescent cytoplasm was distinctly brighter than the background level. Control sections did not contain any level of fluorescence.

Cells were considered double-labeled when they contained both the red fluorescent puncta for mGluR8 and the green fluorescent cytoplasm for TRPV1 (merged image resulted in a yellow fluorescence, Figure 2.2C). Analysis demonstrated that 80.0 ± 23.3

% and 28.5 ± 8.9 % of cells in the L4 DRG were labeled for mGluR8 and TRPV1, respectively. Results also demonstrated that 25.1 ± 7.1 % of cells labeled for mGluR8 were double-labeled for TRPV1 and 71.0 ± 21.9 % of cells labeled for TRPV1 were double-labeled for mGluR8 (Table 2).

Table 2.2: Percent mGluR8- and TRPV1-labeled cells in the DRG Rat # % mGluR8 % mGluR8 % TRPV1 % TRPV1

single- double-labeled single- double-labeled

labeled with TRPV1 labeled with mGluR8

# 2 60.6 25.3 28.3 54.2

# 3 73.6 32.1 37.5 63.0

# 4 105.9 17.9 19.8 95.7

Mean ± S.D. 80.0 ± 23.3 25.1 ± 7.1 28.5 ± 8.9 71.0 ± 21.9

When nuclei were present, cell diameters were measured by placing 2 lines perpendicular to each other through the center of the cell. Line measurements were added

together and divided by 2. The average diameter of mGluR8-labeled cells was 30.9 µm

(range of 12.5 - 58.5 µm) and for TPRV1-labeled cells was 23 µm (range 12.5 - 39.0 µm)

(Figure 2.3). Double-labeled cells had an average diameter of 23.3 µm (range 12.5 – 39.0

µm).

Figure 2.3: Diameters of single- and double-labeled DRG neurons Diameters of mGluR8-(A) and TRPV1-(B) single-labeled and double-labeled (C) DRG neurons.

GROUP III MGLUR AGONIST L-AP-4 ATTENUATES CAP-INDUCED NOCICEPTIVE BEHAVIORS

In the behavioral studies, we first examined the effects of L-AP-4 on naïve rats.

Separate groups of rats were injected with one of the following: 0.1, 1.0 or 10.0 µM L-

AP-4, or PBS and tested for sensitivity to heat every 10 min. Compared to baseline, there was no significant change in thermal PWL following these injections, (although L-AP-4 did tend to increase PWL), demonstrating that administration of L-AP-4 in this dose range had little or no effect in the naïve state (Figure 2.4).

Next, the effects of L-AP-4 on CAP-induced nociception were examined.

Separate groups of rats received consecutive intraplantar injections of one of the following drug combinations with a 10 min interval between the 1st and 2nd drug injection: 10 µM L-AP-4 (30 µl, n = 8) + 0.05 % CAP (30 µl); PBS (30 µl, n = 9) + 0.05

% CAP (30 µl). Our lab has previously determined that 2 consecutive injections of PBS do not result in Lifting/Licking (L/L) or flinching (data not shown) thus double injections did not contribute to the nociceptive behaviors observed here. The PBS + CAP group responded robustly with L/L and flinching behaviors during the first 5 min following

CAP injection (Figure 2.5C and D). Responses returned to baseline 40 to 50 min post- injection. Rats that received L-AP-4 prior to CAP showed attenuated nociceptive behaviors: L-AP-4 significantly reduced L/L during the first 5 min interval and significantly reduced the total time engaged in this behavior (Figure 2.5A and C); however, while there was a downward trend, the L-AP-4-induced reduction in flinching did not reach significance (Figure 2.5B and D).

Figure 2.4: L-AP-4, a group III agonist, has no effect in the naïve state Intraplantar administration of L-AP-4 (0.1, 1.0, and 10.0 µM) in the naïve rat does not produce any significant change in paw withdrawal latency (PWL) to heat when compared to baseline (p=0.196) and intraplantar PBS (p=0.291). Two Way Repeated Measures ANOVA.

In order to demonstrate receptor specificity of L-AP-4, this group III agonist was co-injected with a selective group III antagonist, UBP. A separate group of rats received intraplantar injections with the following drug combination with a 10 min interval between the 1st and 2nd drug injections: cocktail of 100 µM UBP + 10 µM L-AP-4 (30

µl, n = 9), 0.05 % CAP (30µl). UBP blocked the inhibitory effect of L-AP-4 such that this group showed behavior that was not significantly different from the group receiving

CAP alone (Figure 2.5A). UBP significantly reversed the L-AP-4 inhibitory effect on

CAP during the 0 - 10 min interval (Figure 2.5C).

In order to show that L-AP-4 was acting locally, a separate group of rats received

10 µM L-AP-4 (30 µl, n = 8) in one hind paw + PBS (30 µl) followed 10 min later by

0.05 % CAP (20 µl) in the contralateral hind paw. This group showed behavioral

responses that were no different from the PBS + CAP group, indicating L-AP-4 was not having a systemic effect (data not shown).

Figure 2.5: L-AP-4 produces an anti-hyperalgesic effect on CAP-induced nociception Intraplantar administration of CAP (0.05 %) produces lifting/licking (A, total time spent lifting/licking in 1 hr; C, time course of lifting/licking) and flinches (B, total flinches in 1 hr; D, time course of flinching). L-AP-4 (10 µM) significantly reduces the time spent lifting/licking (A, *p < 0.05, significantly different from all other groups, one way ANOVA, Holm-Sidak post hoc test) and there is a tendency for a reduction in the number of flinches (B). L-AP-4 (10 µM) + UBP (100 µM), a group III antagonist, demonstrates selective group III activation by L-AP-4 since UBP inhibits the anti-hyperalgesic effects of L-AP-4 (C, +p<0.05, significantly different from L-AP-4 + CAP, two way RM ANOVA, Holm-Sidak post hoc test).

If group III mGluRs were tonically active, blocking activity with an antagonist would result in nociceptive behaviors (Carlton et al., 2001). To determine if group III mGluRs were tonically active, rats received an intraplantar injection of 100 µM UBP (30

µl, n = 7) alone. This injection did not result in the generation of nociceptive behaviors, demonstrating that group III mGluRs are not tonically active (data not shown).

GROUP III MGLUR ACTIVATION MODULATES THE CYCLIC AMP (CAMP)/PROTEIN KINASE A (PKA) PATHWAY

In order to examine whether group III mGluR activation inhibits the cAMP/PKA pathway, the AC activator, forskolin (FSK), was used. We hypothesized that FSK would induce heat sensitization that could be blocked by group III mGluR activation. Separate groups of rats received intraplantar injections of one of the following drug(s): PBS alone

(20 µl, n = 6), 10 µM FSK alone (20 µl, n = 6), or 10 µM L-AP-4 + 10 µM FSK (20 µl, n

= 6). Compared to the PBS group, PWL to heat was significantly lowered in the FSK group at both the 30 min (data not shown) and 60 min time points (Figure 2.6).

Administration of L-AP-4 reversed the effects of FSK such that the L-AP-4 + FSK group was not significantly different from the PBS group (Figure 2.6).

Figure 2.6: L-AP-4 inhibits FSK-induced heat sensitization Intraplantar administration of FSK (10 µM) significantly lowers PWL, compared to PBS at both the 30 min (data not shown) and 60 min time points (shown above). Administration of L-AP-4 (10 µM) + FSK (10 µM) reverses the effects of FSK, bringing PWL back to baseline. *p < 0.05, significantly different from all other groups, one way ANOVA, Holm-Sidak post hoc test.

Discussion

The present study examined the effects of group III mGluR activation on CAP- induced nociception in the periphery. Our results demonstrate that 1) mGluR8 is localized on unmyelinated axons in digital nerves, 2) mGluR8 and TRPV1 are co- localized in rat DRGs, 3) intraplantar administration of L-AP-4, a selective group III mGluR agonist, has an anti-hyperalgesic effect on CAP-induced nociceptive behaviors, and 4) group III mGluR activation inhibits forskolin-induced heat sensitization.

Our data demonstrate for the first time, the localization of group III mGluR8 in peripheral unmyelinated axons, presumably nociceptors, which would allow mGluR8 agonists and/or antagonists to have a direct effect on nerve terminals. We confirmed TRPV1 localization on small diameter sensory neurons (Carlton and Coggeshall, 2001; Carlton et al., 2009), suggesting that a direct interaction between group III mGluRs and TRPV1 is possible. Analysis indicated approximately 40 % of digital axons labeled for mGluR8 in the periphery, while 80 % of cells labeled for mGluR8 in the DRG. There are several possible explanations for this disparate finding. First, mGluR8-labeled cells in the DRG may have been overestimated due to the fact that background staining was variable in each case. Second, the cells contained within the DRG send axons to various regions of the body, including viscera, joints, and muscles in addition to cutaneous tissue, many of which could express mGluR8. Therefore, those DRG cells involved in cutaneous innervations make up only a subpopulation of the total mGluR8 population. Third, in contrast to paraformaldehyde (LM fixative for DRG), conformational changes caused by glutaraldehyde (EM fixative for digital nerves) can cause tighter cross-linking of the proteins which could reduce antibody-antigen recognition, resulting in fewer axons being

labeled. Fourth, the receptor is not continuously expressed along the length of the axon

(Carlton and Coggeshall, 2002). Thus, the location of the cut/section may or may not display label even though the axon is in fact labeled. Finally, it is unknown if postganglionic sympathetic neurons express mGluR8 so we cannot exclude the possibility that some of the labeled unmyelinated axons represent sympathetic fibers.

Therefore, due to these caveats, the estimates of labeled axons in the digital nerves are conservative compared to estimates in the DRG.

Our results provide the first anatomical evidence for the co-localization of mGluR8 and TRPV1 receptors in the rat DRG. The percentage of DRG cells that single- labeled for each receptor is consistent with previous reports (Guo et al., 1999; Carlton et al., 2004; Carlton and Hargett, 2007; Carlton et al., 2009; Hoffman et al., 2010). These data provide further support for a possible direct interaction between mGluR8 and

TRPV1 receptors. Additionally, mGluR8 is only one of four group III mGluRs; thus it is highly possible that there is a higher percentage of co-localization of TRPV1 with at least two other group III mGluRs (mGluR4 and 7). The results confirm that group III is also expressed in SGCs (Carlton and Hargett, 2007), however their function in this glial population remains unknown (Pocock and Kettenmann, 2007; D'Antoni et al., 2008).

While mGluR1α and mGluR2/3 have been localized on keratinocytes (Genever et al.,

1999), expression of mGluR8 by these cells has not been investigated. Since TRPV1 is expressed by keratinocytes (Ständer et al., 2004), we cannot completely rule out that the effect of L-AP-4 on nociceptors could be indirect.

In the present study, L-AP-4 was chosen because it is a potent nonselective agonist for group III mGluR subtypes. Although nonselective, studies demonstrate

greater affinity of this drug at mGluR8, mGluR4 and mGluR6 (localization appears to be limited to retina cells [Nakajima et al., 1993; Nomura et al., 1994]) compared to mGluR7.

Receptor potency for L-AP-4 appears to be as follows: mGluR8> mGluR4>mGluR6>>mGluR7 (Conn and Pin, 1997; Saugstad et al., 1997; Wu et al.,

1998; Cartmell and Schoepp, 2000; Yang, 2005; Selvam et al., 2007; Niswender et al.,

2008).

The lack of an L-AP-4 effect in naïve is consistent with studies that demonstrated sub-cutaneous injections of this drug, in the mM dose range, had no effect on PWL (Jin et al., 2009) or paw withdrawal threshold (PWT) (Walker et al., 2001). The anti- hyperalgesic effects of group III mGluR activation have been demonstrated through central administration of the agonists DCPG and L-AP-4 (Neugebauer et al., 2000;

Marabese et al., 2007a; Zhang et al., 2009). In the isolated spinal cord preparation, L-AP-

4 application on the spinal cord dose-dependently inhibited CAP-induced ventral root potentials (Ault and Hildebrand, 1993). However, the present study demonstrates a local anti-hyperalgesic effect can also be evoked following peripheral administration. Our findings are consistent with a previous report that peripheral group III mGluR activation attenuates bee-venom induced hyperalgesia (Chen et al., 2010). These findings suggest that a regimen that includes both a central and peripheral administration of a group III mGluR agonist could be advantageous in the treatment of pain.

The direct inhibition of TRPV1, through administration of TRPV1 antagonists, has been shown to produce hyperthermia in rats (Swanson et al., 2005b; Gavva et al.,

2007b; Wong and Gavva, 2009) and humans (Gavva et al., 2007a; Honore et al., 2009).

In the human studies, onset of hyperthermia occurred following one or more doses of a

TRPV1 antagonist but attenuated with repeated dosing. While there has been some progress in the development of TRPV1 antagonists that do not induce hyperthermia

(Watabiki et al., 2011), other issues remain. For example, in a randomized healthy volunteer trial, the thermosensory profile of the subjects was shifted, causing patients to perceive hotter temperatures as optimal temperatures (Rowbotham et al., 2011). Thus targeting the TRPV1 receptor directly leads to unwanted side effects. However, modulating downstream effectors of TRPV1 such as cAMP/PKA, that are negatively coupled to group III mGluR activity, could possibly avoid these adverse effects.

These two receptor families, TRPV1 and group III mGluRs, most likely interact at the level of intracellular downstream effectors and of particular interest is PKA. TRPV1 activity (Bhave et al., 2002; Jeske et al., 2008) and its membrane insertion (Rathee et al.,

2002) appear to be modulated through phosphorylation by PKA and other kinases

(Cesare et al., 1999; Numazaki et al., 2002; Bhave et al., 2003; Zhang et al., 2005). In contrast, group III mGluRs are negatively coupled to AC, thus inhibiting cAMP formation and cAMP-dependent PKA activity (Neugebauer and Carlton, 2002). This group III mGluR-induced inhibition may decrease the phosphorylation of TRPV1 thereby inhibiting the enhancement of TRPV1 activity and/or its insertion into the plasma membrane.

Alternatively, group III mGluR activation may modulate TRPV1 through other mechanisms. First, basal vesicular cycling (Chavis et al., 1998) and glutamate release

(Kumar et al., 2010) have been shown to decrease following activation of group III mGluRs. This could counteract the membrane depolarization caused by TRPV1-induced glutamate release (Ueda et al., 1993; Jin et al., 2009). Second, in a related fashion, this

reduced extracellular glutamate may curtail activation of group I mGluRs (Bhave et al.,

2001) and ionotropic glutamate receptors (iGluRs) N-Methyl-D-aspartate (NMDA) (Du et al., 2003), alpha-amino-3-hydroxy-5-methylisoxazolone-4-propionic acid (AMPA)

(Carlton et al., 1995; Coggeshall and Carlton, 1998) and kainate receptors (Du et al.,

2006) known to be present on peripheral nociceptors. Third, group III mGluR activation may inhibit voltage-gated Ca2+ channels, reducing neuronal excitability (Takahashi et al.,

1996; Perroy et al., 2000; Capogna, 2004; Guo and Ikeda, 2005). Fourth, activation of group III mGluRs may lead to opening of K+ channels, both background K+ channels and

G-protein coupled inwardly rectifying K+ (GIRK) channels (Saugstad et al., 1997; Cain et al., 2008; Niswender et al., 2008). Potassium efflux from the cell results in a more negative membrane potential, thus the opening of K+ channels could decrease excitability enough to prevent depolarization. However, based on our results in the present study, the interaction between TRPV1 and group III mGluRs most likely includes down regulation of the cAMP/PKA pathway.

