University of Louisville ThinkIR: The University of Louisville's Institutional Repository Electronic Theses and Dissertations 5-2019 Glycine receptor expression across identified etinalr ganglion cell types. Ian Scot Pyle University of Louisville Follow this and additional works at: https://ir.library.louisville.edu/etd Part of the Medicine and Health Sciences Commons, Molecular and Cellular Neuroscience Commons, Molecular Biology Commons, and the Other Neuroscience and Neurobiology Commons Recommended Citation Pyle, Ian Scot, "Glycine receptor expression across identified retinal ganglion cell types." (2019). Electronic Theses and Dissertations. Paper 3187. https://doi.org/10.18297/etd/3187 This Doctoral Dissertation is brought to you for free and open access by ThinkIR: The University of Louisville's Institutional Repository. It has been accepted for inclusion in Electronic Theses and Dissertations by an authorized administrator of ThinkIR: The University of Louisville's Institutional Repository. This title appears here courtesy of the author, who has retained all other copyrights. For more information, please contact [email protected]. GLYCINE RECEPTOR EXPRESSION ACROSS IDENTIFIED RETINAL GANGLION CELL TYPES By Ian Scot Pyle B.A., Hanover College (Hanover, IN), 2008 M.S., University of Louisville (Louisville, KY), 2013 M.S., University of Louisville (Louisville, KY), 2016 A Dissertation Submitted to the Faculty of the School of Medicine of the University of Louisville in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy in Anatomical Sciences and Neurobiology Department of Anatomical Sciences and Neurobiology University of Louisville School of Medicine Louisville, KY May 2019 Copyright 2019 by Ian Scot Pyle All rights reserved GLYCINE RECEPTOR EXPRESSION ACROSS IDENTIFIED RETINAL GANGLION CELL TYPES By Ian Scot Pyle B.A., Hanover College (Hanover, IN), 2008 M.S., University of Louisville (Louisville, KY), 2013 M.S., University of Louisville (Louisville, KY), 2016 A Dissertation Approved on April 9, 2019 by the following Dissertation Committee: _________________________________________ Maureen McCall, Ph.D. _________________________________________ Bart Borghuis, Ph.D. _________________________________________ Ron Gregg, Ph.D. _________________________________________ William Guido, Ph.D. _________________________________________ Jeffrey Petruska, Ph.D. ii DEDICATION I dedicate this dissertation to my two lovely daughters Ella and Lilia and to my beautiful wife Miki whose unconditional love and fervent support was my reprieve during this time in my life. iii ACKNOWLEDGMENTS Foremost, I thank my mentor, Dr. Maureen McCall. She is the epitome of work ethic; a passionate and professional scientist whose wisdom and attention to detail is consistent while she leads by example and demonstrates the most diligent work hours among her students and colleagues. Intentionally or unintentionally, Maureen constantly pushed me to think harder, work smarter, be more skeptical, and trust my data. Also, my research and success were only possible with help from: Drs. Gobinda Pangeni, Chi Zhang, James Fransen, Katie Fransen, Jennifer Noel, Archana Jalligampala, Aaron Rising, Nazarul Hasan, Sean Masterson, Rui Ji and Ms. Madeline Mitchell and Ms. Maha Jabbar. Furthermore, I thank my committee members, Drs. Bart Borghuis, Ron Gregg, Bill Guido, and Jeffrey Petruska. I thank my parents for their love and support. I would not be successful today if it weren’t for their philosophy of raising a lifelong learner and assert independence. They were gracious in providing me with exceptional educational opportunities both in the classroom and via traveling the world. Finally, I thank my beautiful, loving and gracious wife, Miki. The journey of graduate school is challenging, arduous, time-consuming, and outright brutal. Miki supported me during this time with unconditional love while simultaneously raising our two daughters Ella and Lilia, a feat unsurpassed by any human. Ultimately, both of us accomplished this journey together, and I will forever be in her gratitude. iv ABSTRACT GLYCINE RECEPTOR EXPRESSION ACROSS IDENTIFIED RETINAL GANGLION CELL TYPES Ian Scot Pyle April 9, 2019 Retinal ganglion cells (RGCs) represent the culmination of all retinal signaling and their output forms the substrate for vision throughout the rest of the brain. About 40 different RGC types have been defined by differences in their visually evoked responses, morphology, and genetic makeup. These responses arise from interactions between inhibition and excitation throughout the retinal circuit (Franke et al., 2017; Masland, 2012; Sanes & Masland, 2015; Werblin, 2011). Unlike most other areas of the central nervous system (CNS), the retina utilizes