Neuronal Plasticity in the Enteric Nervous System Motor Pathways Chae Ran Lim A thesis in fulfilment of the requirement for the degree of Masters by Research School of Medical Sciences Faculty of Medicine March 2014 Chae Ran Lim (z3258672) Neuronal Plasticity in ENS Motor Pathways Originality Statement ‘I hereby declare that this submission is my own work and to the best of my knowledge it contains no materials previously published or written by another person, or substantial proportions of material which have been accepted for the award of any other degree or diploma at UNSW or any other educational institution, except where due acknowledgement is made in the thesis. Any contribution made to the research by others, with whom I have worked at UNSW or elsewhere, is explicitly acknowledged in the thesis. I also declare that the intellectual content of this thesis is the product of my own work, except to the extent that assistance from others in the project's design and conception or in style, presentation and linguistic expression is acknowledged.’ Signed …………………………………………….............. Date ………12th September 2014…………………………. 2 Chae Ran Lim (z3258672) Neuronal Plasticity in ENS Motor Pathways Table of contents Originality Statement ........................................................................................................ 2 Abstract ............................................................................................................................. 6 Acknowledgements ........................................................................................................... 8 List of figures and tables ................................................................................................. 10 List of abbreviations ........................................................................................................ 11 List of relevant publications or presentations used in thesis ........................................... 13 Introduction ..................................................................................................................... 14 The intrinsic nervous system of the gut ....................................................................... 14 The structure of the ENS ............................................................................................. 15 The myenteric plexus ............................................................................................... 16 The submucous plexus ............................................................................................. 16 Morphology of the enteric neurons .......................................................................... 18 Neuro-coding concept in the ENS ........................................................................... 20 Neurochemistry and neurotransmitters of enteric neurons ...................................... 20 Electrophysiological characteristics of enteric neurons ........................................... 21 Functions of the GI tract .............................................................................................. 22 Control of secretion into the lumen of the GI tract .................................................. 23 Control of the motor functions of the GI tract ......................................................... 23 Immune functions of the GI tract ............................................................................. 26 Inflammatory bowel disease and functional bowel disorders ..................................... 28 GI tract control of satiety ......................................................................................... 30 Aims of the study ............................................................................................................ 33 Methods ........................................................................................................................... 36 Preparation setup and timeline .................................................................................... 36 Threshold tests ......................................................................................................... 39 Electrical stimulation ............................................................................................... 43 3 Chae Ran Lim (z3258672) Neuronal Plasticity in ENS Motor Pathways Video recording ....................................................................................................... 44 Drugs and statistical analysis ................................................................................... 48 Results ............................................................................................................................. 49 Measuring the peristaltic reflex threshold ................................................................... 49 Effects of blockade of fast excitatory neurotransmitter receptors ............................... 51 Hexamethonium blocks nicotinic receptors ............................................................. 51 Mecamylamine blocks nicotinic receptors .............................................................. 55 Blockade of 5-HT3 receptors with granisetron ........................................................ 57 Effects of blockade of slow excitatory neurotransmitter receptors ............................. 58 Blockade of muscarinic M1 receptors with VU 0255035 ....................................... 58 Blockade of all muscarinic receptors with hyoscine ............................................... 61 Effects of blocking both fast and slow excitatory neurotransmitter receptors ............ 61 Effects of other channel blockers and aboral electrical stimulation ............................ 63 Blockade of intermediate calcium activated potassium channel (IKCa++) with Tram-34 ................................................................................................................... 63 ++ Blockade of the IKCa and nicotinic receptors ....................................................... 64 Blockade of neuronal conduction with lidocaine .................................................... 65 Stimulation at the aboral end of the preparation ......................................................... 65 Discussion ....................................................................................................................... 67 Electrical stimulation enhanced the peristaltic motor pattern in the presence of nicotinic receptor blockade.......................................................................................... 68 Blockade of 5-HT3 receptors did not have an effect on recovery ............................... 69 Excitation of AH/sensory neurons may enhance the peristaltic reflex ........................ 71 An M1 muscarinic receptor antagonist did not have an effect on recovery ................ 71 Overcoming experimental problems and issues .......................................................... 72 Future directions .......................................................................................................... 73 Conclusions ..................................................................................................................... 74 4 Chae Ran Lim (z3258672) Neuronal Plasticity in ENS Motor Pathways References ....................................................................................................................... 75 5 Abstract Background: Many bowel disorders and diseases are linked to enteric neuronal dysfunction. Details of the mechanisms underlying neurotransmission within in the enteric nervous system (ENS) are not yet known. Acetylcholine acting on nicotinic receptors mediates the majority of fast synaptic transmission between enteric neurons however, recent studies suggest that the peristaltic motor pattern can occur during nicotinic receptor blockade. Aim: To determine whether the electrical stimulation of the enteric nerves enhances the recovery of the intestinal motility during nicotinic blockade and to determine the mechanisms by which this occurs. Method: Two segments of proximal ileum measuring 5-7cm were extracted from guinea pigs of either sex (232 to 900g) and positioned in an organ bath. Each segment was cannulated and the peristaltic pressure threshold was determined by step-wise increases of the intraluminal pressure from the oral end while pressure recordings were made from the aboral end. The parameters of the peristaltic motor pattern were compared in electrically stimulated (ES: 1Hz; 1ms; 250 pulses) and non-electrically stimulated (non-ES) preparations using an ANOVA with a repeated measures post-hoc test. Video recordings were made throughout the experiment to detect changes in luminal diameter. Drug containing solutions were added to the serosal side of the intestine. Results: The peristaltic pressure threshold was taken as the pressure at which four consecutive propulsive contractions were observed. Nicotinic receptor blockade using hexamethonium (300 µM) or mecamylamine (3 µM) inhibited peristalsis. In the presence of hexamethonium, electrical stimulation at the oral end enhanced recovery of peristalsis with 6 out of 20 preparations showing almost full recovery. Peristalsis also Chae Ran Lim (z3258672) Neuronal Plasticity in ENS Motor Pathways showed some recovery when electrical stimuli were applied in the presence of muscarinic blockade (VU 0255035 (150 nM)) or hyoscine (10 µM)); or IKCa2+ blockade (TRAM-34 (100 µM)). A combination of ligand-gated