Dissertation entitled Neuromodulation in a Nociceptive Neuron in C. elegans By Paul David Edward Williams Submitted to the Graduate Faculty as partial fulfilment of the requirements for the Doctor of Philosophy in Biology Dr. Bruce Bamber, Committee Chair Dr. David Giovannucci, Committee Member Dr. Richard Komuniecki, Committee Member Dr. Guofa Liu, Committee Member Dr. Scott Molitor, Committee Member Dr. Robert Steven, Committee Member Dr. Amanda Bryant-Friedrich, Dean College of Graduate Studies The University of Toledo, May 2018 Copyright 2018 Paul David Edward Williams This document is copyrighted material. Under copyright law, no parts of this document may be reproduced without the expressed permission of the author. An Abstract of Neuromodulation in a Nociceptive Neuron in C. elegans By Paul David Edward Williams Submitted to the Graduate Faculty as partial fulfillment of the requirements for the Doctor of Philosophy in Biology The University of Toledo May 2018 Neuromodulators have the capacity to alter neuronal excitability and synaptic strengths throughout the nervous system, allowing animals to switch between different behavioral states. The mechanisms by which neuromodulators change neuronal physiology, and the impact of those changes on circuit output and overall behavior, remain poorly understood. Neuromodulator-dependent changes in neuronal activity patterns are frequently measured using calcium reporters, since calcium imaging can easily be performed on intact functioning nervous systems. With only 302 neurons, the nematode Caenorhabditis elegans provides a relatively simple, yet powerful system to understand neuromodulation at the level of individual neurons. C. elegans is repelled by 1-octanol, and these aversive responses are modulated by monoamines and neuropeptides. Previously, we identified that serotonin (5-HT) suppresses 1-octanol Ca++ responses and potentiates depolarizations in the ASH in response to 1-octanol. This suggested that the net effect of 5-HT is disinhibitory. Here, I further dissect the pathway used by 5-HT to disinhibit the ASH and identify a Ca++-activated K+ channel known as SLO-1 acting downstream of Ca++. Furthermore, SLO-1 plays a critical role in regulating ASH Ca++ dynamics, with important consequences for aversive behavior and 5-HT modulation. Intriguingly, mutants lacking the SLO-1 accessory proteins DYB-1, BKIP-1 and ISLO-1 have similar phenotypes, suggesting that SLO-1 may need to be localized to specific Ca++ microdomains to properly modulate ASH responses. Finally, I show that other monoamines and neuropeptides effect the ASH Ca++ dynamics, suggesting that Ca++-dependent regulation of ASH signaling and excitability may be a central regulatory theme within the neuron. iii My dissertation is dedicated to my friends and family, specifically my mother and father Geraldine and Keith Williams whose outstanding love and support kept me strong and made this dream a reality. I also dedicate this dissertation to all the teachers and professors who guided me towards and down this path. Finally, to Drs. Bruce Bamber and Richard Komuniecki who made me into the scientist I am today. I thank you all. iv Acknowledgements I would like to thank Drs. Bruce Bamber and Richard Komuniecki for giving me this opportunity and their immense help, guidance and patience that was necessary for the completion of this project. Bruce, I thank you for giving me the opportunity to be a member of your lab and for your immense help, and Rick for your challenging nature to ensure I always stayed on course. I would also like to thank Amanda Korchnak for her aid in strain generation and help during my graduate career. I would also like to acknowledge members of the Bamber, Komuniecki and Steven labs: Dr. Jeff Zahratka, Dr. Robert Layne, Matt Rodenbeck, Jason Wanamaker, Hilary Linzie, Dr. Holly Mills, Dr. Vera Hapiak, Mitchell Oakes, Tobias Clark, Dr. Robert Steven and Alyssa Hoop for their input and help in all aspects of this project. Lastly, I would like to acknowledge my committee, Drs. Bruce Bamber, David Giovannucci, Richard Komuniecki, Guofa Liu, Scott Molitor and Robert Steven. v Table of Contents Abstract .............................................................................................................................. iii Acknowledgements ..............................................................................................................v Table of Contents ............................................................................................................... vi List of Figures .................................................................................................................... xi List of Tables ................................................................................................................... xiii List of Abbreviations ....................................................................................................... xiv List of Symbols .............................................................................................................. xviii 1 Introduction ....................................................................................................................1 1.1 Neuromodulation: A major Regulator of Wellbeing .........................................3 1.2 Monoamines and Neuropeptides Modulate Neuronal Activity .........................4 1.2.1 Serotonin ................................................................................................4 1.2.1.1 5-HT1 Receptors...............................................................................6 1.2.1.2 5-HT2 Receptors...............................................................................8 1.2.1.3 5-HT3, 5-HT4, 5-HT5, 5-HT6 and 5-HT7 Receptors .......................11 1.2.2 Other Neuromodulators .......................................................................14 1.2.2.1 Dopamine .......................................................................................14 1.2.2.2 Norepinephrine and Epinephrine ...................................................16 1.2.2.3 Neuropeptides ................................................................................17 vi 1.3 Calcium Imaging: A Powerful Technique to Measure Neuronal Activity in Intact Nervous Systems ..................................................................................................20 1.3.1 Fluorescent Dyes: Pioneers in Measuring Neuronal Activity..............22 1.3.2 FRET-based Ca++ Indicators ................................................................24 1.3.3 GCaMP: The Best Ca++ Indicator? ......................................................27 1.3.4 Voltage Sensor Proteins .......................................................................32 1.4 C. elegans: A Model Organism for Studying Neuromodulation ..........................35 1.4.1 The C. elegans Nervous System ..........................................................37 1.4.2 The ASH Sensory Neuron ...................................................................39 1.4.3 Monoamines Extensively Modulate ASH Mediated Behaviors in C. elegans .................................................................................................41 1.4.3.1 Serotonin ..................................................................................41 1.4.3.2 Dopamine .................................................................................43 1.4.3.3 Octopamine ..............................................................................44 1.4.3.4 Tyramine ..................................................................................46 1.5 Analyzing the Neuromodulation of Ca++ Signals in C. elegans ...........................49 1.5.1 Ca++ Channels in C. elegans ................................................................49 1.5.1.1 EGL-19 ....................................................................................50 1.5.2 Modulation of ASH Ca++ Signals by Monoamines .............................51 1.5.2.1 5-HT Modulates ASH Ca++ and Depolarization Signals Differentially ............................................................................52 2 Standard Materials and Methods .................................................................................57 vii 2.1 Strains and Worm Maintenance ...........................................................................57 2.2 RNA Interference .................................................................................................59 2.3 Behavioral Assays ................................................................................................60 2.4 Ca++-imaging ........................................................................................................61 2.5 Electrophysiology .................................................................................................64 2.6 Statistical Analysis ...............................................................................................65 3 Development of Innovative Techniques for Pharmacology and Imaging Studies in . C. elegans .........................................................................................................................67 3.1 Results ..................................................................................................................67 3.1.1 Worm Position Dictates Neuron Accessibility ....................................68 3.1.2 Plate Hydration Affects the Quality of Neuron Dissection .................70 3.1.3