both GABA and glycine inhibitory neurotransmitters to refine glutamatergic excitatory signals (Franke & Baden, 2017; Werblin, 2011; C. Zhang, Nobles, & McCall, 2015). Glycine receptors (GlyRs) are heteromers composed of a single β subunit and one of four α subunits, with a stoichiometry of 3β:2α (Grudzinska et al., 2005; Heinze, Harvey, Haverkamp, & Wassle, 2007; Lynch, 2004). All four GlyRα subunits (α1, α2, α3, or α4) are differentially expressed in the retina and subunit specific expression has been defined for bipolar, some amacrine cells and v RGCs (Haverkamp, Muller, Zeilhofer, Harvey, & Wassle, 2004; Heinze et al., 2007). The roles for GlyRα subunit specific inhibition are unknown, although glycinergic input is generally linked to temporal response tuning (Murphy & Rieke, 2006; Nobles, Zhang, Muller, Betz, & McCall, 2012; van Wyk, Wassle, & Taylor, 2009; Wassle et al., 2009; Werblin, 2010). We have surveyed GlyRα subunit expression in a variety of identified RGC types, using GlyRα knockout mice and an rAAV-mediated RNAi to knockdown GlyRα subunit specific expression. We find that the four α RGCs only express GlyRα1. All of the other RGCs we studied express at least two GlyRα subunits. In some RGCs, the GlyR kinetics are similar, whereas in others the kinetics differs. We propose that this diversity will contribute to the richness of retinal inhibitory processing. vi TABLE OF CONTENTS ABSTRACT .......................................................................................................... v LIST OF FIGURES ...............................................................................................ix LIST OF TABLES .................................................................................................xi CHAPTER I .......................................................................................................... 1 INTRODUCTION .................................................................................................. 1 1.1 The Visual system .................................................................................... 1 1.2 The Retina ............................................................................................... 2 1.3 Photoreceptors ......................................................................................... 4 1.4 Photoisomerization................................................................................... 5 1.5 Bipolar Cells ............................................................................................. 6 1.6 Horizontal Cells ........................................................................................ 8 1.7 Amacrine Cells ......................................................................................... 8 1.8 Inhibitory Circuits ..................................................................................... 9 1.9 Retinal Ganglion Cells ............................................................................ 12 1.10 Neurotransmission ............................................................................... 13 1.11 Synaptic Release ................................................................................. 14 1.12 Inhibitory Receptors in the Retina ........................................................ 16 1.13 Glycine Receptors ................................................................................ 17 1.14 Glycine Receptor Kinetics .................................................................... 19 1.15 Differential Expression of GlyRα Subunits in the IPL of the retina ....... 22 II. SPECIFIC AIMS .......................................................................................... 24 Aim 1 – Identify Glycinergic Expression on a Subset of Retinal Ganglion Cells ............................................................................................................. 25 Aim 2 - Examine the GlyRα Expression of ooDS RGCs using an rAAV- Glra4shRNA ................................................................................................. 26 CHAPTER II ....................................................................................................... 27 EXPERIMENTAL MATERIALS AND METHODS ............................................... 27 2.1 Animals .................................................................................................. 27 vii 2.2 Viral Vector Construction and Packaging ............................................... 29 2.3 rAAV injection into Major Optic Nerve Targets of the Brain. ................... 32 2.4 Tissue Dissection and Preparation ........................................................ 33 2.5 Electrophysiology Recording of Retinal Ganglion Cells ......................... 34 2.6 Pharmacological