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C:\Documents and Settings\lproctor.ULL\LocaI Settings\Temporary Internet Files\OLK36\Copyright - thesis.doc CENTRAL SEROTONERGIC CONTROL OF CARDIOVASCULAR REFLEXES A Thesis submitted for the Degree of Doctor of Philosophy to the Faculty of Science of the University of London by Daniel Otto Kellett MA BSc Departments of Physiology & Pharmacology University College London Hampstead Campus Rowland Hill Street London NW3 2PF January 2005 UMI Number: U594502 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. Dissertation Publishing UMI U594502 Published by ProQuest LLC 2013. Copyright in the Dissertation held by the Author. Microform Edition © ProQuest LLC. All rights reserved. This work is protected against unauthorized copying under Title 17, United States Code. ProQuest LLC 789 East Eisenhower Parkway P.O. Box 1346 Ann Arbor, Ml 48106-1346 Abstract Central serotonergic neurones control reflex parasympathetic outflow to the heart, airways and bladder in a number of species, and different 5-HT receptor subtypes are involved in this effect. 5-HTja and 5 -HT3 receptors in the brainstem facilitate these reflexes, whilst 5-HTjb/id, 5-HT2 and 5 -HT4 receptors inhibit them. Recently, central 5 -HT7 receptors have been implicated in bladder reflexes. Experiments on anaesthetised rats showed that the selective 5-HT7 receptor antagonists SB-269970 and SB-656104, given intracistemally (i.e.), attenuated cardiopulmonary, baroreflex and chemoreflex bradycardias. Similarly, the selective 5-HTia receptor antagonist WAY-100635 attenuated cardiopulmonary and chemoreflex (but not baroreflex) bradycardia, whilst robalzotan and (-)-pindolol (antagonists at 5-HT 1 a receptors) had no effect on cardiopulmonary and baroreflex bradycardias respectively. Chemical stimulation of presumed^ serotonergic cell bodies in raphe magnus/pallidus evoked a bradycardia that could not be attenuated either by 5-HT receptor antagonists (given i.v.) or by prior 5-HT depletion. The latter did, however, significantly attenuate cardiovascular reflex sensitivity. Activation of nucleus tractus solitarius (NTS) neurones by the vagus was inhibited by the iontophoretic AMPA receptor antagonist DNQX or by topical SB-269970. Subsequent histology suggested that 5-HT containing terminals do not make close appositions with these neurones. Preliminary data demonstrate that SB-269970 (given i.e.) effectively attenuates the cardiopulmonary reflex in awake rats, but has variable effects on the chemoreflex. The data suggest that 5-HT7 receptors in the NTS are crucially involved in the central transmission of reflex bradycardias, at least in rats. The role of the 5 -HT ia receptor is less clear-cut than in the rabbit, and may reveal a species difference. The origin of 5- HT activating these receptors is unlikely to be the medullary raphe neurones, but may be primary afferents terminating in the NTS. Since recent ultrastructural evidence shows 5-HT terminals and NTS cardiovascular neurones are often separated by astroglial leaflets, astrocytes may be involved in serotonergic- glutamatergic signalling. 2 Contents List of figures .......................................................................................................................5 List of tables ........................................................................................................................8 Abbreviations ..................................................................................................................... 11 Abbreviated compounds ...................................................................................................12 Publications ........................................................................................................................ 15 Acknowledgements ........................................................................................................... 17 1. G eneral introduction ......................................................................................................... 18 1.1. The autonomic nervous system ........................................................................ 18 1.2. The cardiovascular system ................................................................................19 1.3. Cardiovascular reflexes .................................................................................... 33 1 .4. Neuropharmacology of the NTS ..................................................................... 44 1.5. The central serotonergic system .......................................................................49 1.6. 5-HT receptors ................................................................................................ .. 59 1.7. Aims of the thesis..............................................................................................98 2. General methods .................................................................................................100 2.1. Preparation of rats ........................................................................................... 101 2.2. Neuromuscular blockade and stabilisation ................................................... 106 2.3. Raphe stimulation ........................................................................................... 107 2.4. Depletion of 5-HT stores with para-chlorophenylalanine ...........................108 2.5. Single unit electrophysiology .........................................................................108 2.6. Histological processing .................................................................................. I l l 2.7. Data Capture .................................................................................................... 113 2.8. Data analysis .................................................................................................... 115 2.9. Drugs and solutions ......................................................................................... 134 3. The roles of central 5-HT7 a n d 5-H T iA receptors in the reflex ACTIVATION OF CARDIAC VAGAL OUTFLOW...................................................................137 3.1. Introduction ...................................................................................................... 137 3.2. Methods ............................................................................................................139 3 3.3. Results..... 143 3.4. Discussion 202 4. The contribution of 5-HT containing cell groups to autonom ic BASELINES AND REFLEXES..............................................................................................225 4.1. Introduction ......................................................................................................225 4.2. Methods ........................................................................................................... 226 4.3. Results..............................................................................................................230 4.4. Discussion ........................................................................................................ 265 5. Serotonergic a n d glutamatergic m echanism s in the N ucleus T ractus SOLITARIUS...................................................................................................................... 281 5.1. Introduction ......................................................................................................281 5.2. Methods ............................................................................................................282