Pharmacological modulation of processes contributing to spinal hyperexcitability: electrophysiological studies in the rat. By Katherine J Carpenter A thesis submitted to the University of London for the degree of Doctor of Philosophy Department of Pharmacology University College London Gower Street London WC1E6BT ProQuest Number: U642184 All rights reserved INFORMATION TO ALL USERS The quality of this reproduction is dependent upon the quality of the copy submitted. In the unlikely event that the author did not send a complete manuscript and there are missing pages, these will be noted. Also, if material had to be removed, a note will indicate the deletion. uest. ProQuest U642184 Published by ProQuest LLC(2015). Copyright of the Dissertation is held by the Author. All rights reserved. This work is protected against unauthorized copying under Title 17, United States Code. Microform Edition © ProQuest LLC. ProQuest LLC 789 East Eisenhower Parkway P.O. Box 1346 Ann Arbor, Ml 48106-1346 Abstract Two of the most effective analgesic strategies in man are (i) blockade of the NMDA receptor for glutamate, which plays a major role in nociceptive transmission and (ii) augmentation of inhibitory systems, exemplified by the use of ketamine and the opioids respectively. Both are, however, are associated with side effects. Potential novel analgesic targets are investigated here using in vivo electrophysiology in the anaesthetised rat with pharmacological manipulation of spinal neuronal transmission. Three different approaches were used to target NMDA receptors: (i) glycine site antagonists (Mrz 2/571 and Mrz 2/579), (ii) antagonists selective for receptors containing the NR2B subunit (ifenprodil and ACEA-1244), (iii) elevating the levels of N-acetyl-aspartyl- glutamate (NAAG), an endogenous peptide, by inhibition of its degradative enzyme. All agents produced selective inhibitions of noxious-evoked activity of dorsal horn neurones, with differing profiles. Effects of NAAG elevation were enhanced after inflammation and neuropathy, probably a result of agonist activity at the mGlu3 metabotropic glutamate receptor rather than reported NMDA receptor partial agonist properties. The mu opioid receptor agonist methadone has affinity for the NMDA receptor. Spinal application of racemic methadone and the weak opioid optical isomer d-methadone, revealed that the inhibitory effects were predominantly opioid receptor mediated. Functional NMDA receptor blockade was not apparent. Nociceptin/orphanin FQ (NC/OFQ) differs from the classical opioids and can, like NMDA receptor antagonists, attenuate spinal hyperexcitability. Pre-synaptic effects were enhanced after peripheral inflammation. Peripheral administration caused excitations of primary afferent fibres. Three putative antagonists were studied. [Phe^ v( C H2- NH)Gly^]nociceptin-(1-13)-NH2 was as potent as nociceptin in inhibition of neuronal responses and is now recognised as a partial agonist. [N-Phe^]nociceptin(1-13)NH2 was similarly unreliable, but J-113397, proved more fruitful. Protons are implicated in inflammatory pain and peripheral hyperalgesia. I demonstrated activation of primary afferent fibres by peripheral administration of low pH solutions, the first electrophysiological study to do so in vivo. The overall results provide a number of pharmacological strategies that may lead to a better control of pain. The thesis also sheds some light on a fundamental question in neurobiology, in describing both peripheral and central mechanisms that underlie normal transmission and aspects of plasticity in pain pathways. Dedication To Dorothy Carpenter, a wonderful grandmother, who would have been so proud to see this, and to Jo Pearson, who will sadly never read my “Book about Drugs”. Acknowledgements Thanks must go first to Tony, for football chat Monday mornings, for learning about cerise pink and for teaching about lime green. If you had never given that course of lectures in Cambridge, I may never have found out that some scientists can be enthusiastic and passionate not only about their work, but about getting that message across. After all, what use is knowledge if you keep it to yourself? And what use is a lab with no one to work in it....I am indebted to everyone in the lab, old and new. Who would have guessed that I’d outlast Wahida, Vicky AND Louise? (Well, briefly Louise!) Thanks girls for everything...and for saying that you liked every haircut, thanks to Mark for a different perspective and for not noticing any haircuts, Lars for brief encounters, Vesa for longer, Katie for all things clinical, Rie for all things Latino, Sarah for running the London Marathon so that we didn’t have to (surely 3 miles each would have been easier?). Ana Paula and Idil for fresh ideas and generosity, Lucinda