Copyright by Rosann Mae Govea 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 by 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