It is now apparent that TRPV1 activity can be modulated indirectly through a variety of cell surface receptors expressed on nociceptors, resulting in inhibition (via mGluRs as shown here), or in sensitization. For example, activation of the bradykinin B2 receptor or nerve growth factor (NGF) TrkA receptor will sensitize TRPV1 channels, enhancing heat hyperalgesia and heat-evoked currents (Chuang et al., 2001; Ferreira et al., 2004). Furthermore, activation of prostaglandin E2 (PGE2) and PGI2 receptors (EP1 and IP, respectively), will indirectly enhance/sensitize TRPV1 responses (Moriyama et al., 2005). These actions occur through stimulation of PLC and protein kinase C (PKC) signaling pathways (Chuang et al., 2001; Julius and Basbaum, 2001). It is attractive to

pursue Group III mGluRs for drug development because TRPV1 is the ultimate target for so many algogenic substances and second messenger pathways.

Conclusion

In conclusion, the present study identifies several important characteristics of the group III mGluRs. Their activation has an anti-hyperalgesic effect; this effect can be evoked locally at cutaneous nociceptors, and most likely involves down regulation of the

AC/cAMP/PKA pathway. An ideal candidate for pain therapy is one that has few side effects, which might be attainable with a peripherally applied group III mGluR agonist.

Additionally, the use of anti-hyperalgesics is favorable over analgesics since administration of the former inhibits enhanced nociceptive responses while allowing for normal responses to potentially noxious stimuli. Due to the fact that mGluR8 is highly co-localized with TRPV1 and significantly attenuates TRPV1-induced nociceptive behaviors, Group III mGluRs are attractive targets for novel therapies in peripheral pain treatment.

Acknowledgments

We thank Zhixia Ding for her excellent assistance with the electron microscopy.

CHAPTER 3: GROUP III MGLUR8 NEGATIVELY MODULATES TRPA1

ACTIVITY

Introduction

Several lines of evidence indicate that transient receptor potential (TRP) ion channels play an important role in hypersensitivity after injury and/or inflammation. transient receptor potential ankyrin 1 (TRPA1) is a member of a subfamily of TRP ion channels. Similar to other TRP channels, TRPA1 is a calcium permeable non-selective cation channel (Story et al., 2003; Zhang and Liao, 2014). Activators of TRPA1 include a wide variety of chemical compounds such as mustard oil, bradykinin, arachidonic acid, environmental irritants (Bandell et al., 2004; Bautista et al., 2006), formalin (0.01%,

Fluo-4 Ca2+, (McNamara et al., 2007)]), an assortment of pungent plant compounds and extracts including cinnamaldehyde (cinnamon), eugenol (cloves), gingerol (ginger), methyl salicylate (wintergreen) (Bandell et al., 2004), allicin (found in garlic)

(Macpherson et al., 2005; Bautista et al., 2006), D9-tetrahydrocannabinol (THC) (Jordt et al., 2004), as well as noxious cold (< 17 °C) (Story et al., 2003) and mechanical (Bautista et al., 2006; Kwan et al., 2006; Kwan et al., 2009) stimuli. More recently, it was reported that TRPA1 plays a role in acute and chronic itch (Wilson et al., 2011; Wilson et al.,

2013).

Several lines of evidence suggest that TRPA1 plays a role in hypersensitivity associated with a variety of pain states. These include inflammation, neuropathic pain

(resulting from either traumatic injury or metabolic disease), migraine, dental pain, and colicky pain in the gastrointestinal tract (Haas et al., 2011; Bautista et al., 2013; Benemei

et al., 2013; Tsumura et al., 2013; Kaneko and Szallasi, 2014). For example, intraplantar formalin, an inflammatory mediator, or 4-hydroxynoneal (4HNE) an endogenous aldehyde, induces flinching and/or licking/lifting behavior and tactile allodynia that is attenuated by TRPA1 antagonists (McNamara et al., 2007; Trevisani et al., 2007).

However, TRPA1 KO mice do not show any nociceptive responses to 4HNE injection.

At the single fiber level, the TRPA1 antagonist HC-030031 inhibits formalin-induced activity and reduces formalin-induced mechanical sensitivity in C-fibers (Kerstein et al.,

2009). Following peripheral nerve injury, TRPA1 protein levels are increased in the spinal cord and the rats show increased mechanical sensitivity that is blocked by systemic administration of HC-030013 (Zhang et al., 2014a). Therefore, modulation of TRPA1 could prove to be an effective therapeutic target in a variety of pain states.

Previous reports from our lab demonstrate that group II and III metabotropic glutamate receptors (mGluRs) negatively modulate transient potential receptor vanilloid

1 (TPRV1) activity (Carlton et al., 2009; Govea et al., 2012). TRPV1 plays an important role in the development of hyperalgesia (Caterina et al., 2000; Davis et al., 2000;

Bolcskei et al., 2005). Group III mGluRs (mGluR4, 6, 7, and 8) are negatively coupled to adenylyl cyclase and the cAMP/PKA intracellular pathway, and are thus associated with neuronal inhibition. Furthermore, group III mGluRs exert activity-dependent modulation of TRPV1 (Carlton et al., 2011). Several lines of evidence indicate that TRPV1 and

TRPA1 are highly co-localized in DRG neurons (Story et al., 2003; Kobayashi et al.,

2005; Dai et al., 2007), thus we hypothesized that group III mGluRs might also negatively modulate TRPA1. In the present study, we incorporated a multifaceted

approach (calcium imaging, behavior, electrophysiological recording) to investigate the possible interactions between group III mGluR8 and TRPA1.

Methods

ANIMAL USE AND CARE

All experiments were approved by the University Animal Care and Use

Committee at the University of Texas Medical Branch (Galveston, TX) and followed the

International Association for the Study of Pain guidelines for the ethical care and use of laboratory animals (Zimmermann, 1983). Adult male Sprague-Dawley rats (150 - 300 g,

Harlen, Houston, TX) were used in all experiments. Steps were taken to minimize both the number of animals used and their discomfort.

DRUG PREPARATIONS

Mustard oil (MO) was made fresh daily. The stock solution of MO was diluted using 1% DMSO (vehicle) in order to produce a working solution of 10 mM MO. α- methyl-3-methyl-4-phosphonophenylglycine (UBP1112 [UBP], Tocris), a selective group

III antagonist and (S)-3,4-dicarboxyphenylglycine (DCPG, Tocris), a selective group III mGluR8 agonist were dissolved in 1 N NaOH to produce a 100 mM stock solution. Rp- cyclic 3′,5′-hydrogen phosphorothioate adenosine triethylammonium salt (Rp-cAMPS,

Santa Cruz Biotech), a cell permeable PKA inhibitor, was dissolved in dH2O to produce a

10 mM stock solution. All stock solutions were diluted with PBS (vehicle) unless otherwise noted and the pH was adjusted to 7.4.

DRUGS: TOPICAL APPLICATION AND INJECTION

MO was applied topically, using a sterile Q-tip, to the plantar surface of the rat hind paw. All other drugs were delivered via intraplantar injection using a 28-gauge needle attached to a 50 µl Hamilton syringe with PE20 tubing. Rats were not anesthetized since it would potentially alter their initial responses to MO. The needle punctured the plantar surface of the hind paw and was guided through the subcutaneous space to a site proximal to the pads. All drugs were administered to the same place in the chosen hind paw and all injected volumes were kept consistent within each paradigm, unless otherwise noted. Each animal was only used once in an experimental paradigm and the researcher was unaware of the drug combinations being given to an animal.

HABITUATION FOR BEHAVIORAL TESTING

Upon arrival at the animal care facility, animals were acclimated for at least 3 days prior to being placed into an experimental group. They were housed in groups of three in plexiglass cages with soft bedding and were kept on a reversed light/dark cycle

(12hr/12hr). All experiments were performed between 8AM to 5PM, during the animal’s dark cycle. Care was taken to ensure minimal lighting conditions during transport, habituation, and testing. Rats were habituated for mechanical threshold by placing them on a wire screen platform in plexiglass cages for 1 hr followed by stimulation using the

3.41 von Frey filament three times, on each of 2 days.

TESTING FOR MUSTARD OIL-INDUCED BEHAVIORS AND MECHANICAL SENSITIVITY

Application of MO to the hind paw induced flinching behavior. This behavior made the assessment of mechanical threshold using von Frey filaments difficult; however, the flinching behavior ceased 30 min post-MO application and it was at this

time that mechanical threshold testing began. Prior to any drug treatment, rats were baselined using the Dixon Up-Down method (Chaplan et al., 1994) then changes in mechanical threshold were assessed beginning 30 min post-MO at 10 min intervals for 30 min. Mechanical threshold was plotted as a percent change of baseline.

ELECTROPHYSIOLOGICAL RECORDINGS

In vitro skin-nerve preparation

Rats (300 g) were killed with an overdose of CO2. For both hind paws, the glabrous skin from the ankle to the tips of the toes was dissected free with the medial and lateral plantar nerves still attached. The preparation was placed corium side up in an organ bath and superfused (15 ml/min, 34 ˚C) with an oxygenated synthetic interstitial fluid (SIF, in mM: NaCl, 123; KCl, 3.5; MgSO4, 0.7; CaCl2, 2.0; Na gluconate, 9.5;

NaH2PO4, 1.7; Glucose, 5.5; Sucrose, 7.5; and HEPES, 10; pH 7.45 ± 0.05). All muscle and tendon tissues were removed from the preparation. The plantar nerves were guided to a separate chamber containing a bottom layer of SIF and a superficial layer of mineral oil.

The plantar nerves were desheathed and delicately teased apart on a mirror stage. Small nerve bundles were repeatedly teased apart until single unit activity was obtained by recording with a gold wire electrode.

Thermal and chemical testing procedures

Only units responding to mechanical probing of the glabrous skin with a blunt glass rod with a clearly defined receptive field were studied in detail.

THERMAL STIMULATION. Radiant heat (unit was built in house) was applied to each receptive field by a lamp placed beneath the organ bath. The light beam was focused

through the bottom of the organ bath onto the epidermal surface of the glabrous skin. A thermocouple was placed into the corium above the light beam to measure intracutaneous temperature. A standard heat ramp from 34 ˚C to 51 ˚C in 10 s was applied to each unit from the epidermal side. The threshold for heat response was defined as the temperature evoking the second spike after initiation of the heat ramp.

MECHANICAL STIMULATION. A dual mode lever system (Aurora Scientific Inc.,

Ontario Canada) was used to determine mechanical threshold and discharge rate. The system has a motor-driven stylus that delivered a force in the form of either a continuous ascending ramp (from 0-250 mN in 20 s) or a square wave suprathreshold stimulus (250 mN for 10 s) to determine mechanical threshold and discharge rate, respectively. The stylus (1.5 mm diameter) was placed in the receptive field of the unit on the corium side.

The first spike occurring above mean background discharge rate once the ramp force was activated was considered threshold for that unit. Mechanical discharge rate was determined by measuring the discharge rate in response to the square wave mechanical stimulus. The mean background discharge rate for each fiber was determined by measuring the mean discharge rate for 10 s prior to any mechanical stimulation.

CHEMICAL STIMULATION. A small hollow cylinder (hereafter referred to as a

“well”, (5 mm diameter) was placed over the receptive field of each unit. The SIF in the well was replaced with the drug(s) made as described above, but SIF was used for final dilutions (pH 7.4).

CONDUCTION VELOCITY. The conduction velocity of each unit was determined by monopolar electrical stimulation (1 ms duration, 1 Hz) at the most mechanosensitive site in the receptive field for each unit using a Teflon-coated steel electrode (5 MΩ

impedance, 250 µm shaft diameter). Conduction velocity was calculated by determining the latency of the action potential and the distance from the stimulating electrode to the recording site.

Spike Sorting

Action potentials were acquired and analyzed with a CED 1401 (Cambridge, UK) using Spike 2 (v7.0) software. Single spike units were isolated from multi-unit recordings, using template matching. A minimum number of 8 spikes was needed for a template to be created. If the amplitude of a spike was no greater than 25 % of the template amplitude it was considered a match. If 60 % of the points of an action potential over-laid one of the templates it was considered a match.

CALCIUM IMAGING

Cell Culture

Rats (150 g) rats were killed with an overdose of CO2. The L4-L6 DRG were removed bilaterally and placed into a culture dish containing ice-cold oxygenated dissecting solution (in mM: 130 NaCl, 5 KCl, 2 KH2PO4, 6 MgCl2, 1.5 CaCl2, 10 glucose, 10 HEPES, pH adjusted to 7.2 with NaOH base. Osmolality was adjusted with sucrose to 300 mOsm (Xu and Huang, 2002). The DRG were minced into small pieces.

DRG fragments were digested in 0.5 mg/ml trypsin and 1 mg/ml collagenase D in a warm bath (37 ˚C) shaker for 1 hr and then rinsed with extracellular solution (ES), containing the same components as dissecting solution with the following adjustments: 1 mM MgCl2 and 2.5 mM CaCl2. The DRG cells were mechanically dissociated using fire polished glass pipettes until the solution was clear. Dissociated cells (0.1 ml) were plated on poly

D-lysine coated glass coverslips inside 35 mm culture dishes. ES (2 ml) was added to each culture dish 15 min after cells were plated, and cells were maintained for 30 min at room temperature before adding Fura-2 AM. DRGs from 3 to 4 rats were used for each treatment paradigm.

Cytosolic Ca2+ imaging

DRG cells were incubated in 3 µM Fura-2 AM in ES containing 0.2% pluronic acid F-127 at room temperature for 45 min, rinsed with ES, and allowed to stabilize in the dark for 1 hr at room temperature. Ratiometric Ca2+ imaging was performed using an inverted fluorescent Nikon microscope equipped with a 340 nm and 380 nm excitation filter changer, a 510 nm long-pass emission filter, and a 12-bit digital monochrome cooled CCD camera controlled by Metafluor software v6.5 (Molecular Devices).

Fluorescent images and ratios were acquired every 5 s and processed using MetaMorph software (Molecular Devices). The ES in the dish was replaced with the drug(s) made as described above, but ES was used for final dilutions (pH 7.4). Cells were considered responsive if the F340/F380 ratio change was >20% during drug application. All experiments were performed at room temperature.

DATA ANALYSIS

Data were expressed as mean ± SEM unless otherwise noted and evaluated using

SigmaStat (Systat software). For the behavioral studies, paired t-tests and one way

ANOVA analyses were used. For the unit recordings, paired and unpaired t-tests, Chi-

Square and Fischer Exact analyses were used when appropriate. For the calcium imaging studies, parametric (one-way ANOVA followed by Holm Sidak test), nonparametric

(one-way ANOVA on ranks followed by Dunn’s Method or Student-Newman-Keuls), or

Chi-square analyses were used when appropriate. *p < 0.05 was considered significant.

Results

DCPG REDUCES THE NUMBER OF MO-RESPONSIVE CELLS

Ca2+ imaging of acutely dissociated DRG cells was used to determine co- localization of mGluR8 and TRPA1on cells since TRPA1 antibodies with high specificity were not available. At the start of each experiment a 2 min baseline of Ca2+ activity was recorded for each cell, then cells were exposed to one of the following conditions: 30 µM

MO; 10 µM DCPG + 30 µM MO; 1 µM DCPG + 30 µM MO; or 0.3 µM DCPG +30 µM

MO.

Addition of 10 µM DCPG resulted in an initial delay in Ca2+ mobilization followed by enhanced Ca2+ mobilization compared to MO alone (Figure 3.1C).

Compared to baseline, 30 µM MO induced robust Ca2+ mobilization in DRG neurons

(Figure 3.1C). Unexpectedly, results demonstrated that 10 µM DCPG + MO induced more Ca2+ mobilization and 1 µM and 0.3 µM DCPG tended to reduce Ca2+ mobilization compared to MO alone (Figure 3.1B, one way ANOVA, *p < 0.05). Both 10.0 µM and

0.3 µM DCPG significantly reduced the number of viable DRG cells responding to MO

(Figure 3.1A, Chi-Square, *p < 0.05). These findings provide a strong line of evidence for functional coupling and co-localization of mGluR8 and TRPA1.

