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EAAT5 in the Rod Bipolar Cell and Oscillatory Potentials in Diabetes

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EAAT5 in the Rod Bipolar Cell and Oscillatory Potentials in Diabetes Washington University in St. Louis Washington University Open Scholarship Arts & Sciences Electronic Theses and Dissertations Arts & Sciences Spring 5-15-2020 Electrophysiology of the Inner Retina in Health and Disease: EAAT5 in the Rod Bipolar Cell and Oscillatory Potentials in Diabetes Gregory William Bligard Washington University in St. Louis Follow this and additional works at: https://openscholarship.wustl.edu/art_sci_etds Part of the Neuroscience and Neurobiology Commons, and the Ophthalmology Commons Recommended Citation Bligard, Gregory William, "Electrophysiology of the Inner Retina in Health and Disease: EAAT5 in the Rod Bipolar Cell and Oscillatory Potentials in Diabetes" (2020). Arts & Sciences Electronic Theses and Dissertations. 2168. https://openscholarship.wustl.edu/art_sci_etds/2168 This Dissertation is brought to you for free and open access by the Arts & Sciences at Washington University Open Scholarship. It has been accepted for inclusion in Arts & Sciences Electronic Theses and Dissertations by an authorized administrator of Washington University Open Scholarship. For more information, please contact [email protected]. WASHINGTON UNIVERSITY IN ST. LOUIS Division of Biology and Biomedical Sciences Neurosciences Dissertation Examination Committee: Peter D. Lukasiewicz, Chair Erik D. Herzog Timothy E. Holy Daniel Kerschensteiner Steven J. Mennerick Electrophysiology of the Inner Retina in Health and Disease: EAAT5 in the Rod Bipolar Cell and Oscillatory Potentials in Diabetes by Gregory W. Bligard A dissertation presented to The Graduate School of Washington University in partial fulfillment of the requirements for the degree of Doctor of Philosophy May 2020 St. Louis, Missouri © 2020, Gregory W. Bligard Table of Contents List of Figures ............................................................................................................................ iv Acknowledgments ..................................................................................................................... vi Abstract ..................................................................................................................................... ix Chapter 1: Background and Introduction ..................................................................................... 1 1.1 The Neural Circuitry of the Inner Retina ...................................................................... 1 1.2 Excitatory Amino Acid Transporters ........................................................................... 4 1.3 Electroretinography in Diabetic Retinopathy ............................................................... 6 1.4 Objectives of the Dissertation ...................................................................................... 7 Chapter 2: The Glutamate Transporter EAAT5 Mediates Rod Bipolar Cell Autoinhibition ......... 9 2.1 Background ................................................................................................................. 9 2.2 Materials and Methods .............................................................................................. 12 2.2.1 Animal Protocols .............................................................................................................. 12 2.2.2 Retinal Dissection ............................................................................................................. 13 2.2.3 Whole-Cell Recordings ..................................................................................................... 13 2.2.4 Morphological Identification ............................................................................................. 14 2.2.5 Solutions and Drugs .......................................................................................................... 14 2.2.6 Light Stimulation .............................................................................................................. 16 2.2.7 Data Analysis ................................................................................................................... 16 2.2.8 Modeling .......................................................................................................................... 16 2.3 Results ....................................................................................................................... 17 2.3.1 The Mode of Retinal Presynaptic Inhibition Does Not Switch with Light Intensity. ........... 17 2.3.2 GABA and Glycine Mediate Alloinhibition onto Rod Bipolar Cells. ................................. 20 2.3.3 EAAT5 Mediates Autoinhibition onto Rod Bipolar Cells. ................................................. 24 2.3.4 EAAT5 Inhibition Modulates Signaling to AII Amacrine Cells. ........................................ 31 2.4 Discussion ................................................................................................................. 35 Chapter 3: Functional Deficits Precede Structural Lesions in Diabetic Retinopathy ................... 40 3.1 Background ............................................................................................................... 40 3.2 Materials and Methods .............................................................................................. 45 3.2.1 Animals ............................................................................................................................ 45 3.2.2 Electroretinography .......................................................................................................... 46 ii 3.2.2 Statistical Analysis ........................................................................................................... 46 3.3 Results ....................................................................................................................... 47 3.4 Discussion ................................................................................................................. 56 Chapter 4: Summary and Future Directions ............................................................................... 59 4.1 EAAT5 ...................................................................................................................... 59 4.2 Diabetic Retinopathy ................................................................................................. 61 References ................................................................................................................................ 64 iii List of Figures Figure 1.1: The retina is a well-organized, laminar structure composed of five distinct classes of neurons. ...................................................................................................................................... 1 Figure 1.2: Rod pathways in the mouse retina utilize cone circuitry. ........................................... 3 Figure 1.3: EAATs are both glutamate transporters and chloride channels................................... 5 Figure 2.1: Conventional inhibition is mediated by a variety of sources..................................... 10 Figure 2.2: EAAT5-mediated presynaptic inhibition is gated by glutamate and has two potential sources ...................................................................................................................................... 12 Figure 2.3: Differential interference contrast photomicrograph of a pipette and rod bipolar cell (RBC) ....................................................................................................................................... 14 Figure 2.4: Example RBC IPSCs. .............................................................................................. 18 Figure 2.5: The mode of light-evoked inhibition to rod bipolar cells does not depend on light intensity. ................................................................................................................................... 19 Figure 2.6: Inhibition onto cone bipolar cells is exclusively conventional. ................................. 20 Figure 2.7: Cartoon depicting intracellular and puff application of receptor antagonists. ........... 21 Figure 2.8: Puff application of GABA/glycine blockers successfully blocks the light-evoked IPSC. ........................................................................................................................................ 22 Figure 2.9: Results of intracellular and puff application of receptor antagonists. ........................ 23 Figure 2.10: Intracellular application of TBOA blocks the response to glutamate. ..................... 24 Figure 2.11: Voltage pulse input. .............................................................................................. 25 Figure 2.12: Voltage pulse example traces with and without GABA/gly blockers...................... 26 Figure 2.13: Voltage pulse example traces with and without TBOA. ......................................... 27 Figure 2.14: EAAT5 is the primary mediator of autoinhibition in RBCs. ................................... 28 Figure 2.15: EAAT5-mediated autoinhibition is dependent on synaptic activation. ................... 29 Figure 2.16: EAAT5-mediated autoinhibition is dependent on glutamate release. ...................... 30 iv Figure 2.17: EAAT5-mediated autoinhibition is dependent on synaptic activation and glutamate release: Summary ...................................................................................................................... 31 Figure 2.18: Outputs of in-silico model of the RBC – AII amacrine
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