for topping up the blonde quotient....and biggest love to the original blonde, Lizzy, we’ve got some stories eh? Thanks also to my project students, for their good times and hard work, Mansi, Sayan, Mash and Mei Mei. Love and thanks to all my family (for caring about the details or gracefully ignoring them), Emilie and Helen (you didn’t know what you had started with the shoe and handbag thing), Kirstie, Rog, Beth and the pharmacology crew for a good excuse to drink, and all the rugby girls for keeping me sane and reminding me of my age. Finally, all my love to Rich, for giving more than all of the above. Contents Chapter 1. Introduction 1.1 Classification of primary afferent fibres___________________ 10 1.1.1 Functional classification denotes response properties________________ 10 1.1.2 Physiological properties of the axon can be classified_________________ 11 1.1.3 Anatomical characterisation illuminates the pathways each fibre enters_ 12 1.1.4 Histochemical classification reveals heterogeneity among nociceptors___12 1.2 Pharmacology of peripheral nociceptor terminals__________14 1.2.1 The kinins are potent algogenic and proinflammatory peptides_________ 14 1.2.2 Inflammatory mediators interact to sensitise nociceptors_____________ 15 1.2.3 The sympathetic nervous system _________________________________ 16 1.2.4 Peptide transmitters are released from peripheral terminals___________ 16 1.2.5 Protons can activate and sensitise nociceptors______________________ 17 1.2.6 The capsaicin receptor, a putative endogenous sensor for noxious heat 20 1.2.7 A sensory neurone specific, TTX-resistant sodium channel___________ 21 1.2.8 P2X3, an ATP-gated ion channel _________________________________ 21 1.2.9 Adenosine is generated by hydrolysis of ATP _______________________ 22 1.2.10 Peripheral opioids_____________________________________________ 22 1.2.11 Peripheral glutamate _______________________________________24 1.2.12 Nociceptors are equipped to integrate many signals_________________25 1.3. Primary afferent fibre termination and transmitter profiles 26 1.3.1 A (3-fibres __________________________ 26 1.3.2 Aô-fibres______________________________________________________ 27 1.3.3 C-fibres________________________________________________ 28 1.3.4 Transmitter profile of C-fibre afferents______________________________ 29 1.4 Primary afferent fibre projection characteristics____________31 1.5 Excitatory pharmacology of spinal processing _____________ 33 1.5.1 Peptides_____________________________________________________ 33 1.5.2 Glutamate____________________________________________________ 35 1.5.3 Mechanisms of spinal hyperexcitability _____________________________40 1.6 Inhibitory pharmacology of spinal processing _____________44 1.6.1 Inhibitory amino acids ___________________________________________ 44 1.6.2 Opioids_______________________________________________________ 44 1.6.3 Nociceptin/orphanin FQ__________________________________________ 48 1.6.4 Cannabinoids __________________________________________________ 49 1.6.5 G a l a n i n __________________________________________________ 49 1.6.6 Purines_______________________________________________________ 50 1.6.7 Inflammation induces changes in inhibitory systems___________________50 1.7 Aims of Study _________________________________________ 52 References_______________________________________________53 Chapter 2. Methods___________________________________________81 2.1 Anaesthesia and surgery____________________ 82 2.2 Electrophysiological recordings_________________________ 82 2.2.1 Data capture___________________________________________________ 83 2.2.2 Isolating a neurone______________________________________________85 2.2.3 Characterisation of isolated neurone_______________________________ 85 2.3 Drug application_______________________________________ 88 2.3.1 Application of drugs for electrical studies ___________________________ 8 8 2.3.2 Peripheral administration of agents________________________________ 89 2.4 Analysis of results_____________________________________ 89 2.4.1 Graphical representation of results________________________________ 89 2.4.2 Statistical analysis of results______________________________________ 90 2.5 Models of chronic pain__________________________________90 2.5.1 Carrageenan-induced inflammation_______________________________ 90 2.5.3 Spinal nerve ligation____________________________________________ 91 2.6 Drugs_________________________________________________92 References 93 Chapters. NMDA receptor blockade____________________________ 94 3.1 Introduction___________________________________________ 95 3.1.1 NMDA receptor structure_________________________________________ 95 3.1.2 NMDA receptor function_________________________________________