Figure 3.1: DCPG Dose-Response Relationship

DCPG significantly reduced the percent of cells responding to MO (A). There was a trend for DCPG to reduce total Ca2+ mobilization induced by MO but significance was not reached (B). B is a summary graph representing total Ca2+ mobilization from 0-800 sec from C. Time course showing the Ca2+ response of cells to various drug combinations (C). 30 µM MO (n=6 animals), 30 µM MO + 10 µM DCPG (n=5 animals), 30 µM MO + 1 or 0.3 µM DCPG (n=4 animals); *p < 0.05, significantly different vs MO, Chi-Square analysis (A) and one way ANOVA (B).

DCPG ATTENUATES MO-INDUCED MECHANICAL SENSITIVITY

In order to determine whether activation of mGluR8 could block TRPA-induced mechanical sensitivity, rats were separated into the following groups: Vehicle only (20

µl, n=7), Vehicle (20 µl) + 10 mM MO (n=10), or 30 µM DCPG (20 µl) + 10 mM MO

(n=10) with 10 min between 1st and 2nd drug deliveries. Application 10 mM MO enhanced mechanical sensitivity evidenced by a significant reduction in paw withdrawal

threshold (PWT, Figure 3.2, inset, paired t-test, *p < 0.05). Conversely, rats that received

DCPG prior to MO application did not display changes in mechanical sensitivity for their

PWT was not different from baseline (Figure 3.2, 2nd column). In order to demonstrate receptor specificity, DCPG was co-injected with a group III selective antagonist,

UBP1112. Thus, a separate group of rats received intraplantar 30 µM DCPG + 300 µM

UBP1112 (20 µl, n=5) 10 min prior to application of 10 mM MO. UBP1112 blocked the inhibitory effect of DCPG such that there was a significant reduction in PWT (one way

ANOVA, *p < 0.05) and this group was not different from MO alone (Figure 3.2, 3rd column).

In order to show that DCPG was having a local versus a systemic effect, a separate group of rats received an intraplantar injection of 30 µM DCPG (20 µl, n=5) in one hind paw and Vehicle (20 µl) followed 10 min later by 10 mM MO application to the contralateral hind paw. This group (Figure 3.2, 4th column) also developed mechanical sensitivity that was no different from the MO alone group, indicating that the effects of

DCPG were local in the injected hind paw and not systemic.

Figure 3.2: DCPG inhibits MO-induced mechanical sensitivity in vivo A 2 min exposure to MO induced mechanical sensitivity, significantly reducing the PWT from 15 g to 6 g (inset, *p < 0.05, paired t-test). 30 µM DCPG significantly reversed MO-induced mechanical sensitivity such that the DCPG treatment group was not significantly different from baseline. The group III antagonist UBP1112 reversed the DCPG effect. The inhibitory effect of DCPG was local since administration of DCPG in one paw and application of MO on the contralateral (contra) paw resulted in a PWT that was not significantly different from MO alone. *p < 0.05, significantly different from baseline, one way ANOVA followed by Student-Newman-Keuls. Numbers in the bars represent the N for each group

DCPG ATTENUATES MO-INDUCED NOCICEPTOR ACTIVITY AND MECHANICAL SENSITIVITY

In order to confirm that the behavioral results reflected what was occurring at the single fiber level, we recorded from primary afferent nociceptors using the in vitro glabrous skin-nerve preparation. We recorded from c-mechano (CM, n = 25), c-

mechanoheat (CMH, n = 3), and Aδ (n = 13) units in 9 naive rats. The mean background discharge rate for all units was 0.23 ± 0.03 imp/sec. The average conduction velocity for the c fibers was 0.62 ± .05 and for Aδ fibers was 3.07 ± 0.71 m/s. Once the receptive field of a nociceptor was identified the unit activity, mechanical threshold and mechanical discharge rate were measured before and after a 2 min exposure to either 30

µM MO or 10 µM DCPG + 30 µM MO (Figure 3.3 C-D). Nociceptor activity significantly increased from 0.18 ± 0.08 to 0.48 ± 0.1 imp/sec following exposure to MO

(Figure 3.3A, inset, paired t-test, *p < 0.05). DCPG returned nociceptor activity to baseline (Figure 3.3A, t-test, *p < 0.05). Mechanical threshold decreased from 138.99 ±

16.66 to 92.28 ± 15.95 mN following exposure to MO (Figure 3.4A, inset, paired t-test

*p < 0.05). DCPG reversed MO-induced decrease in mechanical threshold beyond baseline, from 69 % to 204 % of baseline (Figure 3.4A, t-test *p < 0.05, C-D). There was a trend for an increase in mechanical discharge rate, 2.03 ± 0.45 to 2.41 ± 0.69 imp/sec to a supra-threshold stimulus following exposure to MO (Figure 3.5A, paired t-test, *p <

0.05, C-D). The number of fibers that increased vs decreased in mechanical discharge rate was examined following exposure to either MO or MO + DCPG. DCPG significantly reduced the number of fibers that increased mechanical discharge rate in response to a supra-threshold stimuli compared to MO (Figure 3.5B, Chi-Square, *p < 0.05).

Figure 3.3: DCPG attenuates MO-induced nociceptor activity in vitro MO increases nociceptor activity at the single fiber level (inset in A, *p < 0.05paired t- test). DCPG returns the enhanced nociceptor discharge rate to baseline levels. Single spike and raw data traces from the same unit pre MO (B) post MO (C). Responses to MO+DCPG (D) were recorded from a different unit. *p < 0.05, significantly different from MO alone, Mann-Whitney test). Numbers in the bars represent the N for each group.

Figure 3.4: DCPG attenuates the MO-induced decrease in mechanical threshold in vitro MO sensitizes the nociceptors, significantly reducing their mechanical threshold (inset in A, *p < 0.05, t-test). DCPG reverses the MO effect, bringing mechanical threshold to above baseline levels. *p < 0.05, significantly different from MO alone, Mann-Whitney test. Single spike and raw data traces from the same unit pre MO (B) post MO (C). Responses to MO+DCPG (D) were recorded from a different unit.

Figure 3.5: DCPG reduces the number of mechanically sensitive fibers in vitro There was a trend for MO to increase the discharge rate to mechanical stimulation: mean discharge rates were 2.03 ± 0.45 imp/sec pre MO and 2.41 ± 0.69 imp/sec post MO but this increase did not reach significance (A). However, when comparing the number of fibers with increased vs decreased discharge rates following MO, DCPG significantly reduced the number of fibers that were excited by MO (B). Single spike and raw data traces from the same unit pre MO (C) post MO (C). Responses to MO+DCPG (E) were recorded from a different unit. *p < 0.05 significantly different from MO alone, Chi- Square analysis (B).

2+ PKA INHIBITION ATTENUATES MO-INDUCED CA MOBILIZATION

In order to investigate whether TRPA1 can be modulated by PKA activity we used a PKA inhibitor, RpCAMPS. Using calcium imaging, a 2 min baseline of Ca2+

activity was obtained, and then cells were exposed for 6 min to one of the following conditions: 30 µM MO; 10 µM RpCAMPS + 30 µM MO; or 1 µM RpCAMPS + 30 µM

MO. Compared to MO alone, RpCAMPS (10 µM and 1 µM) induced a delay in Ca2+ mobilization (Figure 3.6A) and this resulted in a significant reduction in total Ca2+ mobilization (Figure 3.6B, one-way ANOVA, *p < 0.05). Furthermore, 10 µM

RpCAMPs reduced the total number of viable cells responding to MO, however this did not reach significance (Figure 3.6C, Chi-Square analysis, *p < 0.05).

Figure 3.6: RpCAMPS attenuates MO-induced Ca2+ mobilization Application of 1 or 10 µM RpCAMPS, a PKA inhibitor, significantly attenuated MO- induced Ca2+ mobilization (A and B). There was a trend for RpCAMPS to also reduce the number of MO-responsive cells but this did not reach significance (C). B is a summary graph representing total Ca2+ mobilization from 0-300 sec from A. *p < 0.05, significantly different from MO alone, one way ANOVA followed by Holm-Sidak test (B) and Chi-Square analysis (C).

Discussion

In the present study we investigated the interaction between group III mGluR8 and TRPA1. Findings demonstrate that mGluR8 and TRPA1 are co-localized in sensory neurons of the DRG. Behaviorally, peripheral activation of mGluR8 significantly attenuated TRPA1-induced mechanical sensitivity. Furthermore, activation of mGluR8 significantly attenuates TRPA1-induced activity and mechanical hypersensitivity at the single fiber level. Further analysis suggest that the cAMP/PKA pathway is at least one mechanism by which mGluR8 negatively modulates TRPA1 activity.

MGLUR8 AND TRPA1 ARE EXPRESSED IN THE RAT DRG

TRPA1 is widely expressed in both neuronal and non-neuronal cells. TRPA1 expression has been shown in parts of the gastrointestinal tract and in the lung (Lapointe and Altier, 2011), smooth muscle (bladder) (Du et al., 2008b), skeletal muscle (Lapointe and Altier, 2011), keratinocytes (Atoyan et al., 2009), inner hair cells (Corey et al., 2004), cerebellar neurons (Garrison and Stucky, 2011), and in sensory ganglia (Story et al.,

2003; Nagata et al., 2005). Several lines of evidence indicate > 90 % of TRPA1 positive neurons express TRPV1 while < 66 % of TRPV1 positive neurons express TRPA1 (Story et al., 2003; Kobayashi et al., 2005; Dai et al., 2007). Furthermore, this agrees with Ca2+ imaging studies which demonstrated 93 % of MO-responsive DRG neurons were also

CAP-responsive (Zhang et al., 2014b).

Using a variety of techniques, the distribution of mGluR8 has been shown to be widespread. Prominent mGluR8 expression has been found in numerous brain regions, including the olfactory bulb, piriform cortex, pontine gray, lateral reticular nucleus of the

thalamus and lower levels of expression in cerebral cortex, hippocampus, cerebellum, and mammillary bodies (Saugstad et al., 1997; Turner and Salt, 1999). Later studies have demonstrated mGluR8 expression in the amygdala (Palazzo et al., 2008), periaqueductal grey (PAG) (Marabese et al., 2007b), NTS (Liu et al., 2012), the enteric nervous system

(Tong and Kirchgessner, 2003), spinal cord (Thomas et al., 2001), small to large DRG neurons (Carlton and Hargett, 2007; Govea et al., 2012) as well as in both myelinated and unmyelinated digital axons (Govea et al., 2012).

Initially, it was proposed, from studies in mice, that TRPA1 is expressed in approximately 3.6% of DRG neurons (Story et al., 2003). However, subsequent reports indicate a larger percentage of expression in sensory neurons, ranging from 20 – 57 %

(Jordt et al., 2004; Nagata et al., 2005; Obata et al., 2005). In the present study, MO was used because it is a potent specific agonist for TRPA1 (Jordt et al., 2004; Bautista et al.,

2006). Our findings from Ca2+ imaging studies demonstrate 10 % and 33 % of L4-L6 rat

DRG neurons are responsive to 10 and 30 µM, respectively. These findings are supported by previous reports which demonstrate that the percentage of DRG neurons responsive to

MO using Ca2+ imaging is dose-dependent (Sawada et al., 2007; Caspani et al., 2009;

Barabas et al., 2012; Zhang et al., 2014a).

MGLUR8 AND TRPA1 ARE CO-LOCALIZED IN THE RAT DRG

Since our lab has previously demonstrated that activation of group III mGluRs negatively modulates TRPV1 activity (Govea et al., 2012) and there is a high level of co- localization between TRPV1 and TRPA1, we investigated the potential relationship between TRPA1 and group III mGluR8. In the present study, Ca2+ imaging in DRG neurons allowed us to first assess the co-localization of mGluR8 and TRPA1 and then to

begin to assess the functional relevance of this co-localization. An attenuation of TRPA1 induced activity by mGluR8 activation would suggest expression of TRPA1 and mGluR8 on the same cell, using Ca2+ imaging. While the curves for various group did not completely overlap, there was a significant difference in the number of cells responding.

In the present study DCPG was used because it is a potent specific agonist for mGluR8

(Thomas et al., 2001). Our data demonstrate that DCPG significantly decreased the percent of cells responding to MO, suggesting that activation of mGluR8 inhibits TRPA1 activity in DRG neurons.

MGLUR8 ACTIVATION ATTENUATES TRPA1-INDUCED MECHANICAL SENSITIVITY

Functional relevance of TRPA1 and mGluR8 co-localization was further assessed using a behavior assay. TRPA1 antagonists have been shown to attenuate mechanical sensitivity in a nerve root constriction model (Miyakawa et al., 2014), CFA models

(Petrus et al., 2007; Eid et al., 2008; da Costa et al., 2010; Lennertz et al., 2012;

Montrucchio et al., 2013), the spinal nerve ligation model (Eid et al., 2008), a postoperative pain model (Wei et al., 2012), and an oxaliplatin treatment model (Nassini et al., 2011). (da Costa et al., 2010) demonstrated that treatment with TRPA1 antisense prior to CFA injection prevented CFA-induced mechanical sensitivity. Using a global knockout of TRPA1, (Garrison and Stucky, 2011) demonstrated significantly higher mechanical thresholds in TRPA1 KO compared to WT up to 2 wk post CFA injection.

However, it should be noted that Bautista et al 2006 found no difference in baseline mechanical thresholds between TRPA1 KO and WT mice, suggesting that TRPA1 may not play a role in acute normal responses to mechanical stimuli. Our findings support a role for TRPA1 in mechanical hypersensitivity since acute TRPA1 activation with MO,

an irritant, induced a significant reduction of mechanical threshold compared to baseline.

To date, no studies have investigated the interaction of group III mGluRs and TRPA1.

Our data demonstrate that activation of group III mGluR8 reverses MO-induced mechanical sensitivity.

Previous studies have assessed activity at the single fiber level from the hairy skin

(Garrison and Stucky, 2011). For the purposes of this study, we assessed single fiber activity from the glabrous skin; since in the behavioral assays, mechanical thresholds are determined by probing the glabrous surface of the rat hind paw. Similar to our behavioral data, we found a significant reduction in mechanical threshold following TRPA1 activation with MO. Several electrophysiological studies indicate that TRPA1 antagonist attenuate mechanical hypersensitivity in CFA models (McGaraughty et al., 2010;

Lennertz et al., 2012), in an osteoarthritis model (McGaraughty et al., 2010), and in a nerve root constriction model (Miyakawa et al., 2014). Our data indicate that a similar effect is seen when an mGluR8 agonist is applied with MO.

MGLUR8 MODULATES TRPA1 ACTIVITY THROUGH THE CAMP/PKA PATHWAY

The interaction of these two receptors most likely occurs at the level of second messengers, the cAMP/PKA pathway. (Wang et al., 2008) demonstrated that a PKA activator, forskolin (FSK), potentiates MO-induced currents in DRG neurons, suggesting that PKA can enhance TRPA1 activity. In a later study, (Schmidt et al., 2009) showed that FSK, at high concentrations (1mM) significantly enhanced nocifensive responses.

