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Ital: ind-1.13“ u , . .5- . 7!...21/111 . 7.“ 3.13. 3.. a: . 1.3:; This is to certify that the dissertation entitled PHENOTYPIC CHARACTERIZATION OF PROGRESSIVE RETINAL ATROPHY IN THE CARDIGAN WELSH CORGI WITH A MUTATION IN THE PDE6A GENE presented by NALINEE TUNTIVANICH has been accepted towards fulfillment of the requirements for the Doctoral degree in Comparative Medicine and Integrative Biology Maifi Professor’s Signature August, 2006 Date MSU is an Affirmative Action/Equal Opportunity Institution LIBRARY Michigan State University PLACE IN RETURN BOX to remove this checkout from your record. TO AVOID FINES return on or before date due. MAY BE RECALLED with earlier due date if requested. DATE DUE DATE DUE DATE DUE 2/05 p:/C|RC/DateDue.indd-p.1 PHENOTYPIC CHARACTERIZATION OF PROGRESSIVE RETINAL ATROPHY IN THE CARDIGAN WELSH CORGI WITH A MUTATION IN THE PDE6A GENE By Nalinee Tuntivanich A DISSERTATION Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Comparative Medicine and Integrative Biology 2006 ABSTRACT PHENOTYPIC CHARACTERIZATION OF PROGRESSIVE RETINAL ATROPHY IN THE CARDIGAN WELSH CORGI WITH A MUTATION IN THE PDE6A GENE By Nalinee Tuntivanich The purpose of this study was to perform a detail characterization of the electroretinographic and morphological aspects of progressive retinal atrophy (PRA) in the Cardigan Welsh corgi (CWC) due to a mutation in the gene encoding the alpha subunit of the rod cyclic GMP phosphodiesterase (PDE6A), and to investigate expression of the canine PDE6A gene in other tissues. Homozygous PDE6A mutant, heterozygous PDE6A carrier, and homozygous PDE6A normal dogs were used in this study. Short flash and long flash electroretinograms (ERG) were recorded as well as pharmacological dissections of the abnormal ERG responses of the PDE6A mutant dogs. Morphological characterization of the mutant retina was performed by light and electron microscopy. Retinal cell counting and layer thickness measurement were performed in addition to immunohistochemical staining using specific cell markers. The PDE6A mutant dogs had very reduced scotopic ERG responses with elevated amplitude threshold at all ages investigated, starting from 2 weeks of age. Qualitative analysis of the leading edge of the rod-mediated a—wave showed absent or substantially reduced and delayed rod response with loss of sensitivity. This concurred with results from the Naka-Rushton analysis. Application of 2-amino-4-phosphonobutyric acid (APB) to block ON-bipolar activity failed to enhance the a-wave from the mutant dog at 6 weeks of age strongly suggesting a severe loss of photoreceptor response prior to retinal maturation. Reduction of photopic a-wave response with elevated amplitude threshold present from 3 weeks of age onwards indicated an initial development of cone responses followed by a gradual deterioration. Flicker and long flash ERGs also showed a decrease in amplitudes indicating decreased responses from inner retinal cells. Early morphological changes were present in the rod outer segments (083) at 3 weeks of age. By around 4 weeks of age there was evidence of apoptotic cell death affecting the rod photoreceptor cells. Reduction of the OS layer thickness was shown at 5 weeks of age, at which age a loss of rod 085 was shown by rhodopsin immunohistochemistry. Significant loss of cone 085 was apparent by 7 weeks of age, demonstrated by red/cone opsin staining, with the remaining inner segments (IS) stunted. At this age, there was a notable decrease in the number of photoreceptor nuclei row and number of rod nuclei per unit length of the retina while the number of cones was maintained. By 60 days of age only one row of photoreceptor nuclei remained in the mutant retina. Morphological alterations were also observed in rod bipolar cells, horizontal cells and Mt‘rller cells. Expression of PDE6A amplicons by RT-PCR was not only found in normal control retina but also in iris, choroid, pituitary gland, pineal gland, normal and mutant kidney, normal and mutant small intestine and mutant retina at 5 weeks of age. The PDE6A transcript in the kidney appeared to be shorter than that from the retina and in the mutant dog an alternatively spliced transcript was found that had a skipped exon. The exon skipping had the result that the premature termination codon was excluded and the reading frame restored. Further investigation would be required to see if the predicted shortened PDE6A product is translated in kidney and to investigate its possible function. My deepest appreciation is extended to my family for their support, patience, love and understanding shown to me throughout this endeavor. To my father, Mr. Vorasak Tuntivanich my mother, Professor Pranee Tuntivanich my sister, Miss Vareemon Tuntivanich my husband, Mr. Kittipob Smitalamba iv ACKNOWLEDGMENTS I wish to acknowledge those who have contributed to my completion of this milestone in my professional career. I would like to express my sincere appreciation to my committee members: Dr Arthur Weber, Dr John Fyfe, Dr Matti Kiupel and Dr James Render. I am deeply grateful for their kind guidance. I sincerely appreciate the support from the Thai government, Chulalongkom University and the faculty of Veterinary science, Chulalongkorn University, Thailand, in particular, the department of Surgery. I am thankful for the support from the administrative people in the Comparative Medicine and Integrative Biology program; Dr Vilma Yuzbasiyan-Gurkan, Victoria Hoelzer-Maddox, collaborators; Naheed Khan, Andy Fischer, technicians; Ralph Common, Thomas Wood, Scott Marsh, Nicole Grosjean, Dionne Rodgers, Christine Harman and Walter Bobrowski. I am appreciative to Dr Cheri Johnson and Lisa Allen for taking care of the dog colony at the MSU Vivarium, Michelle Curcio for molecular guidance, Juan Steibel for statistical consultance, Dr