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Neurophaema.Cological Studies on the Interactions of Some Putative Peptide Transmitters with the Nigrostriatal and Mesolimbic Dopamine Systems

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Neurophaema.Cological Studies on the Interactions of Some Putative Peptide Transmitters with the Nigrostriatal and Mesolimbic Dopamine Systems NEUROPHAEMA.COLOGICAL STUDIES ON THE INTERACTIONS OF SOME PUTATIVE PEPTIDE TRANSMITTERS WITH THE NIGROSTRIATAL AND MESOLIMBIC DOPAMINE SYSTEMS by Robert Denham Pinnock B.Sc. A thesis submitted in support of the Degree of Doctor of Philosophy of the University of Southampton. September, 198O. INDEX ABSTRACT (iv) ACKNOWLEDGEMENTS (v) ABBREVIATIONS ( CHAPTER ONE. INTRODUCTION 1. Substance P 1 1.1. Isolation and structure 1 1.2. Distribution of substance P in the brain 1 1.3. Structure activity studies in peripheral preparations 1 1.4. Structure activity relationships in the CNS 2 1.5. Metabolism of substance P 3 1.6. Mechanism of action 4 1.7. Possible role of substance P in the CNS 5 2. Melanocyte stimulating hormone release inhibitory factor (MIF) 8 2.1. General pharmacology of MIF 8 2.2. Interactions of MSH and MIF with dopamine 8 3 Thyrotropin releasing hormone (TRH) 12 3.1. General pharmacology of TRH 12 3.2. TRH in the CNS 12 k. Opioid peptides I6 4.1. Opioid peptides in the CNS 16 U.2. Interactions between dopamine and opiates I6 5. Dopamine 19 5.1. Dopamine, distribution and pathways in the CNS 19 5.2. Biochemistry of dopamine release, receptor binding and postsynaptic actions I9 5.3. lontophoretic dopamine pharmacology in the caudate nucleus and nucleus accumbens 20 5.4. Effect of drugs on the cell bodies of the nigro striatal tract 21 5.5. Electrophysiology of tlie mesolimbic and nigrostriatal pathways " • . 22 1.5.6. Behavioural effects of dopamine 23 (i) CHAPTER TWO. METHODS 2.1. Electrophysiology 27 2.1.1. Preparation of animals 27 2.1.2. Recording and iontophoresis 27 2.1.3. Release of drugs from micropipettes 29 2.1.4. Stimulation and identification of neurones 30 2.1.5. Analysis of data 31 2.1.6. Verification of electrode positions 31 2.2. Behaviour 33 2.2.1. Preparation of animals for chronic cannulae 33 2.2.2. Intracerebral and peripheral injections 34 2.2.3. Experimental procedure 3I+ 2.2.4. Analysis of data 35 2.2.5. Histological procedures 35 CHAPTER THREE. RESULTS 3.1. Electrophysiology and iontophoresis 36 3.1.1. Iontophoresis of substance P and analogues 36 a 3.1.2. Substance P behavioural studies 38 3.2.1. Effects of MIF on the firing rate of neurones 40 3.2.2. MIF, behavioural studies 4l 3.3.1. TRH, iontophoretic studies 43 3.3.2. TRH, behavioural studies 44 3.4.1. Angiotensin II, iontophoretic studies 45 3.5.1. Opidd peptides, behavioural studies 46 3.5.2, Opioid peptides, iontophoretic studies 47 3.6.1. lontophoretic studies with dopamine agonists and )|8 antagonists in the substantia nigra 3.6.2. lontophoretic action of sulpiride in the caudate nucleus 51 3.7.1. Electrical stimulation of the substantia nigra ')p 3.7.2. Electrical stimulation of the striatum 3.7.3. Position of stimulating and recording electrodes in the brain 53 3.7.^. Position of cannulae in the brain '•,5 3.8.1. Release of drugs from micropipettes 3.9.1. Potency of substance P fragments and putative antagonists in the peripheral assays (ii) 3-9.2. Potency of opiate agonists and antagonists on peripheral preparations 54 CHAPTER FOUR. DISCUSSION 4.1. Substance P 55 4.2. MSH release inhibiting factor (MIF) 6l 4.3. Thyrotropin releasing hormone (TRH) 64 4.4. Opioid peptides 6j 4.5. Dopamine 70 4.6. Identification of nigrostriatal and striatonigral neurones 82 SUMMARY 84 FORMULAE OF DRUGS 1. Amino acid sequence of peptides 8j 2. Structures of non peptide drugs 88 BIBLIOGRAPHY 93 (iii) UNIVERSITY OF SOUTHAMPTON ABSTRACT FACULTY OF SCIENCE PHYSIOLOGY AND PHARMACOLOGY Doctor of Phil osophy NEUROPHARMACOLOGICAL STUDIES OF THE INTERACTION OF SOME PUTATIVE PEPTIDE TRANSMITTERS WITH THE NIGROSTRIATAL AND MESOLIMBIC DOPAMINE SYSTEMS, by Robert Denham Pinnock The experiments described in this thesifare an investigation of the inter- actions of some peptides with dopamine in areas of the rat brain known to be components of the ascending dopamine pathways. Most attention was paid to drug interactions at the cellular level using conventional iontophoretic techniques in three brain regions, the substantia nigra, the caudate nucleus and nucleus accumbens. Behavioural studies of the locomotor activity pro- duced by some of these peptides after injection into the nucleus accumbens or ventral tegmentum of chronically cannulated rats were also carried out. By using a series of substance P fragments it was found that the 1-9 N-terminal of the molecule previously found inactive has similar iontophor- etic effects in the substantia nigra and similar behavioural actions in the ventral tegmentum as substance P. Two hypothalamic releasing hormones, TRH and MIF and some analogues of them were found to be inactive after ionto- phoretic application into any of the