Furthermore, live-cell imaging in transfected HEK293T cells demonstrates that FSK induces translocation of functional TRPA1 to the surface (Schmidt et al., 2009), suggesting that the PKA pathway plays a role in enhancing surface expression. Agonist

activation of TRPA1 can also induce translocation to the surface in both HEK293T cells and trigeminal ganglia (TG) neurons (Schmidt et al., 2009). Taken together these studies suggest that the cAMP/PKA pathway is an important target for TRPA1 modulation. Thus negative modulators of the cAMP/PKA pathway would be ideal candidates for TRPA1 modulation. Activation of group III mGluRs leads to down regulation of andenylyl cyclase and the cAMP/PKA pathway (Niswender and Conn, 2010). Similar to DCPG, inhibition of PKA by RpcAMPS attenuates MO-induced Ca2+ mobilization, suggesting that down-regulation of the cAMP/PKA pathway could be an important in negatively modulating TRPA1. A larger decrease in Ca2+ mobilization is seen in the presence of

RpcAMPS compared to DCPG; however, it should be noted that RpcAMPS most likely inhibits all PKA while DCPG would only inhibit PKA downstream of the mGluR. Since, activation of mGluR8 leads to a down-regulation of adenylyl cyclase and the cAMP/PKA pathway, it is possible that this is sufficient to prevent enhancement of TRPA1 activity and/or translocation to the surface. These findings suggest that the cAMP/PKA pathway is one potential mechanism by which mGluR8 negatively modulates TRPA1 activity.

Alternatively, there are other mechanisms by which mGluR8 may modulate

TRPA1 activity. Several studies indicate that activation of group III mGluRs inhibits vesicular cycling (Chavis et al., 1998) and glutamate release (Kumar et al., 2010;

Erdmann et al., 2012). For example, in the hippocampus that mGluR8 inhibition was found to primarily function through inhibition of vesicle release (decreasing Ca2+ affinity of the release sensor), rather than inhibition of Ca2+ entry (Erdmann et al., 2012). This inhibitory action by mGluR8 may prevent TRPA1 activation-induced Ca2+ release

(Kosugi et al., 2007; Sun et al., 2009; Yokoyama et al., 2011).

Conclusion

Understanding how TRPA1 is modulated is clinically relevant. Emerging evidence suggest TRP channels underlie various pain syndromes; in particular, studies suggests that a gain of function mutation in TRPA1 underlies familial episode pain syndrome (FEPS) (Nilius et al., 2005; Kremeyer et al., 2010; Bennett and Woods, 2014).

Persons that suffer from FEPS exhibit severe episodes of pain, which start at infancy, that are primarily localized to the upper part of the body. These pain episodes, lasting 60-90 min, can be triggered by fatigue, hunger or cold and are not alleviated by typical analgesics. Furthermore, these patients display a hypersensitivity the TRPA1 agonist,

MO. Interestingly, TRPA1 inhibitors appear to alleviate symptoms. Our findings, which demonstrate that activation of mGluR8 negatively modulates TRPA1 activity, suggest that mGluR8 is an attractive target for pain therapies.

CHAPTER 4: EXPANDED DISCUSSION

TRPV1 and TRPA1 are often referred to as prominent integrators of painful stimuli. Activation of various inflammatory mediator receptors, such as BK2 and PAR2 induces multiple second messenger pathways leading to phosphorylation of both TRPV1 and TRPA1 and trafficking of TRPA1 to the cell surface membrane (Premkumar and

Ahern, 2000; Dai et al., 2004; Patapoutian et al., 2009; Schmidt et al., 2009). A compelling amount of data have been generated concerning the interplay of these two receptors.

TRPV1/TRPA1 interactions

Several lines of evidence suggest that TRPV1 and TRPA1 interact (Ruparel et al., 2008; Staruschenko et al., 2010; Fischer et al., 2014; Spahn et al., 2014). Numerous reports demonstrate that these receptors are highly co-localized, with over 90% of

TRPA1 positive sensory neurons also expressing TRPV1 (Story et al., 2003; Kobayashi et al., 2005; Dai et al., 2007). In rat sensory neurons and in an expression system (CHO cells), TRPV1 and TRPA1 heteromeres appear to form as they can be co-precipitated

(Staruschenko et al., 2010; Fischer et al., 2014). Also noted in this study is the presence of TRPA1-TRPA1 and TRPV1-TRPV1 homomeres.

Several lines of evidence indicate that TRPA1 regulates TRPV1 activity (Spahn et al., 2014). In this study, TRPV1/TRPA1 transfected HEK cells that were pretreated with

MO displayed an enhanced Ca2+ mobilization response to CAP compared to cells without

MO pretreatment. Interestingly, in DRG neurons that received the following in sequence:

MO, washout, TRPA1 antagonist, CAP; the CAP-induced current was enhanced compared to control. Further analysis demonstrated that inhibition of either AC or PKA prevented TRPA1-induced potentiation of TRPV1 current. These findings might suggest that activation of TRPA1 triggered downstream modulators of TRPV1 that were not inhibited by the presence of the TRPA1 antagonist. Similarly, TRPV1 has been shown to regulate TRPA1 activity (Kosugi et al., 2007; Staruschenko et al., 2010). For example,

TRPA1 current is greater in WT compared to TRPV1 KO, suggesting a positive interaction between the two receptors (Staruschenko et al., 2010). Additionally in substantia gelatinosa, neurons pretreated with MO followed by exposure to CAP + MO showed an enhanced MO-current compared to that in neurons pretreated with MO followed by a second exposure to MO (Kosugi et al., 2007). These findings suggest that

TRPV1 regulates TRPA1 activity and/or that TRPV1 plays a role in stabilizing functional

TRPA1 surface expression.

In contrast to these findings, TRPV1 and TRPA1 have been found to cross- desensitize each other (Akopian et al., 2007; Kosugi et al., 2007; Ruparel et al., 2008;

Salas et al., 2009). Pretreatment with CAP resulted in an attenuation of both CAP- or

MO-induced calcitonin gene related peptide (CGRP) release compared to control in rat hind paw skin biopsies (Ruparel et al., 2008). Similarly, pretreatment with MO resulted in attenuation of both MO- or CAP-induced CGRP release compared to control. In vivo behavioral assays demonstrated similar effects: pretreatment with CAP attenuated both

CAP- or MO-induced nocifensive behaviors and pretreatment with MO attenuated both

MO- and CAP-induced nocifensive behaviors. There are two possibilities for an attenuated response after either TRPA1 or TRPV1 activation; first, the CGRP pool could

have been diminished, resulting in less CGRP available for the 2nd stimulation; second, the TRPA1 or TRPV1 channel could have been cross-desensitized from the 1st stimulation. Another study reported enhanced MO-induced currents in TRPA1 expressing

CHO cells compared to TRPA1/TRPV1 co-expressing CHO cells (Salas et al., 2009).

Altogether these studies demonstrate that TRPV1 and TRPA1 form complexes and in these complexes, channel properties are different when compared to each channel expressed in the absence of the other. Further studies will be needed to unravel the complexity of the TRPV1:TRPA1 interaction.

TRP channel modulation

TRPV1 SENSITIZATION

Sensitization of TRPV1 has been shown to occur through phosphorylation by several kinases: PKA, PKC, Ca2+/CaM-dependent kinase II (CaMKII), or Src kinase

(Premkumar and Ahern, 2000; Vellani et al., 2001; Bhave et al., 2002; Numazaki et al.,

2002; Bhave et al., 2003; Mohapatra and Nau, 2003; Tominaga and Tominaga, 2005;

Jeske et al., 2008; Jeske et al., 2009). A-Kinase Anchoring Protein 150 (AKAP150) is a scaffolding protein that has been found to mediate PKA- and PKC-induced phosphorylation of TRPV1 (Jeske et al., 2008; Jeske et al., 2009).

Phosphorylation by PKC, at two sites: Ser-502 and Ser-800, appears to play an important role in TRPV1 sensitization, particularly bradykinin-, anadamide-, and PAR2- induced sensitization (Premkumar and Ahern, 2000; Numazaki et al., 2002; Dai et al.,

2004). In one study, TRPV1 was found to co-localize with PAR2 in DRG neurons (Dai et al., 2004). Activation of PAR2, which induces release of substance P and CGRP,

enhanced CAP-induced currents in DRG cells demonstrating functional relevance of this interaction. Furthermore, this PAR2-induced enhancement of CAP current was blocked by PKC inhibitors. Additionally, studies indicate that bradykinin enhances TRPV1 activity in a PKC dependent manner, since PKC inhibition prevents bradykinin-induced

TRPV1 sensitization (Cesare and McNaughton, 1996; Cesare et al., 1999; Premkumar and Ahern, 2000).

TRPV1 DESENSITIZATION

Desensitization of TRPV1 can occur through repetitive heat or agonist activation

(Tominaga et al., 1998). Several reports suggest Ca2+ dependent dephosphorylation of

TRPV1 (Tominaga and Tominaga, 2005). For example, CAP-induced desensitization of

TRPV1 has been shown to result from rapid influx of intracellular Ca2+ (Cholewinski et al., 1993; Koplas et al., 1997; Rosenbaum et al., 2004), which activates calcineurin and calmodulin (CaM). Inhibition of calcineurin or disruption of the CaM binding site attenuates TRPV1 desensitization (Docherty et al., 1996; Tominaga and Tominaga,

2005).

Recently, β-arrestin-2 has been identified as a scaffolding protein for TRPV1 that prevents PKA phosphorylation (Por et al., 2012). In this study, sensory neurons from β- arrestin-2 KO animals displayed significantly attenuated TRPV1-desensitization compared to WT. Further analysis revealed that β-arrestin-2 regulated PKA phosphorylation of TRPV1 at two known phosphorylation sites (Ser-116, Thr-370). A later study demonstrated that phosphodiesterase PDE4D5 is particularly important for β- arrestin-2-mediated dephosphorylation of TRPV1 (Por et al., 2013). Furthermore, phosphorylation of both Thr-370 on TRPV1 and Thr-382 on β-arrestin-2 are essential for

association of TRPV1 and β-arrestin-2 on the surface membrane, and this associate could be part of a negative feedback mechanism.

TRPA1 SENSITIZATION

Several mediators have been shown to sensitize TRPA1. Studies suggest that

TRPA1 activity may be sensitized by PAR2 and bradykinin (both GPCRs) through phospholipase C (PLC) (Dai et al., 2007; Wang et al., 2008). PAR2 and TRPA1 were found to be co-localized in DRG neurons (Dai et al., 2007). Findings demonstrate that inhibition of PLC resulted in an inhibition of BK-induced potentiation of MO-induced currents (Wang et al., 2008). PLC has also been shown to potentiate MO-induced currents (Dai et al., 2007). Interestingly, PLC downstream effectors (DAG and PKC) do not appear to be associated with the PLC-induced sensitization of TRPA1. Rather, evidence suggests that PIP2 inhibits TRPA1 and thus when GPCR activation of PLC hydrolyzes PIP2 to IP3 the inhibition of TRPA1 by PIP2 is released, resulting in TRPA1 current potentiation (Dai et al., 2007).

TRPA1 DESENSITIZATION

While TRPA1 can undergo desensitization after agonist activation or by TRPV1 activation (described above) (Akopian et al., 2007; Dai et al., 2007; Ruparel et al., 2008), mechanisms are still being unraveled. For example, in HEK cells expressing TRPA1,

MO-induced currents decreased with repeated MO application (Dai et al., 2007).

Interestingly, CAP-induced desensitization of TRPA1 appears to be Ca2+ dependent/calcineurin-independent while MO-induced desensitization of TRPA1 appears to be Ca2+-independent (Ruparel et al., 2008).

In contrast, one study suggests a lack of desensitization of TRPA1 upon agonist activation (Dunham et al., 2008). In this study, responses from primary afferents from both naïve and CFA-inflamed animals did not desensitize following repeated TRPA1 agonist exposure (5 min interval).

TRPA1 AND COLD SENSITIVITY

While the role of TRPA1 in cold hypersensitivity following nerve injury, inflammation, or chemotherapy is well established (Ta et al., 2010; Cavaletti, 2014), its role as a cold receptor is controversial (Story et al., 2003; Babes et al., 2004; Bandell et al., 2004; Jordt et al., 2004; Nagata et al., 2005; Bautista et al., 2006; Kwan et al., 2006;

Sawada et al., 2007; Zurborg et al., 2007). In one study using TRPA1 KO mice there is no difference between KO and WT in latency to paw lift in a cold plate assay with temperatures decreasing in 5 degree intervals from 20 to -10 °C (Bautista et al., 2006).

Moreover, Ca2+ imaging analysis demonstrated no significant difference in TG neurons from either WT or TRPA1 KO in the magnitude of response when the temperature was reduced from 23 to 6 °C. Furthermore, the percent of cells responding to the temperature reduction was similar between WT and KO, with a small percentage of these cold responders insensitive to menthol (TRPM8). In contrast to these findings, another study demonstrates a significant reduction in the number of paw lifts in TRPA1 KO compared to WT using a cold plate assay (5 °C) (Kwan et al., 2006). Similar results were found in a later study, where the data demonstrate a significant reduction in nocifensive behaviors

(shivering/paw lifts) in TRPA1 KO compared to WT mice in a cold plate assay at 0 °C

(Karashima et al., 2009). However, WT mice displayed jumping behavior that was

significantly reduced in KO mice (only 3 of 25 KO mice), and a significant difference was seen in latency to first jump between KO and WT.

Fueling the controversy, one report suggests that cold-induced TRPA1 activation was secondary to cold-induced release of intracellular Ca2+ stores (Zurborg et al., 2007).

However, a later study demonstrates cold-induced TRPA1 currents in an intracellular

Ca2+ free environment, using BAPTA, a Ca2+ chelator (Karashima et al., 2009).

One study done in HEK cells reports that there is a decrease in MO-current following a decrease in bath temperature from 22 to 6 °C (Jordt et al., 2004). In line with this, findings from our skin-nerve study demonstrate that acute irritation with MO did not enhance cold responses. In this paradigm, we assessed cold sensitivity for 10 sec after a frozen SIF pellet was placed into the well. At 10 sec, the temperature dropped, on average, from 34 to 17.3 ± 6.2 ˚C. Overall, our results indicated a decrease in the cold response of nociceptors after MO was applied (Figure 4.1). This decrease in response might be attributed to cold-induced desensitization of TRPA1.

Figure 4.1: MO decrease cold response in primary afferents Application of MO to primary afferents induced a significant reduction in cold response from 2.36 ± 0.65 imp/sec before MO to 0.95 ± 0.23 imp/sec after MO. n=13 fibers from 8 animals. *p < 0.05, significantly different from Pre MO, Wilcoxon Signed Rank test.

Using the skin-nerve preparation to record directly from primary afferents, our findings demonstrate that 36 % of all fibers were MO responsive and of these 63 % were also cold responsive. These findings are in agreement with previous reports (Sawada et al., 2007; Caspani et al., 2009).

One study, using Ca2+ imaging, demonstrated that 28.9 % of DRG cells were MO responsive. Of these MO responsive cells 119/135 (~88%) were also activated by deep cooling, described as less than 15.5 ± 0.52 ˚C (Sawada et al., 2007). A second study also using Ca2+ imaging, found that 45 % of DRG cells were MO responsive. Of these MO

responsive cells, only 18 % were also cold responsive (temperature decreased from ~30

°C to ~10 °C) (Caspani et al., 2009).

Of all fibers studied, 59% were cold responsive and of these 62 % of were cold responsive after pretreatment with MO. Interestingly after treatment with DCPG and MO only to 13 % of fibers were cold responsive (Figure 4.2). These findings suggest that mGluR8 inhibits TRPA1 expressing nociceptors.

Figure 4.2: DCPG reduced the number of cold responsive fibers Following 30 µM MO treatment, 62 % (8 out of 13) of cold responsive fibers were still responsive to cold. After treatment with 30 µM DCPG + 30 µM MO, only 13% (2 out of 8) of cold responsive fibers were still activated by cold temperatures. *p < 0.05 significantly different from MO alone, Fisher Exact test.

