University of Nebraska Medical Center DigitalCommons@UNMC Theses & Dissertations Graduate Studies Summer 8-17-2018 Properties of Synaptic Transmission from Rods and Cones in The Outer Plexiform Layer of The Vertebrate Retina Xiangyi Wen University of Nebraska Medical Center Follow this and additional works at: https://digitalcommons.unmc.edu/etd Part of the Neurosciences Commons Recommended Citation Wen, Xiangyi, "Properties of Synaptic Transmission from Rods and Cones in The Outer Plexiform Layer of The Vertebrate Retina" (2018). Theses & Dissertations. 290. https://digitalcommons.unmc.edu/etd/290 This Dissertation is brought to you for free and open access by the Graduate Studies at DigitalCommons@UNMC. It has been accepted for inclusion in Theses & Dissertations by an authorized administrator of DigitalCommons@UNMC. For more information, please contact [email protected]. PROPERTIES OF SYNAPTIC TRANSMISSION FROM RODS AND CONES IN THE OUTER PLEXIFORM LAYER OF THE VERTEBRATE RETINA by Xiangyi Wen A DISSERTATION Presented to the Faculty of the University of Nebraska Graduate College in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy Pharmacology & Experimental Neuroscience Graduate Program Under the Supervision of Professor Wallace B. Thoreson University of Nebraska Medical Center Omaha, NE August, 2018 Supervisory Committee: Wallace B. Thoreson, Ph.D. Woo-Yang Kim, Ph.D. Anna Dunaevsky, Ph.D. George Rozanski, Ph.D. Huangui Xiong, M.D. Ph.D. This work is dedicated to my family. i Acknowledgements I would like to thank my mentor Dr. Wallace (Wally) B. Thoreson for his careful instruction and support throughout my graduate study. Wally patiently answered any questions about science and non-science, discussed and designed detailed steps with me. His precise attitude to science guided me into the world of science and my future career. I would also like to thank my dissertation committee: Dr. Woo-Yang Kim, Ph.D., Dr. Anna Dunaevsky, Ph.D., Dr. George Rozanski, Ph.D., and Dr. Huangui Xiong, M.D. Ph.D. for their advice and support with my thesis project. I would like to thank all the laboratory members who directly or indirectly helped me to finish my work. In particular, I would like to thank Dr. Matthew (Matt) J. Van Hook for his great help in experiments and especially in learning new techniques. He is a great teacher for electrophysiological techniques and helped me to be familiar with the environment when I joined our lab. He also contributed to some of the data shown in Figure 8. I would like to thank Dr. Minghui Chen and Grant Saltzgaber for their kind help with TIRFM experiments. I would also like to thank Dr. Karlene Cork, Dr. Ted Warren, Justin Grassmeyer, Cody Barta, Asia Cahill, and Cassandra Hays for discussion and support in helping to obtain my degree. I would like to thank Dr. Scott Nawy from the Department of Ophthalmology & Visual Sciences for discussion and suggestions for my projects. I would like to thank Tom Bargar from the Electron Microscopy Core Facility (EMCF) for his help with electron microscopy imaging in Figures 2 and 17. I am grateful to the late Dr. Richard Hallworth from Creighton University for assistance with the FRAP experiments in Figure 3. ii I would also like to thank staff members within the Departments of Ophthalmology & Visual Sciences and Pharmacology & Experimental Neuroscience for their kind help with ordering and other paperwork. Finally, I want to thank my family for their support for my years of education. iii Table of Contents Acknowledgements ........................................................................................................ i Abstract .......................................................................................................................... 1 Chapter 1 Introduction .................................................................................................. 4 I. Retinal Anatomy .................................................................................................. 4 II. Photoreceptor anatomy ........................................................................................ 6 a. Photoreceptors .................................................................................................. 6 b. Ultrastructure of photoreceptor ribbon synapses ............................................... 7 c. Proteins at the ribbon synapse ........................................................................ 11 III. Phototransduction ...........................................................................................14 a. Rhodopsin cascade ......................................................................................... 14 b. Cone opsins and their spectral sensitivity ........................................................ 16 IV. Synaptic release from photoreceptors .............................................................17 a. Ribbon release ................................................................................................ 17 b. Exocytosis and endocytosis ............................................................................ 21 c. CICR and non-ribbon release from rods .......................................................... 24 V. Retinal circuits ....................................................................................................26 VI. Lateral feedback signals in the OPL ................................................................30 Chapter 2 Endocytosis sustains release at photoreceptor ribbon synapses by restoring fusion competence...................................................................................... 40 I. Abstract: .............................................................................................................40 II. Introduction: ........................................................................................................40 III. Materials and Methods ....................................................................................43 a. Animal care and use ........................................................................................ 43 iv b. Retinal slices ................................................................................................... 43 c. Patch-clamp electrophysiology ........................................................................ 44 d. Reagents ......................................................................................................... 45 e. Photoreceptor isolation .................................................................................... 46 f. TIRFM experiments ......................................................................................... 46 g. Statistical Analysis ........................................................................................... 49 IV. Results ............................................................................................................49 a. Paired pulse depression is extended by inhibiting endocytosis ........................ 49 b. TIRFM imaging of single vesicles .................................................................... 55 c. Impact of dynasore on ribbon-mediated release visualized by TIRFM ............. 64 V. Discussion ..........................................................................................................70 Chapter 3 Kiss-and-run is a significant contributor to synaptic exocytosis and endocytosis in photoreceptors .................................................................................. 78 I. Abstract: .............................................................................................................78 II. Introduction: ........................................................................................................79 III. Materials and Methods: ...................................................................................81 a. Animal care and use ........................................................................................ 81 b. Reagents ......................................................................................................... 81 c. Photoreceptor isolation .................................................................................... 82 d. Capacitance measurements of endocytosis .................................................... 82 e. Whole-terminal fluorescence measurements ................................................... 84 f. In vitro dye fluorescence measurements ......................................................... 85 g. Dye release measurements in retinal slices ..................................................... 85 v h. Electron microscopy ........................................................................................ 86 i. TIRFM experiments ......................................................................................... 87 j. Statistical Analysis ........................................................................................... 89 IV. Results: ...........................................................................................................89 a. Rapid endocytosis in photoreceptors. .............................................................. 89 b. Small dyes were loaded preferentially into synaptic terminals. ........................ 92 c. Dyes were loaded into synaptic vesicles.......................................................... 96 d. Impact of endocytosis inhibitors on uptake of large and small dyes ................