Role of nuclear metabotropic glutamate receptor 5 in rat models of persistent pain Kathleen Vincent Department of Psychology Faculty of Science McGill University Montreal, Quebec, Canada December 2017 A thesis submitted to McGill University Faculty of Graduate and Postdoctoral Studies Office in partial fulfillment for the degree of Doctor of Philosophy in the Department of Psychology © Kathleen Vincent, 2017 i Table of Contents Table of Figures ............................................................................................................................................ v Abbreviations ............................................................................................................................................. vii Abstract ....................................................................................................................................................... xi Résumé...................................................................................................................................................... xiii Acknowledgements .................................................................................................................................... xv Contributions of authors .......................................................................................................................... xvii Introduction ................................................................................................................................................ 1 Background .............................................................................................................................................. 1 Metabotropic glutamate receptor structure and subtypes .................................................................... 3 Group I mGluR splice variants ............................................................................................................. 4 Group I mGluR-linked intracellular messengers ..................................................................................... 5 DAG-mediated signalling pathway ...................................................................................................... 7 IP3-mediated signalling pathway ......................................................................................................... 8 JNK pathway ........................................................................................................................................ 8 G protein-mediated calcium oscillations ............................................................................................. 9 G protein-independent signalling ...................................................................................................... 10 Group I mGluR scaffolding interactions ................................................................................................ 11 Group I mGluR agonists ......................................................................................................................... 13 mGluR5 antagonists and inverse agonists ............................................................................................ 16 Localization of mGluRs in the SCDH ...................................................................................................... 20 Expression and regulation of mGluR5 following injury ......................................................................... 21 Intracellular GPCRs ................................................................................................................................ 23 Nuclear mGluR5 .................................................................................................................................... 25 Activating nuclear mGluR5 .................................................................................................................... 27 Excitatory amino acid transporters ................................................................................................... 27 Membrane permeability .................................................................................................................... 30 Nuclear mGluR5 implications for pain .................................................................................................. 31 Involvement of EAATs in pain ............................................................................................................... 33 EAAT inhibitors: Development and subtype selectivity .................................................................... 33 ii EAAT inhibitors and nociception ....................................................................................................... 35 EAAT expression following injury ...................................................................................................... 35 EAATs inhibitors in animals with persistent pain .............................................................................. 37 Background on methodologies selected ............................................................................................... 42 Animal models of neuropathic pain .................................................................................................. 42 Animal models of inflammatory pain ................................................................................................ 45 Testing methods for assessing pain hypersensitivity ........................................................................ 48 Methods .................................................................................................................................................... 53 Animals .................................................................................................................................................. 53 Neuropathic model: Spared nerve injury (SNI) ..................................................................................... 54 Inflammatory model: Complete Freund’s adjuvant (CFA) .................................................................... 55 Materials ............................................................................................................................................... 56 Drug administration .............................................................................................................................. 56 Nociceptive testing ................................................................................................................................ 57 Glutamate-induced pain behaviors ................................................................................................... 57 Mechanical allodynia testing ............................................................................................................. 57 Conditioned place preference – SNI .................................................................................................. 58 Conditioned place preference – CFA ................................................................................................. 59 Immunohistochemistry ......................................................................................................................... 60 Tissue preparation ............................................................................................................................. 60 Staining protocol................................................................................................................................ 60 Cell counting ...................................................................................................................................... 61 Subcellular fractionation and western blot analysis ............................................................................. 62 Tissue collection ................................................................................................................................ 62 Western blot ...................................................................................................................................... 63 Microdialysis Sampling .......................................................................................................................... 64 Statistical analysis.................................................................................................................................. 64 Results ....................................................................................................................................................... 67 mGluR5 is increased in nuclear-enriched SCDH fractions of rats with neuropathic pain .................... 67 mGluR5 is increased in nuclear-enriched SCDH fractions of rats with inflammatory pain .................. 70 Nuclear mGluR5 increased by one day following SNI surgery .............................................................. 74 iii Nuclear mGluR5 increased by one day following CFA injection ........................................................... 76 Spinal glutamate induces pain behaviors in rats .................................................................................. 79 Glutamate induces greater Fos expression in the SCDH of rats with persistent pain .......................... 82 Glutamate induces greater Jun expression in the SCDH of rats with persistent pain .......................... 84 Blocking neuronal glutamate uptake is antinociceptive in animals with neuropathic pain ................. 86 Blocking