three brain areas. These compounds also failed to potentiate hyperactivity produced by amphetamine. The pep- tide BWI8OC thought to be an opiate agonist produced inhibitions of cell activity in the nucleus accumbens. Behaviourally it caused a long term hyperactivity after injections into either the nucleus accumbens or ventral tegmentum. None of the effects could be reversed by classical antagonists such as nalaxone or putative peptide antagonists. By using both atypical neuroleptics and semirigid dopamine analoguos the study was extended to find if there was any evidence from iontophoretic studies to support a multiple dopamine receptor hypothesis. ADTN was found to produce a more powerful inhibition of firing on nigral compact a neuronor. than has been reported for other dopamine agonists. The atypical neuro- leptic sulpiride blocked the effects of dopamine both on these iicuronoG .'uid on those in the caudate nuclues. Our results with this and another neuro- leptic fluphenazine imply that both are acting at a common site. These results are discussed with regard to the multiple dopamine receptor thoories. (iv) ACKNOWLEDGEMENTS I am very grateful to Dr. G.N. Woodruff for his supervision and suggestions over the past three years. I would also like to thank Dr. M.J. Turnbull for his interest in the peptide studies. Professor K.A. Munday in whose department the electrophysiology experiments were performed and ICI Ltd., for allowing the use of their laboratories for the behavioural studies. I am also grateful to Dr. J.A. Poat for measurement of tritiated dopamine and noradenaline levels in the testing of release from micropipettes, N.N. Petter for measuring MIF levels in similar experiments, J.W. Grocott for the data from peripheral studies with substance P analogues and J. Shaw for the data from peripheral studies with opiate agonists and antagonists. thanks are extended to Mr. N. Dale for photography of figures, the electronics department for maintenance of equipment, to the staff of the animal house for consistently providing animals in good condition, * and to Mrs. M.P. Reilly for her patient and very efficient typing of this thesis. I am also indebted to Dr. J.A. Poat for correcting the first draft. Finally my thanks go to my parents for their encouragement and financial support and most of all to my wife without whose co-operation the project would have been incomplete. (v) ABBREVIATIONS Ach Acetylcholine ADTN 2-Amino-6,7-d.ihydroxy-1,2,3,4-tetrahydronaphthalene C Centigrade CNS Central Nervous System cAMP Cyclic adenosine 3',5'-monophosphate cGMP Cyclic guanosine 3',5'-monophosphate DA Dopamine 1-Dopa 1-3,^,-Dihydroxyphenylalanine DLH DL Homocysteic acid Flu Fluphenazine Fig Figure g gram GABA Gammaamino butyric acid 5-HT 5-hydroxy tryptamine Hz Hertz I.P. Intraperitoneal I.V. Intravenous Kg Kilogram yg Microgram yi Microlitre pmol Micromole M Molar ml Millilitre mM Millimolar mg Milligram mV Millivolt yv Microvolt m Megohm nA Nanoamp M Noradrenaline nmol Kanomole 3,^-diOHNom 3,4,-dihydroxynomifensine nM Nanomolar NaCl Sodium Chloride 6-OHDA 6-hydroxydopamine B.C. Subcutaneous S.E.M. Standard error of the mean (vi) ABBREVIATIONS (contd.) sue Substantia nigra compacta SNCDcell Substantia nigra compacta dopamine cell SNR Substantia nigra reticulata y micron (vii) CHAPTER 1. INTRODUCTION 1.1 SUBSTANCE P 1.1.1. Isolation and structure Substance P is an undecapeptide with the following structure : H-Arg-Pro-Lys-Pro-Gln-Gln-Phe-Phe-Gly-Leu-Met-NHg Although an impure form which contracts the isolated guinea pig ileum (GPl) was first extracted from the horse hypothalamus in 1931 by Gaddum & Von Euler, its amino acid sequence was only determined in 1971 by Chang et al. Since then its physical properties have been examined. Interactions be- tween different substance P molecules in solution do not occur readily and and the peptide does not possess a secondary structure in aqueous or organ- ic solvents until high (10 M) concentrations are reached (Mehlis et al 1975, Mehlis et al 1980). 1.1.2. Distribution of substance P in the brain Substance P is unevenly distributed in the CNS. It was first extracted from the hypothalamus. Its distribution has been studied using bioassay, radioimmunoassay and immunohistochemistry. The highest concentrations of substance P are found in the substantia nigra (11 pmol/mg protein and the lowest concentration in the cerebellum (0.02 pmol/mg protein) (Brownstein et al, 1976). Substance P positive cell bodies, fibres and nerve terminals have been identified by immunohistochemistry (Cuello & Kanazawa 1978). The results from these techniques correlates with the distribution by bioassay (Lembeck & Zetler 1971). Recently attempts to map substance P containing nerve tracts have been made. In these studies specific knife cuts of the nerve tracts caused a loss of immunoreactivity or fluorescence in the area innervated and a build up of fluorescence on the cell body side of the knife cut. Using these techniques several pathways have been proposed (Fig.
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