Electronic Theses and Dissertations UC San Diego Peer Reviewed Title: Pharmacological analysis of the cholinergic and serotonergic signaling systems in Caenorhabditis elegans Author: Cregg, James F. Acceptance Date: 2010 Series: UC San Diego Electronic Theses and Dissertations Degree: Ph. D., UC San Diego Permalink: http://escholarship.org/uc/item/5xh8h237 Local Identifier: b7021783 Abstract: The nervous system is designed to allow an organism to react to its changing environment in a coordinated manner. The simple nervous system of Caenorhabditis elegans has been a model for molecular studies in neurobiology for over four decades. This organism has contributed to our understanding of the molecules of neurotransmission and behavioral genetics. Here we seek to further characterize the molecules and simple circuits which lead to behaviors with hopes to generalize our knowledge from worms to higher organisms. Worms' sinusoidal locomotion is based on the coordinated contraction of body wall muscles (BWM) mediated by cholinergic signaling. Cholinergic signaling at the neuromuscular junction (NMJ) is mediated by the nicotinic acetylcholine receptors (nAChRs), this is also the site of psychotropic activity of the addictive drug nicotine. Our ultimate goal of this research was to elucidate the molecular mechanisms involved in cholinergic signaling to further understand nicotine addiction. We initiated a screen to find effectors of the cholinergic signaling system to further our understanding of the molecular elements involved in neurotransmission. We over- stimulated the BWMs with a cholinergic agonist, nicotine, causing paralysis. Loss of molecules involved with cholinergic neurotransmission ameliorates the nicotine- induced paralysis. Utilizing a RNA interference (RNAi) library to systematically knock down each predicted gene products on chromosome one, we attempt to find new molecules that play a role in cholinergic signaling. This screen produced several hits, here we analyze one, agef-1, an ADP-ribosylation factor's guanine nucleotide exchange factor (Arf-GEF). Advancements in cell physiologic techniques in C. elegans have been revolutionary in extending the capabilities of researchers of this organism. Using calcium imaging, we examine the activity of specific cells in relation to behaviors. A small circuit of neurons and muscles control the egg-laying behavior. This simple design belies its molecular complexity. Egg-laying is controlled by at least eScholarship provides open access, scholarly publishing services to the University of California and delivers a dynamic research platform to scholars worldwide. two neurotransmitters/neuromodulators, acetylcholine (ACh) and serotonin (5-HT)--as well as neuropeptides. We use calcium imaging to describe the response of the egg-laying circuit to pharmacological agents in order to understand the molecular signaling networks underpinning its control Copyright Information: All rights reserved unless otherwise indicated. Contact the author or original publisher for any necessary permissions. eScholarship is not the copyright owner for deposited works. Learn more at http://www.escholarship.org/help_copyright.html#reuse eScholarship provides open access, scholarly publishing services to the University of California and delivers a dynamic research platform to scholars worldwide. UNIVERSITY OF CALIFORNIA, SAN DIEGO Pharmacological Analysis of the Cholinergic and Serotonergic Signaling Systems in Caenorhabditis Elegans A dissertation submitted in partial satisfaction of the requirements for the degree Doctor of Philosophy in Biology by James F. Cregg Committee in charge: Professor Darwin Berg, Chair Professor William Schafer, Co-Chair Professor Anirvan Ghosh Professor Amy Pasquinelli Professor Palmer Taylor 2010 i Copyright James F. Cregg, 2010 All rights reserved. ii The dissertation of James F. Cregg is approved, and it is acceptable in quality and form for publication on microfilm and electronically: Chair Co-Chair University of California, San Diego 2010 iii DEDICATION To my parents, Dr. James M. Cregg and Mrs. Jeannie Cregg, whose unwavering support since before I can remember has allowed me to become a reasonably productive human being. iv TABLE OF CONTENTS Signature Page ....................................................................................................... iii Dedication .............................................................................................................. iv Table of Contents ................................................................................................... v List of Figures ........................................................................................................ ix List of Tables ......................................................................................................... xi Abbreviations in Use .............................................................................................. xii Acknowledgements ................................................................................................ xiii Vita ........................................................................................................................xvii Abstract of the Dissertation .................................................................................... xix Chapter I Introduction ........................................................................................ 1 C. elegans as a Model Nervous System ......................................................... 3 Molecular Signaling in C. elegans ................................................................. 4 Cholinergic Signaling ................................................................................. 5 Serotonergic Signaling ................................................................................ 7 G-protein Signaling .................................................................................... 8 Behavioral Analysis and Screens ................................................................... 11 Locomotion ................................................................................................ 15 Egg-laying .................................................................................................. 17 Neurophysiology in C. elegans ...................................................................... 24 Electrophysiology........................................................................................ 25 v Calcium Imaging ........................................................................................ 26 Conclusion .................................................................................................... 28 Chapter II Molecular Mechanisms of Serotonergic and Cholinergic Control of Egg-laying in C. elegans ....................................................................................... 30 Abstract ......................................................................................................... 31 Introduction ................................................................................................... 32 Results .......................................................................................................... 35 Mutation of 5-HT Receptors has no Effect on ACh Stimulation of the VMs 35 Mutation of all 5-HT Receptors Abolishes 5-HT Induced Activity in the VMs, but Raises Basal Activity ......................................................................... 38 Basal Activity is not Mediated by an Individual 5-HT Receptor .................. 40 Mutation in Any 5-HT Receptor Reduces Sensitivity to 5-HT in VMs ........ 41 Each 5-HT Receptor Confers a Unique VM Response to 5-HT ................... 42 5-HT Inhibits VM Activity through mod-1 and ser-4 .................................. 43 ser-4 is Expressed in the VMs .................................................................... 44 VM Expressed SER-4 Mediates 5-HT Inhibition of the VM ....................... 44 Adaptation to Serotonin is Mediated by SER-4 in the VM .......................... 47 Single 5-HT Receptors that in Isolation Adapt to 5-HT, Synergistically Combine to not Adapt to 5-HT ................................................................ 49 Discussion ..................................................................................................... 50 ACh Excites VM Directly Through nAChRs .............................................. 50 SER-4 Inhibition of VM Activity and Egg-laying ....................................... 52 vi Vulval Muscle Expressed SER-4 is Required for 5-HT Adaptation ............. 54 Materials & Methods ..................................................................................... 56 Chapter III High-throughput RNAi Screen for Molecules Involved in Cholinergic Transmission ................................................................................................ 76 Abstract ......................................................................................................... 77 Introduction ................................................................................................... 78 Results .......................................................................................................... 83 RNAi Depletion Confers Resistance to Nicotine-Induced Paralysis ............ 83 Maximizing the Effect of RNAi .................................................................. 85 Titration of Nicotine to Maximize Valid Hits .............................................