Based on our findings we cannot confidently conclude whether TRPA1 is a cold receptor. Our paradigm was flawed in that it might have been optimum to investigate whether TRPA1 was a cold receptor by utilizing a TRPA1 antagonist in the presence of a

cold stimulus. Furthermore, in our set up we are unable to determine the cold activation threshold. We applied frozen SIF chips of consistent size; however, a peltier cooling system would allow for consistent control of temperature and possible measurement of the temperature threshold for cold activation.

mGluR Signaling

Our studies focused on the signaling pathway mediated by the Gαi/o subunit of the

Group III mGluRs. Upon agonist activation, the Gα subunit dissociates from the GPCR and binds to adenylyl cyclase (AC). Binding to AC leads to inhibition of cAMP and ultimately PKA. However, activation of group III mGluRs can mediate bidirectional signaling via the Gαi/o and Gβγ subunits (El Far and Betz, 2002).

Several studies indicate an interaction, possibly through the Gβγ subunit, with voltage gated Ca2+ channels, thus inhibiting Ca2+ influx (Takahashi et al., 1996; Perroy et al., 2000; Capogna, 2004; Guo and Ikeda, 2005). Interestingly, one study found that mGluR8-induced inhibition does not inhibit Ca2+ influx, but dampens neuronal excitation by decreasing the Ca2+ affinity for the vesicle release sensor (Erdmann et al., 2012). In line with these findings, activation of group III mGluRs decreases vesicular cycling

(Chavis et al., 1998) and glutamate release (Kumar et al., 2010). Inhibition of glutamate release would reduce extracellular glutamate, potentially reducing the likelihood of activation of excitatory group I mGluRs and ionotropic glutamate receptors known to be present on peripheral nociceptors (Carlton et al., 1995; Coggeshall and Carlton, 1998;

Bhave et al., 2001; Du et al., 2003; Du et al., 2006).

Group III mGluRs are able to couple with G-protein regulated inwardly rectifying potassium (GIRK) channels when co-expressed in HEK cells (Saugstad et al., 1996;

Saugstad et al., 1997). Further analysis has revealed that upon activation of group III mGluRs, it is the Gβγ subunit that activates GIRK channels (Niswender et al., 2008).

Activation of GIRK channels results in hyperpolarization of the cell, thus reducing cell excitability (Lüscher and Slesinger, 2010).

Concluding Remarks

In our study, results suggest that group III mGluR8 modulation of TRPV1 is likely to occur through the cAMP/PKA pathway (Figure 2.6). Additionally, our results indicated that group III mGluR8 modulation of TRPA1 may occur through the cAMP/PKA pathway, however, due to differences in inhibition of Ca2+ mobilization in

MO-responsive cells (Figure 3.1 and 3.6), it is likely that down-regulation of cAMP/PKA is not the major player producing this modulation. Nonetheless, activation of group III mGluRs negatively modulated both TRPV1 and TRPA1 activity in vitro and in vivo.

These findings demonstrate the group III mGluRs, mGluR8 in particular, is an attractive target in modulating these major integrators of pain information.

REFERENCES

AAPM AAPM Facts and Figures on Pain. In: AAPM.

Akopian AN, Ruparel NB, Jeske NA, Hargreaves KM (2007) Transient receptor potential

TRPA1 channel desensitization in sensory neurons is agonist dependent and

regulated by TRPV1-directed internalization. J Physiol 583:175-193.

Allchorne AJ, Broom DC, Woolf CJ (2005) Detection of cold pain, cold allodynia and

cold hyperalgesia in freely behaving rats. Mol Pain 1:36.

Anjaneyulu M, Berent-Spillson A, Russell JW (2008) Metabotropic glutamate receptors

(mGluRs) and diabetic neuropathy. Curr Drug Targets 9:85-93.

Atoyan R, Shander D, Botchkareva NV (2009) Non-neuronal expression of transient

receptor potential type A1 (TRPA1) in human skin. J Invest Dermatol 129:2312-

2315.

Atta-ur-Rahman, Harvey K, Siddiqui RA (1999) Interleukin-8: An autocrine

inflammatory mediator. Curr Pharm Des 5:241-253.

Ault B, Hildebrand LM (1993) Effects of excitatory amino acid receptor antagonists on a

capsaicin-evoked nociceptive reflex: a comparison with morphine, clonidine and

baclofen. Pain 52:341-349.

Babes A, Zorzon D, Reid G (2004) Two populations of cold-sensitive neurons in rat

dorsal root ganglia and their modulation by nerve growth factor. Eur J Neurosci

20:2276-2282.

Bandell M, Story GM, Hwang SW, Viswanath V, Eid SR, Petrus MJ, Earley TJ,

Patapoutian A (2004) Noxious cold ion channel TRPA1 is activated by pungent

compounds and bradykinin. Neuron 41:849-857.

Barabas ME, Stucky CL (2013) TRPV1, but not TRPA1, in primary sensory neurons

contributes to cutaneous incision-mediated hypersensitivity. Mol Pain 9:9.

Barabas ME, Kossyreva EA, Stucky CL (2012) TRPA1 is functionally expressed

primarily by IB4-binding, non-peptidergic mouse and rat sensory neurons. PLoS

One 7:e47988.

Basu S, Srivastava P (2005) Immunological role of neuronal receptor vanilloid receptor 1

expressed on dendritic cells. Proc Natl Acad Sci U S A 102:5120-5125.

Bautista DM, Pellegrino M, Tsunozaki M (2013) TRPA1: A gatekeeper for

inflammation. Annu Rev Physiol 75:181-200.

Bautista DM, Jordt SE, Nikai T, Tsuruda PR, Read AJ, Poblete J, Yamoah EN, Basbaum

AI, Julius D (2006) TRPA1 mediates the inflammatory actions of environmental

irritants and proalgesic agents. Cell 124:1269-1282.

Benemei S, De Cesaris F, Fusi C, Rossi E, Lupi C, Geppetti P (2013) TRPA1 and other

TRP channels in migraine. J Headache Pain 14:71.

Bennett DL, Woods CG (2014) Painful and painless channelopathies. Lancet Neurol

13:587-599.

Bertin S et al. (2014) The ion channel TRPV1 regulates the activation and

proinflammatory properties of CD4⁺ T cells. Nat Immunol 15:1055-1063.

Bessou P, Perl ER (1969) Response of cutaneous sensory units with unmyelinated fibers

to noxious stimuli. J Neurophysiol 32:1025-1043.

Bhave G, Karim F, Carlton SM, Gereau RW (2001) Peripheral group I metabotropic

glutamate receptors modulate nociception in mice. Nat Neurosci 4:417-423.

Bhave G, Zhu W, Wang H, Brasier DJ, Oxford GS, Gereau RW (2002) cAMP-dependent

protein kinase regulates desensitization of the capsaicin receptor (VR1) by direct

phosphorylation. Neuron 35:721-731.

Bhave G, Hu HJ, Glauner KS, Zhu W, Wang H, Brasier DJ, Oxford GS, Gereau RW

(2003) Protein kinase C phosphorylation sensitizes but does not activate the

capsaicin receptor transient receptor potential vanilloid 1 (TRPV1). Proc Natl

Acad Sci U S A 100:12480-12485.

Boye Larsen D, Ingemann Kristensen G, Panchalingam V, Laursen JC, Nørgaard Poulsen

J, Skallerup Andersen M, Kandiah A, Gazerani P (2014) Investigating the

expression of metabotropic glutamate receptors in trigeminal ganglion neurons

and satellite glial cells: implications for craniofacial pain. J Recept Signal

Transduct Res 34:261-269.

Brederson JD, Kym PR, Szallasi A (2013) Targeting TRP channels for pain relief. Eur J

Pharmacol 716:61-76.

Burgess PR, Perl ER (1967) Myelinated afferent fibres responding specifically to noxious

stimulation of the skin. J Physiol 190:541-562.

Bäckström P, Hyytiä P (2005) Suppression of alcohol self-administration and cue-

induced reinstatement of alcohol seeking by the mGlu2/3 receptor agonist

LY379268 and the mGlu8 receptor agonist (S)-3,4-DCPG. Eur J Pharmacol

528:110-118.

Bölcskei K, Helyes Z, Szabó A, Sándor K, Elekes K, Németh J, Almási R, Pintér E,

Petho G, Szolcsányi J (2005) Investigation of the role of TRPV1 receptors in

acute and chronic nociceptive processes using gene-deficient mice. Pain 117:368-

376.

Cain SM, Meadows HJ, Dunlop J, Bushell TJ (2008) mGlu4 potentiation of K(2P)2.1 is

dependant on C-terminal dephosphorylation. Mol Cell Neurosci 37:32-39.

Cao Z, Balasubramanian A, Marrelli SP (2014) Pharmacologically induced hypothermia

via TRPV1 channel agonism provides neuroprotection following ischemic stroke

when initiated 90 min after reperfusion. Am J Physiol Regul Integr Comp Physiol

306:R149-156.

Capogna M (2004) Distinct properties of presynaptic group II and III metabotropic

glutamate receptor-mediated inhibition of perforant pathway-CA1 EPSCs. Eur J

Neurosci 19:2847-2858.

Carlton SM (2001) Peripheral excitatory amino acids. Curr Opin Pharmacol 1:52-56.

Carlton SM, Coggeshall RE (2001) Peripheral capsaicin receptors increase in the

inflamed rat hindpaw: a possible mechanism for peripheral sensitization. Neurosci

Lett 310:53-56.

Carlton SM, Coggeshall RE (2002) Inflammation-induced up-regulation of neurokinin 1

receptors in rat glabrous skin. Neurosci Lett 326:29-32.

Carlton SM, Hargett GL (2007) Colocalization of metabotropic glutamate receptors in rat

dorsal root ganglion cells. J Comp Neurol 501:780-789.

Carlton SM, Hargett GL, Coggeshall RE (1995) Localization and activation of glutamate

receptors in unmyelinated axons of rat glabrous skin. Neurosci Lett 197:25-28.

Carlton SM, Du J, Zhou S (2009) Group II metabotropic glutamate receptor activation on

peripheral nociceptors modulates TRPV1 function. Brain Res 1248:86-95.

Carlton SM, Du J, Zhou S, Coggeshall RE (2001) Tonic control of peripheral cutaneous

nociceptors by somatostatin receptors. J Neurosci 21:4042-4049.

Carlton SM, Zhou S, Govea R, Du J (2011) Group II/III metabotropic glutamate

receptors exert endogenous activity-dependent modulation of TRPV1 receptors on

peripheral nociceptors. J Neurosci 31:12727-12737.

Carlton SM, Zhou S, Du J, Hargett GL, Ji G, Coggeshall RE (2004) Somatostatin

modulates the transient receptor potential vanilloid 1 (TRPV1) ion channel. Pain

110:616-627.

Cartmell J, Schoepp DD (2000) Regulation of neurotransmitter release by metabotropic

glutamate receptors. J Neurochem 75:889-907.

Caspani O, Zurborg S, Labuz D, Heppenstall PA (2009) The contribution of TRPM8 and

TRPA1 channels to cold allodynia and neuropathic pain. PLoS One 4:e7383.

Caterina MJ, Schumacher MA, Tominaga M, Rosen TA, Levine JD, Julius D (1997) The

capsaicin receptor: a heat-activated ion channel in the pain pathway. Nature

389:816-824.

Caterina MJ, Leffler A, Malmberg AB, Martin WJ, Trafton J, Petersen-Zeitz KR,

Koltzenburg M, Basbaum AI, Julius D (2000) Impaired nociception and pain

sensation in mice lacking the capsaicin receptor. Science 288:306-313.

Cavaletti G (2014) Chemotherapy-induced peripheral neurotoxicity (CIPN): what we

need and what we know. J Peripher Nerv Syst 19:66-76.

Cavanaugh DJ, Chesler AT, Bráz JM, Shah NM, Julius D, Basbaum AI (2011)

Restriction of transient receptor potential vanilloid-1 to the peptidergic subset of

primary afferent neurons follows its developmental downregulation in

nonpeptidergic neurons. J Neurosci 31:10119-10127.

Cesare P, McNaughton P (1996) A novel heat-activated current in nociceptive neurons

and its sensitization by bradykinin. Proc Natl Acad Sci U S A 93:15435-15439.

Cesare P, Dekker LV, Sardini A, Parker PJ, McNaughton PA (1999) Specific

involvement of PKC-epsilon in sensitization of the neuronal response to painful

heat. Neuron 23:617-624.

Chao LK, Hua KF, Hsu HY, Cheng SS, Lin IF, Chen CJ, Chen ST, Chang ST (2008)

Cinnamaldehyde inhibits pro-inflammatory cytokines secretion from

monocytes/macrophages through suppression of intracellular signaling. Food

Chem Toxicol 46:220-231.

Chaplan SR, Bach FW, Pogrel JW, Chung JM, Yaksh TL (1994) Quantitative assessment

of tactile allodynia in the rat paw. J Neurosci Methods 53:55-63.

Chaudhury S, Bal M, Belugin S, Shapiro MS, Jeske NA (2011) AKAP150-mediated

TRPV1 sensitization is disrupted by calcium/calmodulin. Mol Pain 7:34.

Chavis P, Mollard P, Bockaert J, Manzoni O (1998) Visualization of cyclic AMP-

regulated presynaptic activity at cerebellar granule cells. Neuron 20:773-781.

Chen HS, Qu F, He X, Kang SM, Liao D, Lu SJ (2010) Differential roles of peripheral

metabotropic glutamate receptors in bee venom-induced nociception and

inflammation in conscious rats. J Pain 11:321-329.

Cholewinski A, Burgess GM, Bevan S (1993) The role of calcium in capsaicin-induced

desensitization in rat cultured dorsal root ganglion neurons. Neuroscience

55:1015-1023.

Chuang HH, Prescott ED, Kong H, Shields S, Jordt SE, Basbaum AI, Chao MV, Julius D

(2001) Bradykinin and nerve growth factor release the capsaicin receptor from

PtdIns(4,5)P2-mediated inhibition. Nature 411:957-962.

Coggeshall RE (1992) A consideration of neural counting methods. Trends Neurosci

15:9-13.

Coggeshall RE, Lekan HA (1996) Methods for determining numbers of cells and

synapses: a case for more uniform standards of review. J Comp Neurol 364:6-15.

Coggeshall RE, Carlton SM (1998) Ultrastructural analysis of NMDA, AMPA, and

kainate receptors on unmyelinated and myelinated axons in the periphery. J Comp

Neurol 391:78-86.

Conn PJ, Pin JP (1997) Pharmacology and functions of metabotropic glutamate receptors.

Annu Rev Pharmacol Toxicol 37:205-237.

Conn PJ, Lindsley CW, Jones CK (2009) Activation of metabotropic glutamate receptors

as a novel approach for the treatment of schizophrenia. Trends Pharmacol Sci

30:25-31.

Conn PJ, Battaglia G, Marino MJ, Nicoletti F (2005) Metabotropic glutamate receptors in

the basal ganglia motor circuit. Nat Rev Neurosci 6:787-798.

Corey DP, García-Añoveros J, Holt JR, Kwan KY, Lin SY, Vollrath MA, Amalfitano A,

Cheung EL, Derfler BH, Duggan A, Géléoc GS, Gray PA, Hoffman MP, Rehm

HL, Tamasauskas D, Zhang DS (2004) TRPA1 is a candidate for the

mechanosensitive transduction channel of vertebrate hair cells. Nature 432:723-

730.

Cosens DJ, Manning A (1969) Abnormal electroretinogram from a Drosophila mutant.

Nature 224:285-287.

Costa SK, de Nucci G, Antunes E, Brain SD (1997) Phoneutria nigriventer spider venom

induces oedema in rat skin by activation of capsaicin sensitive sensory nerves.

Eur J Pharmacol 339:223-226.

D'Antoni S, Berretta A, Bonaccorso CM, Bruno V, Aronica E, Nicoletti F, Catania MV

(2008) Metabotropic glutamate receptors in glial cells. Neurochem Res 33:2436-

2443. da Costa DS, Meotti FC, Andrade EL, Leal PC, Motta EM, Calixto JB (2010) The

involvement of the transient receptor potential A1 (TRPA1) in the maintenance of

mechanical and cold hyperalgesia in persistent inflammation. Pain 148:431-437.

