University of Dundee DOCTOR OF PHILOSOPHY Targeting Opioid Receptor Signal Transduction to Produce Sustained Analgesia Bull, Fiona A. Award date: 2015 Link to publication General rights Copyright and moral rights for the publications made accessible in the public portal are retained by the authors and/or other copyright owners and it is a condition of accessing publications that users recognise and abide by the legal requirements associated with these rights. • Users may download and print one copy of any publication from the public portal for the purpose of private study or research. • You may not further distribute the material or use it for any profit-making activity or commercial gain • You may freely distribute the URL identifying the publication in the public portal Take down policy If you believe that this document breaches copyright please contact us providing details, and we will remove access to the work immediately and investigate your claim. Download date: 04. Oct. 2021 Targeting Opioid Receptor Signal Transduction to Produce Sustained Analgesia Fiona A Bull BSc(Hons), MBChB, FRCA Thesis submitted in candidature for the degree of Doctor of Philosophy, University of Dundee, November 2015. i Table of Contents List of Tables -------------------------------------------------------------------------------------- vi List of Figures ------------------------------------------------------------------------------------ vii Abbreviations ----------------------------------------------------------------------------------- xii Acknowledgements -------------------------------------------------------------------------- xvii Candidates declaration ---------------------------------------------------------------------- xix Supervisors Declaration ---------------------------------------------------------------------- xx Abstract ------------------------------------------------------------------------------------------ xxi Chapter 1: Introduction ------------------------------------------------------------------------ 1 1.1 Opium -------------------------------------------------------------------------------------- 2 1.2 Opioid receptors ------------------------------------------------------------------------- 8 1.3 Constitutive activity -------------------------------------------------------------------- 11 1.4 The endogenous opioid system ----------------------------------------------------- 13 1.5 The genetic manipulation of the opioid receptor system --------------------- 16 1.6 MOP knockout mice -------------------------------------------------------------------- 17 1.7 DOP receptor knockout mice -------------------------------------------------------- 19 1.8 KOP receptor knockout mice --------------------------------------------------------- 20 1.9 Structural models of the opioid receptors ---------------------------------------- 20 1.10 The arrestin system ------------------------------------------------------------------- 22 1.11 Beta-arrestin2 knockout mice ------------------------------------------------------ 25 1.12 Pain and nociception ----------------------------------------------------------------- 26 1.13 Genetic influences on rodent nociceptive responses ------------------------- 30 ii 1.14 Tolerance -------------------------------------------------------------------------------- 31 1.15 The RAVE hypothesis ----------------------------------------------------------------- 34 1.16 Opioid receptor desensitisation in tolerance ----------------------------------- 36 1.17 The role of DOP receptors and BAR2 in tolerance ----------------------------- 37 1.18 Pharmacokinetics of morphine in rodents -------------------------------------- 38 1.19 Mesocorticolimbic reward pathway ---------------------------------------------- 38 1.20 Psychomotor effects of morphine ------------------------------------------------- 43 1.20.1 Locomotor activation ----------------------------------------------------------- 43 1.20.2 Reinforcement -------------------------------------------------------------------- 45 1.21 c-Src and the opioid receptors ----------------------------------------------------- 46 1.22 Aims and Hypotheses ---------------------------------------------------------------- 49 Chapter 2: Methods ---------------------------------------------------------------------------- 52 2.1 Breeding Colony Maintenance and Housing -------------------------------------- 53 2.2 Genotyping ------------------------------------------------------------------------------- 54 2.3 Behavioural Studies -------------------------------------------------------------------- 59 2.3.1 Assessing morphine analgesia ------------------------------------------------- 59 2.3.2 Morphine tolerance --------------------------------------------------------------- 60 2.3.3 Psychomotor testing: Locomotor activation and CPP --------------------- 61 2.4 Dorsal Root Ganglion neurone culture preparation ---------------------------- 64 2.5 Immunohistochemistry ---------------------------------------------------------------- 66 2.6 Slice Electrophysiology ---------------------------------------------------------------- 68 2.7 SW620 cell culture ---------------------------------------------------------------------- 71 2.8 Western Blotting ------------------------------------------------------------------------ 71 iii 2.9 Statistical Analysis ---------------------------------------------------------------------- 73 Chapter 3: Analgesia and Tolerance ------------------------------------------------------- 74 3.1 Introduction ------------------------------------------------------------------------------ 75 3.2 Analgesia ---------------------------------------------------------------------------------- 79 3.2.1 The role of MOP receptors ------------------------------------------------------ 79 3.2.2 The Role of DOP receptors ------------------------------------------------------ 83 3.2.3 The role of β-arrestin2 ----------------------------------------------------------- 85 3.3 Tolerance --------------------------------------------------------------------------------- 89 3.3.1 The role of MOP receptors ------------------------------------------------------ 89 3.3.2 A role for DOP receptors in morphine tolerance --------------------------- 92 3.3.3 A role BAR2 in morphine tolerance -------------------------------------------- 94 3.4 Summary ---------------------------------------------------------------------------------- 97 Chapter 4: Psychomotor effects of morphine ----------------------------------------- 103 4.1 Introduction ---------------------------------------------------------------------------- 104 4.2 Locomotor effects of morphine --------------------------------------------------- 108 4.2.1 The role of MOP receptors ---------------------------------------------------- 108 4.2.2 The role of DOP receptors ----------------------------------------------------- 115 4.2.3 The role of β-arrestin2 --------------------------------------------------------- 119 4.3 Conditioned place preference ----------------------------------------------------- 125 4.3.1 The role of MOP receptors in reinforcement ------------------------------ 125 4.3.2 The role of DOP receptors in reinforcement ------------------------------- 130 4.3.3 The role of β-arrestin2 in reinforcement ----------------------------------- 133 4.4 Summary -------------------------------------------------------------------------------- 138 iv Chapter 5: Opioid receptor signalling within the VTA ------------------------------ 144 5.1 Introduction ---------------------------------------------------------------------------- 145 5.2 VTA cell type identification --------------------------------------------------------- 150 5.3 The influence of opioid drugs on IPSC events within the VTA -------------- 154 5.4 DOP receptors within the VTA ----------------------------------------------------- 163 5.5 The role of β-arrestin2 in signalling within the VTA --------------------------- 167 5.6 Effects of naloxone on sIPSC frequency within the VTA --------------------- 171 5.7 Summary -------------------------------------------------------------------------------- 174 Chapter 6: The effects of c-Src on opioid receptor signalling --------------------- 181 6.1 Introduction ---------------------------------------------------------------------------- 182 6.2 PP2 and Dasatinib inhibit c-Src in vitro and in vivo ---------------------------- 184 6.3 The role of c-Src in morphine tolerance ----------------------------------------- 187 6.4 The effects of c-Src inhibition on locomotor activation by morphine ---- 194 6.5 The effects of c-Src inhibition on conditioned place preference to morphine ------------------------------------------------------------------------------------ 200 6.6 The effects of c-Src inhibition on sIPSC frequency in the VTA -------------- 203 6.7 The effects of MEK inhibition on sIPSC frequency within the VTA -------- 206 6.8 Summary -------------------------------------------------------------------------------- 209 Chapter 7: Discussion and Conclusions ------------------------------------------------- 212 7.1 Discussion ------------------------------------------------------------------------------ 213 7.2 Analgesia and Tolerance ------------------------------------------------------------ 218 7.2.1 MOP receptors in analgesia and tolerance -------------------------------- 218 7.2.2 DOP receptors in analgesia and tolerance --------------------------------- 221 v 7.2.3 The role of BAR2 in analgesia and tolerance ------------------------------