UC San Diego UC San Diego Electronic Theses and Dissertations Title Ultrastructure of Melanopsin-Expressing Retinal Ganglion Cell Circuitry in the Retina and Brain Regions that Mediate Light-Driven Behavior Permalink https://escholarship.org/uc/item/8kd9v9xp Author Liu, Yu Hsin Publication Date 2017 Peer reviewed|Thesis/dissertation eScholarship.org Powered by the California Digital Library University of California UNIVERSITY OF CALIFORNIA, SAN DIEGO Ultrastructure of Melanopsin-Expressing Retinal Ganglion Cell Circuitry in the Retina and Brain Regions that Mediate Light-Driven Behavior A dissertation submitted in partial satisfaction of the Requirements for the degree Doctor of Philosophy in Neurosciences by Yu Hsin Liu Committee in charge: Professor Satchidananda Panda, Chair Professor Mark Ellisman, Co-Chair Professor Nicola Allen Professor Brenda Bloodgood Professor Ed Callaway Professor David Welsh 2017 Copyright Yu Hsin Liu, 2017 All rights reserved. This Dissertation of Yu Hsin Liu is approved, and it is acceptable in quality and form for publication on microfilm and electronically: _________________________________________ _________________________________________ _________________________________________ _________________________________________ _________________________________________ Co-Chair _________________________________________ Chair University of California, San Diego 2017 iii TABLE OF CONTENTS Signature Page ..................................................................................................... iii Table of Contents ................................................................................................. iv List of Figures ...................................................................................................... vii Acknowledgements .............................................................................................. ix Vita ...................................................................................................................... xii Abstract of the Dissertation ................................................................................ xiii Chapter 1 General Introduction ........................................................................... 1 1.1 Retinal circuitry .......................................................................................... 2 1.2 Current state of knowledge regarding mRGC circuitry .............................. 7 1.2.1 mRGCs are essential for non-image forming visual processes ...... 7 1.2.2 mRGC subtypes ........................................................................... 10 1.2.3 mRGC intraretinal circuitry ........................................................... 12 1.2.4 mRGC interactions in mRGC-recipient brain regions ................... 13 1.3 References .............................................................................................. 17 Chapter 2 Ultrastructure of the mRGC terminal .................................................. 23 2.1 Abstract ................................................................................................... 23 2.2 Introduction ............................................................................................. 24 2.3 Materials and Methods ........................................................................... 28 Animals ................................................................................................. 28 Vector Construction ............................................................................... 28 Intravitreal Injection ............................................................................... 29 iv Circadian wheel running ........................................................................ 30 Immunohistochemistry and confocal microscopy imaging .................... 30 Tissue preparation for SBEM ................................................................ 31 SBEM staining and imaging .................................................................. 32 Electron microscope tomography .......................................................... 33 Manual segmentation ............................................................................ 33 Automatic segmentation ........................................................................ 35 2.4 Results .................................................................................................... 38 2.4.1 Vector-mediated expression of miniSOG in mRGCs .................... 38 2.4.2 Photooxidation of miniSOG reveals label amenable to SBEM ..... 39 2.4.3 MiniSOG label fills mRGC soma and processes .......................... 41 2.4.4 mRGC interactions in the OPN and SCN ..................................... 44 2.4.5 mRGC boutons in the OPN and SCN .......................................... 45 2.4.6 mRGC axons in OPN and SCN .................................................... 46 2.4.7 mRGC boutons in the LGN .......................................................... 47 2.5 Discussion ............................................................................................... 48 2.6 References .............................................................................................. 62 Chapter 3 Subtype- and Stratification-Specific Connections of mRGCs ............. 65 3.1 Abstract .................................................................................................. 65 3.2 Introduction ............................................................................................. 66 3.3 Materials and Methods ........................................................................... 69 Animals ................................................................................................. 69 Vector Construction ............................................................................... 69 v Intravitreal Injection ............................................................................... 69 Tissue preparation for SBEM ................................................................ 70 SBEM Staining and Imaging ................................................................. 71 Manual segmentation ............................................................................ 72 Automatic segmentation ........................................................................ 75 3.4 Results ..................................................................................................... 76 3.4.1 Retina volumes ........................................................................... 76 3.4.2 mRGC processes cover the majority of the retina surface area ... 78 3.4.3 Dendritic morphology of mRGC processes .................................. 79 3.4.4 Synaptic density of mRGCs ......................................................... 81 3.4.5 mRGC subtype determines intra-retinal interactions .................... 82 3.5 Discussion .............................................................................................. 84 3.5.1 Subtype- or stratification-specificity .............................................. 84 3.5.2 Pre-synaptic players in mRGC circuitry ........................................ 87 3.5.3 Limitations ................................................................................... 89 3.5.4 Auto-segmentation ....................................................................... 91 3.5.5 The past, present, and future ....................................................... 93 3.6 References ........................................................................................... 123 Chapter 4 Conclusion ...................................................................................... 127 4.1 References ........................................................................................... 136 vi LIST OF FIGURES Figure 1.1 Layers of the Retina .......................................................................... 15 Figure 1.2 Morphology of mRGC subtypes ........................................................ 16 Figure 2.1 Vector-mediated expression of miniSOG in mRGCs ......................... 51 Figure 2.2 Circadian photoentrainment is intact in miniSOG-injected mice ........ 52 Figure 2.3 Photooxidation of miniSOG-labeled tissues ...................................... 53 Figure 2.4 MiniSOG label fills mRGC soma ....................................................... 54 Figure 2.5 MiniSOG labels mRGC axons ........................................................... 55 Figure 2.6 Autosegmentation of miniSOG label ................................................. 56 Figure 2.7 mRGC interactions in the SCN and OPN .......................................... 57 Figure 2.8 Sample mRGC boutons in SCN and OPN ........................................ 58 Figure 2.9 mRGC boutons in SCN and OPN ..................................................... 59 Figure 2.10 mRGC axons in SCN and OPN ....................................................... 60 Figure 2.11 mRGC boutons in LGN.................................................................... 61 Figure 3.1 Retinal volumes used in this study .................................................... 97 Figure 3.2 Volume_1 .......................................................................................... 98 Figure 3.3 Volume_2 .......................................................................................... 98 Figure 3.4 Volume_3 ........................................................................................ 100 Figure 3.5