Dai Y, Moriyama T, Higashi T, Togashi K, Kobayashi K, Yamanaka H, Tominaga M,

Noguchi K (2004) Proteinase-activated receptor 2-mediated potentiation of

transient receptor potential vanilloid subfamily 1 activity reveals a mechanism for

proteinase-induced inflammatory pain. J Neurosci 24:4293-4299.

Dai Y, Wang S, Tominaga M, Yamamoto S, Fukuoka T, Higashi T, Kobayashi K, Obata

K, Yamanaka H, Noguchi K (2007) Sensitization of TRPA1 by PAR2 contributes

to the sensation of inflammatory pain. J Clin Invest 117:1979-1987.

Davis JB, Gray J, Gunthorpe MJ, Hatcher JP, Davey PT, Overend P, Harries MH,

Latcham J, Clapham C, Atkinson K, Hughes SA, Rance K, Grau E, Harper AJ,

Pugh PL, Rogers DC, Bingham S, Randall A, Sheardown SA (2000) Vanilloid

receptor-1 is essential for inflammatory thermal hyperalgesia. Nature 405:183-

187.

De Petrocellis L, Harrison S, Bisogno T, Tognetto M, Brandi I, Smith GD, Creminon C,

Davis JB, Geppetti P, Di Marzo V (2001) The vanilloid receptor (VR1)-mediated

effects of anandamide are potently enhanced by the cAMP-dependent protein

kinase. J Neurochem 77:1660-1663.

Docherty RJ, Yeats JC, Bevan S, Boddeke HW (1996) Inhibition of calcineurin inhibits

the desensitization of capsaicin-evoked currents in cultured dorsal root ganglion

neurones from adult rats. Pflugers Arch 431:828-837.

Du J, Koltzenburg M, Carlton SM (2001) Glutamate-induced excitation and sensitization

of nociceptors in rat glabrous skin. Pain 89:187-198.

Du J, Zhou S, Carlton SM (2006) Kainate-induced excitation and sensitization of

nociceptors in normal and inflamed rat glabrous skin. Neuroscience 137:999-

1013.

Du J, Zhou S, Carlton SM (2008a) Group II metabotropic glutamate receptor activation

attenuates peripheral sensitization in inflammatory states. Neuroscience 154:754-

766.

Du J, Zhou S, Coggeshall RE, Carlton SM (2003) N-methyl-D-aspartate-induced

excitation and sensitization of normal and inflamed nociceptors. Neuroscience

118:547-562.

Du S, Araki I, Kobayashi H, Zakoji H, Sawada N, Takeda M (2008b) Differential

expression profile of cold (TRPA1) and cool (TRPM8) receptors in human

urogenital organs. Urology 72:450-455.

Dunham JP, Kelly S, Donaldson LF (2008) Inflammation reduces mechanical thresholds

in a population of transient receptor potential channel A1-expressing nociceptors

in the rat. Eur J Neurosci 27:3151-3160.

Duvoisin RM, Zhang C, Pfankuch TF, O'Connor H, Gayet-Primo J, Quraishi S, Raber J

(2005) Increased measures of anxiety and weight gain in mice lacking the group

III metabotropic glutamate receptor mGluR8. Eur J Neurosci 22:425-436.

Earley S, Gonzales AL, Crnich R (2009) Endothelium-dependent cerebral artery dilation

mediated by TRPA1 and Ca2+-Activated K+ channels. Circ Res 104:987-994.

Eid SR, Crown ED, Moore EL, Liang HA, Choong KC, Dima S, Henze DA, Kane SA,

Urban MO (2008) HC-030031, a TRPA1 selective antagonist, attenuates

inflammatory- and neuropathy-induced mechanical hypersensitivity. Mol Pain

4:48.

El Far O, Betz H (2002) G-protein-coupled receptors for neurotransmitter amino acids:

C-terminal tails, crowded signalosomes. Biochem J 365:329-336.

Erdmann E, Rupprecht V, Matthews E, Kukley M, Schoch S, Dietrich D (2012)

Depression of release by mGluR8 alters Ca2+ dependence of release machinery.

Cereb Cortex 22:1498-1509.

Fendt M, Bürki H, Imobersteg S, van der Putten H, McAllister K, Leslie JC, Shaw D,

Hölscher C (2010) The effect of mGlu8 deficiency in animal models of

psychiatric diseases. Genes Brain Behav 9:33-44.

Fernandes ES, Fernandes MA, Keeble JE (2012) The functions of TRPA1 and TRPV1:

moving away from sensory nerves. Br J Pharmacol 166:510-521.

Ferreira J, da Silva GL, Calixto JB (2004) Contribution of vanilloid receptors to the overt

nociception induced by B2 kinin receptor activation in mice. Br J Pharmacol

141:787-794.

Fischer MJ, Balasuriya D, Jeggle P, Goetze TA, McNaughton PA, Reeh PW, Edwardson

JM (2014) Direct evidence for functional TRPV1/TRPA1 heteromers. Pflugers

Arch 466:2229-2241.

Folbergrová J, Druga R, Haugvicová R, Mares P, Otáhal J (2008) Anticonvulsant and

neuroprotective effect of (S)-3,4-dicarboxyphenylglycine against seizures induced

in immature rats by homocysteic acid. Neuropharmacology 54:665-675.

Garrison SR, Stucky CL (2011) The dynamic TRPA1 channel: a suitable

pharmacological pain target? Curr Pharm Biotechnol 12:1689-1697.

Gavva NR, Bannon AW, Surapaneni S, Hovland DN, Lehto SG, Gore A, Juan T, Deng

H, Han B, Klionsky L, Kuang R, Le A, Tamir R, Wang J, Youngblood B, Zhu D,

Norman MH, Magal E, Treanor JJ, Louis JC (2007a) The vanilloid receptor

TRPV1 is tonically activated in vivo and involved in body temperature regulation.

J Neurosci 27:3366-3374.

Gavva NR et al. (2007b) Repeated administration of vanilloid receptor TRPV1

antagonists attenuates hyperthermia elicited by TRPV1 blockade. J Pharmacol

Exp Ther 323:128-137.

Genever PG, Maxfield SJ, Kennovin GD, Maltman J, Bowgen CJ, Raxworthy MJ, Skerry

TM (1999) Evidence for a novel glutamate-mediated signaling pathway in

keratinocytes. J Invest Dermatol 112:337-342.

Goudet C, Magnaghi V, Landry M, Nagy F, Gereau RW, Pin JP (2009) Metabotropic

receptors for glutamate and GABA in pain. Brain Res Rev 60:43-56.

Govea RM, Zhou S, Carlton SM (2012) Group III metabotropic glutamate receptors and

transient receptor potential vanilloid 1 co-localize and interact on nociceptors.

Neuroscience 217:130-139.

Guo A, Vulchanova L, Wang J, Li X, Elde R (1999) Immunocytochemical localization of

the vanilloid receptor 1 (VR1): relationship to neuropeptides, the P2X3

purinoceptor and IB4 binding sites. Eur J Neurosci 11:946-958.

Guo J, Ikeda SR (2005) Coupling of metabotropic glutamate receptor 8 to N-type Ca2+

channels in rat sympathetic neurons. Mol Pharmacol 67:1840-1851.

Haas ET, Rowland K, Gautam M (2011) Tooth injury increases expression of the cold

sensitive TRP channel TRPA1 in trigeminal neurons. Arch Oral Biol 56:1604-

1609.

Hardie RC (2007) TRP channels and lipids: from Drosophila to mammalian physiology. J

Physiol 578:9-24.

Hargreaves K, Dubner R, Brown F, Flores C, Joris J (1988) A new and sensitive method

for measuring thermal nociception in cutaneous hyperalgesia. Pain 32:77-88.

Harper AA, Lawson SN (1985a) Conduction velocity is related to morphological cell

type in rat dorsal root ganglion neurones. J Physiol 359:31-46.

Harper AA, Lawson SN (1985b) Electrical properties of rat dorsal root ganglion neurones

with different peripheral nerve conduction velocities. J Physiol 359:47-63.

Hoffman EM, Schechter R, Miller KE (2010) Fixative composition alters distributions of

immunoreactivity for glutaminase and two markers of nociceptive neurons,

Nav1.8 and TRPV1, in the rat dorsal root ganglion. J Histochem Cytochem

58:329-344.

Honore P et al. (2009) Repeated dosing of ABT-102, a potent and selective TRPV1

antagonist, enhances TRPV1-mediated analgesic activity in rodents, but

attenuates antagonist-induced hyperthermia. Pain 142:27-35.

Hopkins CR, Lindsley CW, Niswender CM (2009) mGluR4-positive allosteric

modulation as potential treatment for Parkinson's disease. Future Med Chem

1:501-513.

Hsu CL, Yen GC (2007) Effects of capsaicin on induction of apoptosis and inhibition of

adipogenesis in 3T3-L1 cells. J Agric Food Chem 55:1730-1736.

Huang J, Zhang X, McNaughton PA (2006) Modulation of temperature-sensitive TRP

channels. Semin Cell Dev Biol 17:638-645.

Hwang JT, Park IJ, Shin JI, Lee YK, Lee SK, Baik HW, Ha J, Park OJ (2005) Genistein,

EGCG, and capsaicin inhibit adipocyte differentiation process via activating

AMP-activated protein kinase. Biochem Biophys Res Commun 338:694-699.

Hwang SW, Cho H, Kwak J, Lee SY, Kang CJ, Jung J, Cho S, Min KH, Suh YG, Kim D,

Oh U (2000) Direct activation of capsaicin receptors by products of

lipoxygenases: endogenous capsaicin-like substances. Proc Natl Acad Sci U S A

97:6155-6160.

Iida T, Shimizu I, Nealen ML, Campbell A, Caterina M (2005) Attenuated fever response

in mice lacking TRPV1. Neurosci Lett 378:28-33.

Jaquemar D, Schenker T, Trueb B (1999) An ankyrin-like protein with transmembrane

domains is specifically lost after oncogenic transformation of human fibroblasts. J

Biol Chem 274:7325-7333.

Jasmin L, Kohan L, Franssen M, Janni G, Goff JR (1998) The cold plate as a test of

nociceptive behaviors: description and application to the study of chronic

neuropathic and inflammatory pain models. Pain 75:367-382.

Jeske NA, Patwardhan AM, Ruparel NB, Akopian AN, Shapiro MS, Henry MA (2009)

A-kinase anchoring protein 150 controls protein kinase C-mediated

phosphorylation and sensitization of TRPV1. Pain 146:301-307.

Jeske NA, Diogenes A, Ruparel NB, Fehrenbacher JC, Henry M, Akopian AN,

Hargreaves KM (2008) A-kinase anchoring protein mediates TRPV1 thermal

hyperalgesia through PKA phosphorylation of TRPV1. Pain 138:604-616.

Ji G, Zhou S, Carlton SM (2008) Intact Adelta-fibers up-regulate transient receptor

potential A1 and contribute to cold hypersensitivity in neuropathic rats.

Neuroscience 154:1054-1066.

Jin YH, Yamaki F, Takemura M, Koike Y, Furuyama A, Yonehara N (2009) Capsaicin-

induced glutamate release is implicated in nociceptive processing through

activation of ionotropic glutamate receptors and group I metabotropic glutamate

receptor in primary afferent fibers. J Pharmacol Sci 109:233-241.

Jordt SE, Tominaga M, Julius D (2000) Acid potentiation of the capsaicin receptor

determined by a key extracellular site. Proc Natl Acad Sci U S A 97:8134-8139.

Jordt SE, Bautista DM, Chuang HH, McKemy DD, Zygmunt PM, Högestätt ED, Meng

ID, Julius D (2004) Mustard oils and cannabinoids excite sensory nerve fibres

through the TRP channel ANKTM1. Nature 427:260-265.

Julius D (2013) TRP channels and pain. Annu Rev Cell Dev Biol 29:355-384.

Julius D, Basbaum AI (2001) Molecular mechanisms of nociception. Nature 413:203-

210.

Kaneko Y, Szallasi A (2014) Transient receptor potential (TRP) channels: a clinical

perspective. Br J Pharmacol 171:2474-2507.

Karashima Y, Talavera K, Everaerts W, Janssens A, Kwan KY, Vennekens R, Nilius B,

Voets T (2009) TRPA1 acts as a cold sensor in vitro and in vivo. Proc Natl Acad

Sci U S A 106:1273-1278.

Kerstein PC, del Camino D, Moran MM, Stucky CL (2009) Pharmacological blockade of

TRPA1 inhibits mechanical firing in nociceptors. Mol Pain 5:19.

Khatibi NH, Jadhav V, Charles S, Chiu J, Buchholz J, Tang J, Zhang JH (2011)

Capsaicin pre-treatment provides neurovascular protection against neonatal

hypoxic-ischemic brain injury in rats. Acta Neurochir Suppl 111:225-230.

Kim YH, Back SK, Davies AJ, Jeong H, Jo HJ, Chung G, Na HS, Bae YC, Kim SJ, Kim

JS, Jung SJ, Oh SB (2012) TRPV1 in GABAergic interneurons mediates

neuropathic mechanical allodynia and disinhibition of the nociceptive circuitry in

the spinal cord. Neuron 74:640-647.

Kobayashi K, Fukuoka T, Obata K, Yamanaka H, Dai Y, Tokunaga A, Noguchi K (2005)

Distinct expression of TRPM8, TRPA1, and TRPV1 mRNAs in rat primary

afferent neurons with adelta/c-fibers and colocalization with trk receptors. J Comp

Neurol 493:596-606.

Koplas PA, Rosenberg RL, Oxford GS (1997) The role of calcium in the desensitization

of capsaicin responses in rat dorsal root ganglion neurons. J Neurosci 17:3525-

3537.

Kosugi M, Nakatsuka T, Fujita T, Kuroda Y, Kumamoto E (2007) Activation of TRPA1

channel facilitates excitatory synaptic transmission in substantia gelatinosa

neurons of the adult rat spinal cord. J Neurosci 27:4443-4451.

Kremeyer B, Lopera F, Cox JJ, Momin A, Rugiero F, Marsh S, Woods CG, Jones NG,

Paterson KJ, Fricker FR, Villegas A, Acosta N, Pineda-Trujillo NG, Ramírez JD,

Zea J, Burley MW, Bedoya G, Bennett DL, Wood JN, Ruiz-Linares A (2010) A

gain-of-function mutation in TRPA1 causes familial episodic pain syndrome.

Neuron 66:671-680.

Kumar N, Laferriere A, Yu JS, Poon T, Coderre TJ (2010) Metabotropic glutamate

receptors (mGluRs) regulate noxious stimulus-induced glutamate release in the

spinal cord dorsal horn of rats with neuropathic and inflammatory pain. J

Neurochem 114:281-290.

Kwan KY, Glazer JM, Corey DP, Rice FL, Stucky CL (2009) TRPA1 modulates

mechanotransduction in cutaneous sensory neurons. J Neurosci 29:4808-4819.

Kwan KY, Allchorne AJ, Vollrath MA, Christensen AP, Zhang DS, Woolf CJ, Corey DP

(2006) TRPA1 contributes to cold, mechanical, and chemical nociception but is

not essential for hair-cell transduction. Neuron 50:277-289.

100

Lapointe TK, Altier C (2011) The role of TRPA1 in visceral inflammation and pain.

Channels (Austin) 5:525-529.

Lee SH, Lee SY, Son DJ, Lee H, Yoo HS, Song S, Oh KW, Han DC, Kwon BM, Hong

JT (2005) Inhibitory effect of 2'-hydroxycinnamaldehyde on nitric oxide

production through inhibition of NF-kappa B activation in RAW 264.7 cells.

Biochem Pharmacol 69:791-799.

