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 .............................................. 87 Pilot Screen of Chromosome One ............................................................... 89 Re-screening Putative Hits .......................................................................... 91 Resistance to Non-Cholinergic NT Receptor Agonists ................................ 92 Nicotine Resistance on Plates ..................................................................... 94 Testing the Mutants for Nicotine and Levamisole Resistance ...................... 95 Discussion ..................................................................................................... 96 Genes Identified in the Screen of Chromosome One ................................... 96 Mutant Phenotypes ..................................................................................... 97 Materials & Methods ..................................................................................101 Chapter IV Characterization of the Nicotine Resistance Factor agef-1 .............115 Abstract..........................................................................................................116 Introduction....................................................................................................116 vii Results ...........................................................................................................120 Identification of agef-1 in RNAi Screens ....................................................120 Description of agef-1 and AGEF-1 .............................................................121 agef-1 Expression .......................................................................................122 Cholinergic Agonist Resistance ..................................................................123 Aldicarb and Muscimol Resistance Testing ................................................123 Brefeldin A, Arf-GEF inhibition .................................................................124 Effect of agef-1 (RNAi) on the Expression Level of an nAChR Subunit .....126 Dominant Negative to Eliminate AGEF-1 Activity in BWM ......................128 Discussion......................................................................................................132 Materials & Methods .....................................................................................146 Appendix I Putative Nicotine Resistance Factors Identified in Screening of Chromosome One .......................................................................................146 Appendix II Getting the Most Out of an RNAi Screen ............................................154 References .............................................................................................................158 viii LIST OF FIGURES Chapter II Figure 2.1 Egg-laying System Diagram ................................................................ 65 Figure 2.2 VM Stimulation by ACh ...................................................................... 67 Figure 2.3 Serotonin Strongly Stimulates VM Activity ......................................... 68 Figure 2.4 5-HT Activity Induced in Single 5-HT Mutants ................................... 69 Figure 2.5 Dosage Curve for Quadruple Mutants .................................................. 70 Figure 2.6 SER-4 and MOD-1 Inhibit VM Activity in Response to Serotonin .....