Lee YS, Lee JA, Jung J, Oh U, Kaang BK (2000) The cAMP-dependent kinase pathway

does not sensitize the cloned vanilloid receptor type 1 expressed in xenopus

oocytes or Aplysia neurons. Neurosci Lett 288:57-60.

Leem JW, Willis WD, Chung JM (1993) Cutaneous sensory receptors in the rat foot. J

Neurophysiol 69:1684-1699.

Lennertz RC, Kossyreva EA, Smith AK, Stucky CL (2012) TRPA1 mediates mechanical

sensitization in nociceptors during inflammation. PLoS One 7:e43597.

Levine JD, Fields HL, Basbaum AI (1993) Peptides and the primary afferent nociceptor.

J Neurosci 13:2273-2286.

Linden AM, Johnson BG, Peters SC, Shannon HE, Tian M, Wang Y, Yu JL, Köster A,

Baez M, Schoepp DD (2002) Increased anxiety-related behavior in mice deficient

for metabotropic glutamate 8 (mGlu8) receptor. Neuropharmacology 43:251-259.

Liu XH, Han M, Zhu JX, Sun N, Tang JS, Huo FQ, Li J, Xu FY, Du JQ (2012)

Metabotropic glutamate subtype 7 and 8 receptors oppositely modulate cardiac

nociception in the rat nucleus tractus solitarius. Neuroscience 220:322-329.

Lüscher C, Slesinger PA (2010) Emerging roles for G protein-gated inwardly rectifying

potassium (GIRK) channels in health and disease. Nat Rev Neurosci 11:301-315.

101

Macpherson LJ, Geierstanger BH, Viswanath V, Bandell M, Eid SR, Hwang S,

Patapoutian A (2005) The pungency of garlic: activation of TRPA1 and TRPV1

in response to allicin. Curr Biol 15:929-934.

Malek N, Pajak A, Kolosowska N, Kucharczyk M, Starowicz K (2015) The importance

of TRPV1-sensitisation factors for the development of neuropathic pain. Mol Cell

Neurosci 65C:1-10.

Marabese I, de Novellis V, Palazzo E, Scafuro MA, Vita D, Rossi F, Maione S (2007a)

Effects of (S)-3,4-DCPG, an mGlu8 receptor agonist, on inflammatory and

neuropathic pain in mice. Neuropharmacology 52:253-262.

Marabese I, Rossi F, Palazzo E, de Novellis V, Starowicz K, Cristino L, Vita D, Gatta L,

Guida F, Di Marzo V, Maione S (2007b) Periaqueductal gray metabotropic

glutamate receptor subtype 7 and 8 mediate opposite effects on amino acid

release, rostral ventromedial medulla cell activities, and thermal nociception. J

Neurophysiol 98:43-53.

McDougall JJ (2011) Peripheral analgesia: Hitting pain where it hurts. Biochim Biophys

Acta 1812:459-467.

McGaraughty S, Chu KL, Perner RJ, Didomenico S, Kort ME, Kym PR (2010) TRPA1

modulation of spontaneous and mechanically evoked firing of spinal neurons in

uninjured, osteoarthritic, and inflamed rats. Mol Pain 6:14.

McNamara CR, Mandel-Brehm J, Bautista DM, Siemens J, Deranian KL, Zhao M,

Hayward NJ, Chong JA, Julius D, Moran MM, Fanger CM (2007) TRPA1

mediates formalin-induced pain. Proc Natl Acad Sci U S A 104:13525-13530.

102

Minke B, Wu C, Pak WL (1975) Induction of photoreceptor voltage noise in the dark in

Drosophila mutant. Nature 258:84-87.

Miyakawa T, Terashima Y, Takebayashi T, Tanimoto K, Iwase T, Ogon I, Kobayashi T,

Tohse N, Yamashita T (2014) Transient receptor potential ankyrin 1 in spinal

cord dorsal horn is involved in neuropathic pain in nerve root constriction rats.

Mol Pain 10:58.

Mohapatra DP, Nau C (2003) Desensitization of capsaicin-activated currents in the

vanilloid receptor TRPV1 is decreased by the cyclic AMP-dependent protein

kinase pathway. J Biol Chem 278:50080-50090.

Montrucchio DP, Córdova MM, Santos AR (2013) Plant derived aporphinic alkaloid S-

(+)-dicentrine induces antinociceptive effect in both acute and chronic

inflammatory pain models: evidence for a role of TRPA1 channels. PLoS One

8:e67730.

Moriyama T, Higashi T, Togashi K, Iida T, Segi E, Sugimoto Y, Tominaga T, Narumiya

S, Tominaga M (2005) Sensitization of TRPV1 by EP1 and IP reveals peripheral

nociceptive mechanism of prostaglandins. Mol Pain 1:3.

Nagata K, Duggan A, Kumar G, García-Añoveros J (2005) Nociceptor and hair cell

transducer properties of TRPA1, a channel for pain and hearing. J Neurosci

25:4052-4061.

Nakajima Y, Iwakabe H, Akazawa C, Nawa H, Shigemoto R, Mizuno N, Nakanishi S

(1993) Molecular characterization of a novel retinal metabotropic glutamate

receptor mGluR6 with a high agonist selectivity for L-2-amino-4-

phosphonobutyrate. J Biol Chem 268:11868-11873.

103

Nakamura Y, Une Y, Miyano K, Abe H, Hisaoka K, Morioka N, Nakata Y (2012)

Activation of transient receptor potential ankyrin 1 evokes nociception through

substance P release from primary sensory neurons. J Neurochem 120:1036-1047.

Nassini R, Gees M, Harrison S, De Siena G, Materazzi S, Moretto N, Failli P, Preti D,

Marchetti N, Cavazzini A, Mancini F, Pedretti P, Nilius B, Patacchini R, Geppetti

P (2011) Oxaliplatin elicits mechanical and cold allodynia in rodents via TRPA1

receptor stimulation. Pain 152:1621-1631.

Neugebauer V, Carlton SM (2002) Peripheral metabotropic glutamate receptors as drug

targets for pain relief. Expert Opin Ther Targets 6:349-361.

Neugebauer V, Chen PS, Willis WD (2000) Groups II and III metabotropic glutamate

receptors differentially modulate brief and prolonged nociception in primate STT

cells. J Neurophysiol 84:2998-3009.

Nilius B, Voets T, Peters J (2005) TRP channels in disease. Sci STKE 2005:re8.

Niswender CM, Conn PJ (2010) Metabotropic glutamate receptors: physiology,

pharmacology, and disease. Annu Rev Pharmacol Toxicol 50:295-322.

Niswender CM, Johnson KA, Luo Q, Ayala JE, Kim C, Conn PJ, Weaver CD (2008) A

novel assay of Gi/o-linked G protein-coupled receptor coupling to potassium

channels provides new insights into the pharmacology of the group III

metabotropic glutamate receptors. Mol Pharmacol 73:1213-1224.

Nomura A, Shigemoto R, Nakamura Y, Okamoto N, Mizuno N, Nakanishi S (1994)

Developmentally regulated postsynaptic localization of a metabotropic glutamate

receptor in rat rod bipolar cells. Cell 77:361-369.

104

Numazaki M, Tominaga T, Toyooka H, Tominaga M (2002) Direct phosphorylation of

capsaicin receptor VR1 by protein kinase Cepsilon and identification of two target

serine residues. J Biol Chem 277:13375-13378.

Obata K, Katsura H, Mizushima T, Yamanaka H, Kobayashi K, Dai Y, Fukuoka T,

Tokunaga A, Tominaga M, Noguchi K (2005) TRPA1 induced in sensory neurons

contributes to cold hyperalgesia after inflammation and nerve injury. J Clin Invest

115:2393-2401.

Palazzo E, Fu Y, Ji G, Maione S, Neugebauer V (2008) Group III mGluR7 and mGluR8

in the amygdala differentially modulate nocifensive and affective pain behaviors.

Neuropharmacology 55:537-545.

Patapoutian A, Tate S, Woolf CJ (2009) Transient receptor potential channels: targeting

pain at the source. Nat Rev Drug Discov 8:55-68.

Patil ST, Zhang L, Martenyi F, Lowe SL, Jackson KA, Andreev BV, Avedisova AS,

Bardenstein LM, Gurovich IY, Morozova MA, Mosolov SN, Neznanov NG,

Reznik AM, Smulevich AB, Tochilov VA, Johnson BG, Monn JA, Schoepp DD

(2007) Activation of mGlu2/3 receptors as a new approach to treat schizophrenia:

a randomized Phase 2 clinical trial. Nat Med 13:1102-1107.

Pałucha A, Tatarczyńska E, Brański P, Szewczyk B, Wierońska JM, Kłak K, Chojnacka-

Wójcik E, Nowak G, Pilc A (2004) Group III mGlu receptor agonists produce

anxiolytic- and antidepressant-like effects after central administration in rats.

Neuropharmacology 46:151-159.

Perl ER (1968) Myelinated afferent fibres innervating the primate skin and their response

to noxious stimuli. J Physiol 197:593-615.

105

Perroy J, Prezeau L, De Waard M, Shigemoto R, Bockaert J, Fagni L (2000) Selective

blockade of P/Q-type calcium channels by the metabotropic glutamate receptor

type 7 involves a phospholipase C pathway in neurons. J Neurosci 20:7896-7904.

Petrus M, Peier AM, Bandell M, Hwang SW, Huynh T, Olney N, Jegla T, Patapoutian A

(2007) A role of TRPA1 in mechanical hyperalgesia is revealed by

pharmacological inhibition. Mol Pain 3:40.

Pocock JM, Kettenmann H (2007) Neurotransmitter receptors on microglia. Trends

Neurosci 30:527-535.

Por ED, Gomez R, Akopian AN, Jeske NA (2013) Phosphorylation regulates TRPV1

association with β-arrestin-2. Biochem J 451:101-109.

Por ED, Bierbower SM, Berg KA, Gomez R, Akopian AN, Wetsel WC, Jeske NA (2012)

β-Arrestin-2 desensitizes the transient receptor potential vanilloid 1 (TRPV1)

channel. J Biol Chem 287:37552-37563.

Premkumar LS, Ahern GP (2000) Induction of vanilloid receptor channel activity by

protein kinase C. Nature 408:985-990.

Premkumar LS, Abooj M (2013) TRP channels and analgesia. Life Sci 92:415-424.

Prober DA, Zimmerman S, Myers BR, McDermott BM, Kim SH, Caron S, Rihel J,

Solnica-Krezel L, Julius D, Hudspeth AJ, Schier AF (2008) Zebrafish TRPA1

channels are required for chemosensation but not for thermosensation or

mechanosensory hair cell function. J Neurosci 28:10102-10110.

Purves D, Augustine GJ, Fitzpatrick D, Katz LC, LaMantia A-S, McNamara JO,

Williams SM (2001) Neuroscience, 2nd Edition: Sinauer Associates.

106

Rathee PK, Distler C, Obreja O, Neuhuber W, Wang GK, Wang SY, Nau C, Kress M

(2002) PKA/AKAP/VR-1 module: A common link of Gs-mediated signaling to

thermal hyperalgesia. J Neurosci 22:4740-4745.

Ren W, Palazzo E, Maione S, Neugebauer V (2011) Differential effects of mGluR7 and

mGluR8 activation on pain-related synaptic activity in the amygdala.

Neuropharmacology 61:1334-1344.

Robbins MJ, Starr KR, Honey A, Soffin EM, Rourke C, Jones GA, Kelly FM, Strum J,

Melarange RA, Harris AJ, Rocheville M, Rupniak T, Murdock PR, Jones DN,

Kew JN, Maycox PR (2007) Evaluation of the mGlu8 receptor as a putative

therapeutic target in schizophrenia. Brain Res 1152:215-227.

Rosenbaum T, Gordon-Shaag A, Munari M, Gordon SE (2004) Ca2+/calmodulin

modulates TRPV1 activation by capsaicin. J Gen Physiol 123:53-62.

Rowbotham MC, Nothaft W, Duan WR, Wang Y, Faltynek C, McGaraughty S, Chu KL,

Svensson P (2011) Oral and cutaneous thermosensory profile of selective TRPV1

inhibition by ABT-102 in a randomized healthy volunteer trial. Pain 152:1192-

1200.

Ruparel NB, Patwardhan AM, Akopian AN, Hargreaves KM (2008) Homologous and

heterologous desensitization of capsaicin and mustard oil responses utilize

different cellular pathways in nociceptors. Pain 135:271-279.

Salas MM, Hargreaves KM, Akopian AN (2009) TRPA1-mediated responses in

trigeminal sensory neurons: interaction between TRPA1 and TRPV1. Eur J

Neurosci 29:1568-1578.

107

Saugstad JA, Segerson TP, Westbrook GL (1996) Metabotropic glutamate receptors

activate G-protein-coupled inwardly rectifying potassium channels in Xenopus

oocytes. J Neurosci 16:5979-5985.

Saugstad JA, Kinzie JM, Shinohara MM, Segerson TP, Westbrook GL (1997) Cloning

and expression of rat metabotropic glutamate receptor 8 reveals a distinct

pharmacological profile. Mol Pharmacol 51:119-125.

Sawada Y, Hosokawa H, Hori A, Matsumura K, Kobayashi S (2007) Cold sensitivity of

recombinant TRPA1 channels. Brain Res 1160:39-46.

Scherer M, Reichl SU, Augustin M, Pogatzki-Zahn EM, Zahn PK (2010) The assessment

of cold hyperalgesia after an incision. Anesth Analg 110:222-227.

Schmidt M, Dubin AE, Petrus MJ, Earley TJ, Patapoutian A (2009) Nociceptive signals

induce trafficking of TRPA1 to the plasma membrane. Neuron 64:498-509.

Schoepp DD (2001) Unveiling the functions of presynaptic metabotropic glutamate

receptors in the central nervous system. J Pharmacol Exp Ther 299:12-20.

Schoepp DD, Conn PJ (2002) Metabotropic glutamate receptors. Pharmacol Biochem

Behav 74:255-256.

Selvam C, Goudet C, Oueslati N, Pin JP, Acher FC (2007) L-(+)-2-Amino-4-

thiophosphonobutyric acid (L-thioAP4), a new potent agonist of group III

metabotropic glutamate receptors: increased distal acidity affords enhanced

potency. J Med Chem 50:4656-4664.

Siemens J, Zhou S, Piskorowski R, Nikai T, Lumpkin EA, Basbaum AI, King D, Julius D

(2006) Spider toxins activate the capsaicin receptor to produce inflammatory pain.

Nature 444:208-212.

108

Smart D, Gunthorpe MJ, Jerman JC, Nasir S, Gray J, Muir AI, Chambers JK, Randall

AD, Davis JB (2000) The endogenous lipid anandamide is a full agonist at the

human vanilloid receptor (hVR1). Br J Pharmacol 129:227-230.

Southall MD, Li T, Gharibova LS, Pei Y, Nicol GD, Travers JB (2003) Activation of

epidermal vanilloid receptor-1 induces release of proinflammatory mediators in

human keratinocytes. J Pharmacol Exp Ther 304:217-222.

Spahn V, Stein C, Zöllner C (2014) Modulation of transient receptor vanilloid 1 activity

by transient receptor potential ankyrin 1. Mol Pharmacol 85:335-344.

Staruschenko A, Jeske NA, Akopian AN (2010) Contribution of TRPV1-TRPA1

interaction to the single channel properties of the TRPA1 channel. J Biol Chem

285:15167-15177.

Stock K, Garthe A, de Almeida Sassi F, Glass R, Wolf SA, Kettenmann H (2014) The

capsaicin receptor TRPV1 as a novel modulator of neural precursor cell

proliferation. Stem Cells 32:3183-3195.

Story GM, Peier AM, Reeve AJ, Eid SR, Mosbacher J, Hricik TR, Earley TJ, Hergarden

AC, Andersson DA, Hwang SW, McIntyre P, Jegla T, Bevan S, Patapoutian A

(2003) ANKTM1, a TRP-like channel expressed in nociceptive neurons, is

activated by cold temperatures. Cell 112:819-829.

Stucky CL, Dubin AE, Jeske NA, Malin SA, McKemy DD, Story GM (2009) Roles of

transient receptor potential channels in pain. Brain Res Rev 60:2-23.

Ständer S, Moormann C, Schumacher M, Buddenkotte J, Artuc M, Shpacovitch V,

Brzoska T, Lippert U, Henz BM, Luger TA, Metze D, Steinhoff M (2004)

109

Expression of vanilloid receptor subtype 1 in cutaneous sensory nerve fibers, mast

cells, and epithelial cells of appendage structures. Exp Dermatol 13:129-139.

Sun B, Bang SI, Jin YH (2009) Transient receptor potential A1 increase glutamate

release on brain stem neurons. Neuroreport 20:1002-1006.

Swanson CJ, Bures M, Johnson MP, Linden AM, Monn JA, Schoepp DD (2005a)

Metabotropic glutamate receptors as novel targets for anxiety and stress disorders.

Nat Rev Drug Discov 4:131-144.

Swanson DM, Dubin AE, Shah C, Nasser N, Chang L, Dax SL, Jetter M, Breitenbucher

JG, Liu C, Mazur C, Lord B, Gonzales L, Hoey K, Rizzolio M, Bogenstaetter M,

Codd EE, Lee DH, Zhang SP, Chaplan SR, Carruthers NI (2005b) Identification

and biological evaluation of 4-(3-trifluoromethylpyridin-2-yl)piperazine-1-

carboxylic acid (5-trifluoromethylpyridin-2-yl)amide, a high affinity TRPV1

(VR1) vanilloid receptor antagonist. J Med Chem 48:1857-1872.

Szallasi A (1994) The vanilloid (capsaicin) receptor: receptor types and species

differences. Gen Pharmacol 25:223-243.

Ta LE, Bieber AJ, Carlton SM, Loprinzi CL, Low PA, Windebank AJ (2010) Transient

Receptor Potential Vanilloid 1 is essential for cisplatin-induced heat hyperalgesia

in mice. Mol Pain 6:15.

Takahashi T, Forsythe ID, Tsujimoto T, Barnes-Davies M, Onodera K (1996)

Presynaptic calcium current modulation by a metabotropic glutamate receptor.

Science 274:594-597.

Thomas NK, Wright RA, Howson PA, Kingston AE, Schoepp DD, Jane DE (2001) (S)-

3,4-DCPG, a potent and selective mGlu8a receptor agonist, activates

110

metabotropic glutamate receptors on primary afferent terminals in the neonatal rat

spinal cord. Neuropharmacology 40:311-318.

Tominaga M, Tominaga T (2005) Structure and function of TRPV1. Pflugers Arch

451:143-150.

Tominaga M, Caterina MJ, Malmberg AB, Rosen TA, Gilbert H, Skinner K, Raumann

BE, Basbaum AI, Julius D (1998) The cloned capsaicin receptor integrates

multiple pain-producing stimuli. Neuron 21:531-543.

Tong Q, Kirchgessner AL (2003) Localization and function of metabotropic glutamate

receptor 8 in the enteric nervous system. Am J Physiol Gastrointest Liver Physiol

285:G992-G1003.

Tong Q, Ouedraogo R, Kirchgessner AL (2002) Localization and function of group III

metabotropic glutamate receptors in rat pancreatic islets. Am J Physiol Endocrinol

Metab 282:E1324-1333.

Torebjörk HE (1974) Afferent C units responding to mechanical, thermal and chemical

stimuli in human non-glabrous skin. Acta Physiol Scand 92:374-390.

Traub RJ, Mendell LM (1988) The spinal projection of individual identified A-delta- and

C-fibers. J Neurophysiol 59:41-55.

Trevisani M, Siemens J, Materazzi S, Bautista DM, Nassini R, Campi B, Imamachi N,

Andrè E, Patacchini R, Cottrell GS, Gatti R, Basbaum AI, Bunnett NW, Julius D,

Geppetti P (2007) 4-Hydroxynonenal, an endogenous aldehyde, causes pain and

neurogenic inflammation through activation of the irritant receptor TRPA1. Proc

Natl Acad Sci U S A 104:13519-13524.

111

Tsumura M, Sobhan U, Sato M, Shimada M, Nishiyama A, Kawaguchi A, Soya M,

Kuroda H, Tazaki M, Shibukawa Y (2013) Functional expression of TRPM8 and

TRPA1 channels in rat odontoblasts. PLoS One 8:e82233.

Turner JP, Salt TE (1999) Group III metabotropic glutamate receptors control

corticothalamic synaptic transmission in the rat thalamus in vitro. J Physiol 519 Pt

2:481-491.

Ueda M, Kuraishi Y, Satoh M (1993) Detection of capsaicin-evoked release of glutamate

from spinal dorsal horn slices of rat with on-line monitoring system. Neurosci

Lett 155:179-182.

Vellani V, Mapplebeck S, Moriondo A, Davis JB, McNaughton PA (2001) Protein kinase

C activation potentiates gating of the vanilloid receptor VR1 by capsaicin,

protons, heat and anandamide. J Physiol 534:813-825.

Walker K, Reeve A, Bowes M, Winter J, Wotherspoon G, Davis A, Schmid P, Gasparini

F, Kuhn R, Urban L (2001) mGlu5 receptors and nociceptive function II. mGlu5

receptors functionally expressed on peripheral sensory neurones mediate

inflammatory hyperalgesia. Neuropharmacology 40:10-19.

Wang S, Dai Y, Fukuoka T, Yamanaka H, Kobayashi K, Obata K, Cui X, Tominaga M,

Noguchi K (2008) Phospholipase C and protein kinase A mediate bradykinin

sensitization of TRPA1: a molecular mechanism of inflammatory pain. Brain

131:1241-1251.

Watabiki T, Kiso T, Kuramochi T, Yonezawa K, Tsuji N, Kohara A, Kakimoto S, Aoki

T, Matsuoka N (2011) Amelioration of neuropathic pain by novel transient

112

receptor potential vanilloid 1 antagonist AS1928370 in rats without hyperthermic

effect. J Pharmacol Exp Ther 336:743-750.

Watanabe M, Ueda T, Shibata Y, Kumamoto N, Ugawa S (2015) The role of TRPV1

channels in carrageenan-induced mechanical hyperalgesia in mice. Neuroreport

26:173-178.

Wei H, Karimaa M, Korjamo T, Koivisto A, Pertovaara A (2012) Transient receptor

potential ankyrin 1 ion channel contributes to guarding pain and mechanical

hypersensitivity in a rat model of postoperative pain. Anesthesiology 117:137-

148.

West MJ (1999) Stereological methods for estimating the total number of neurons and

synapses: issues of precision and bias. Trends Neurosci 22:51-61.

Willis WD (2007) The somatosensory system, with emphasis on structures important for

pain. Brain Res Rev 55:297-313.

Wilson SR, Gerhold KA, Bifolck-Fisher A, Liu Q, Patel KN, Dong X, Bautista DM

(2011) TRPA1 is required for histamine-independent, Mas-related G protein-

coupled receptor-mediated itch. Nat Neurosci 14:595-602.

Wilson SR, Nelson AM, Batia L, Morita T, Estandian D, Owens DM, Lumpkin EA,

Bautista DM (2013) The ion channel TRPA1 is required for chronic itch. J

Neurosci 33:9283-9294.

Wong GY, Gavva NR (2009) Therapeutic potential of vanilloid receptor TRPV1 agonists

and antagonists as analgesics: Recent advances and setbacks. Brain Res Rev

60:267-277.

113

Wu S, Wright RA, Rockey PK, Burgett SG, Arnold JS, Rosteck PR, Johnson BG,

Schoepp DD, Belagaje RM (1998) Group III human metabotropic glutamate

receptors 4, 7 and 8: molecular cloning, functional expression, and comparison of

pharmacological properties in RGT cells. Brain Res Mol Brain Res 53:88-97.

Xu GY, Huang LY (2002) Peripheral inflammation sensitizes P2X receptor-mediated

responses in rat dorsal root ganglion neurons. J Neurosci 22:93-102.

Xu X, Wang P, Zhao Z, Cao T, He H, Luo Z, Zhong J, Gao F, Zhu Z, Li L, Yan Z, Chen

J, Ni Y, Liu D (2011) Activation of transient receptor potential vanilloid 1 by

dietary capsaicin delays the onset of stroke in stroke-prone spontaneously

hypertensive rats. Stroke 42:3245-3251.

Yang ZQ (2005) Agonists and antagonists for group III metabotropic glutamate receptors

6, 7 and 8. Curr Top Med Chem 5:913-918.

Yokoyama T, Ohbuchi T, Saito T, Sudo Y, Fujihara H, Minami K, Nagatomo T, Uezono

Y, Ueta Y (2011) Allyl isothiocyanates and cinnamaldehyde potentiate miniature

excitatory postsynaptic inputs in the supraoptic nucleus in rats. Eur J Pharmacol

655:31-37.

Young RL, Cooper NJ, Blackshaw LA (2008) Anatomy and function of group III

metabotropic glutamate receptors in gastric vagal pathways. Neuropharmacology

54:965-975.

Zhai J, Tian MT, Wang Y, Yu JL, Köster A, Baez M, Nisenbaum ES (2002) Modulation

of lateral perforant path excitatory responses by metabotropic glutamate 8

(mGlu8) receptors. Neuropharmacology 43:223-230.

114

Zhang E, Liao P (2014) Brain transient receptor potential channels and stroke. J Neurosci

Res.

Zhang HM, Chen SR, Pan HL (2009) Effects of activation of group III metabotropic

glutamate receptors on spinal synaptic transmission in a rat model of neuropathic

pain. Neuroscience 158:875-884.

Zhang W, Liu Y, Zhao X, Gu X, Ma Z (2014a) The effect of intrathecal administration

TRPA1 antagonists in a rat model of neuropathic pain. Anesth Analg 119:179-

185.

Zhang X, Huang J, McNaughton PA (2005) NGF rapidly increases membrane expression

of TRPV1 heat-gated ion channels. EMBO J 24:4211-4223.

Zhang X, Koronowski KB, Li L, Freeman BA, Woodcock S, de Groat WC (2014b)

Nitro-oleic acid desensitizes TRPA1 and TRPV1 agonist responses in adult rat

DRG neurons. Exp Neurol 251:12-21.

Zimmermann M (1983) Ethical guidelines for investigations of experimental pain in

conscious animals. Pain 16:109-110.

Zurborg S, Yurgionas B, Jira JA, Caspani O, Heppenstall PA (2007) Direct activation of

the ion channel TRPA1 by Ca2+. Nat Neurosci 10:277-279.

Zygmunt PM, Petersson J, Andersson DA, Chuang H, Sørgård M, Di Marzo V, Julius D,

Högestätt ED (1999) Vanilloid receptors on sensory nerves mediate the

vasodilator action of anandamide. Nature 400:452-457.

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VITA

Rosann Govea was born in 1985 in Fort Worth, Texas to Pete and Lucy Govea.

She has one older sister, Roxann, and two younger sisters, Lucinda and Mercedes.

Rosann graduated summa cum laude from the High School of Medical Professions at

North Side High School. Upon graduation, Rosann joined her older sister, Roxann, at

UT-Dallas in Richardson Texas. In 2006, with encouragement from her cousin, Dr.

Martin Farias III (1973-2008), she applied and was accepted as McNair Scholar at UNT-

Health Science Center. Here Rosann was able to experience first-hand what it was like working in a research lab. In 2006, she began working in the Genotyping Core at UT-

Southwestern in the lab of Dr. Luis Parada. She would continue to work here until moving to Galveston, Texas in 2009. In 2007, Rosann was able to volunteer for a semester in Dr. Michael Kilgard’s lab at UTD. She graduated from UTD in 2008 with a double major in Neuroscience and Biology. After graduation, Rosann decided to take a year to take various courses outside of the sciences including American Literature and

American Sign Language.

In 2009, Rosann was accepted into PREP (Post-Baccalaureate Research

Education Program) at UTMB. As a PREP student, Rosann was able to take a few graduate level courses while she began working in Dr. Susan Carlton’s lab. In 2010, she applied and was accepted into the Graduate School of Biomedical Sciences at UTMB.

She later joined the Neuroscience Graduate Program and entered candidacy in 2012.

Rosann’s dissertation work establishes group III mGluR8 as an attractive target for

TRPV1 and TRPA1 associated pain treatments. As a graduate student she was able to

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attend numerous local, national (even in Hawaii!), and international (Milan, Italy!) scientific meetings to present data.

Rosann received travel awards to attend the American Pain Society meeting (2011 and 2014) and the International Association for the Study of Pain meeting (2012). In

2014 she received the Michael Tacheeni Scott Endowed Scholarship from UTMB. In the later years of her graduate career she had the opportunity to serve a teaching apprenticeship for the Neuroscience and Human Behavior course for the medical students.

PUBLICATIONS

ARTICLES IN PEER-REVIEWED JOURNALS:

Govea RM, Zhou S, Carlton SM Group III metabotropic glutamate receptors and TRPV1 co-localize and interact on peripheral nociceptors. Neuroscience, 217:130-139, 2012

Carlton, SM, Zhou, S., Govea, RM, Du, J. Group II/III metabotropic glutamate receptors exert endogenous activity- dependent modulation of TRPV1 receptors on peripheral nociceptors. Journal of Neuroscience, 31:12727-12737, 2011.

ARTICLES IN PREPARATION:

Govea RM, Zhou S, Carlton SM Group III mGluR8 negatively modulates TRPA1-induced activity. To be submitted to Pain.

ABSTRACTS:

Govea RM, Hargett GL, Carlton SM Group III mGluR8 activation attenuates MO (TRPA1)-induced activity, November 2014, Society for Neuroscience, Washington D.C.

Govea RM, Hargett GL, Carlton SM Group III mGluR8 activation attenuates MO (TRPA1)-induced nociceptive behaviors, May 2014, American Pain Society, Tampa, Florida.

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Govea RM, Carlton SM Group III mGluR8 activation attenuates TRPA1-induced calcium mobilization, November 2013, Society for Neuroscience, San Diego, California

Govea RM, Carlton SM Group III mGluRs negatively modulate TRPA1 on primary afferents, May 2013, Fourth International Congress on Neuropathic Pain, Toronto, Canada

Govea RM, Carlton SM Group III mGluRs interact with TRAP1 on primary afferents, May 2013, American Pain Society, New Orleans, LA

Govea RM, Carlton SM Group III mGluR activation attenuates TRPA1-induced nociceptive behaviors, October 2012 Society for Neuroscience, New Orleans, LA

Govea RM, Zhou S, Carlton SM Group III mGluRs inhibit forskolin-induced protein kinase A activation. August 2012 International Association for the Study of Pain, Milan, Italy

Govea RM, Zhou S, Carlton SM Intracellular effectors in the pathway allowing mGluR8 modulation of TRPV1 function. May 2012 American Pain Society, Honolulu, HI

Govea RM, Carlton SM Direct interaction between group III mGluRs and TRPV1 on peripheral nociceptors. Nov 2011 Society for Neuroscience, Washington, D.C.

Govea RM, Carlton SM TRPV1 and mGluR8 are co-localized in rat DRG. May 2011 American Pain Society, Austin, TX

Govea RM, Zhou S, Carlton SM Antihyperalgesic effects of the group III mGluR agonist LAP-4 on capsaicin- induced nociception. Nov 2010 Society for Neuroscience, San Diego, CA

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Permanent address: 3504 N. Houston, Fort Worth, Texas 76106 This dissertation was typed by Rosann Mae Govea.

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