Paul Sieving for ERG consultance, people from comparative ophthalmology laboratory and staffs of the department of Small Animal Clinical Sciences, College of Veterinary Medicine, MSU for their supports. Thanks to friends, the Thai Student Association at MSU and others whose names are not mentioned but their supports are unforgettable. I would like to acknowledge the funding sources of this project. They include Companion Animal Funds, MSU, Purebred Dog Endowment Fund, MSU, MidWest Eye Banks, Fight for Sight and National Institutes of Health. Finally, I would like to give a special acknowledgment to Dr Simon Petersen-Jones who has served as my advisor. His encouragement, constant support and kindness are not only needed for me to finally bring this dissertation to completion but also broaden my scientific experience. I am sincerely grateful for his significant role in this phase of my life. vi TABLE OF CONTENTS LIST OF TABLES ........................................................................................................... xiii LIST OF FIGURES ......................................................................................................... xiv CHAPTER 1 INTRODUCTION 1.1. Ocular embryology .............................................................................................. 1 1.2. Ocular anatomy .................................................................................................... 4 1.2.1. Retinal pigment epithelium ......................................................................... 7 1.2.2. Photoreceptor layer ..................................................................................... 8 1.2.3. Outer limiting membrane .......................................................................... 11 1.2.4. Outer nuclear layer .................................................................................... 12 1.2.5. Outer plexiform layer ................................................................................ 12 1.2.6. Inner nuclear layer .................................................................................... 14 1.2.6.a. Amacrine cells ............................................................................ 15 1.2.6.b. Bipolar cells ................................................................................ 16 l.2.6.e. Mtiller cells ................................................................................ 17 1.2.6.d. Horizontal cells ........................................................................... 17 l.2.6.e. Interplexiform cells ..................................................................... 18 1.2.7. Inner plexiform layer ................................................................................ 19 1.2.8. Ganglion cell layer .................................................................................... 19 1.2.9. Nerve fiber layer ....................................................................................... 21 1.2.10. Inner limiting membrane ........................................................................... 21 1.3. The Central visual pathway. .............................................................................. 22 1.4. The phototransduction pathway and visual cycle .............................................. 24 1.5. Electroretinography ............................................................................................ 30 1.6. Inherited retinal dystrophies in humans ............................................................. 37 1.7. Inherited retinal degenerations in dogs and cats ................................................. 39 1.8. Laboratory animal models for inherited retinal dystrophies .............................. 50 CHAPTER 2 DETAILED ELECTRORETINOGRAPHIC CHARACTERIZATION OF THE PDE6A DOG PHENOTYPE 2.1. Introduction ........................................................................................................ 53 2.2. Materials and methods 2.2.1. Animals ...................................................................................................... 54 2.2.2. Electroretinographic recording 2.2.2.a. Anesthesia .................................................................................. 54 2.2.2.b. Recording electrode placement and animal positioning ............ 55 2.2.2.c. Electroretinographic recording .................................................. 56 vii 2.2.3. Electroretinographic test protocols 2.2.3.3. Intensity-series electroretinography 2.2.3.a.(i) Scotopic intensity-series electroretinography .......... 56 2.2.3.a.(ii) Photopic intensity-series electroretinography ............ 57 2.2.3.b. Flicker electroretinography 2.2.3.b.(i). Rod flicker ERG ........................................................ 58 2.2.3.b.(ii). Cone flicker ERG ...................................................... 58 2.2.3.c. Electroretinography using blue flashes ....................................... 58 2.2.3.d. Long flash electroretinography ................................................... 58 2.2.4. Data analysis 2.2.4.a. The mean a- and b-wave amplitude and implicit time ............... S9 2.2.4.b. Flicker amplitude and implicit time ............................................ 60 2.2.4.c. Analysis of by using Naka-Rushton function equation ............. 60 2.2.4.d. Criterion threshold of means of ERG scotopic and photopic amplitudes ................................................................... 61 2.2.4.e. Rod-isolated responses .............................................................. 62 2.2.4.f. Oscillatory potentials ................................................................. 62 2.2.4. g. Photopic Negative Response ..................................................... 63 2.3. Results 2.3.1. Scotopic ERG responses 2.3.1.a. Scotopic ERG amplitude ........................................................... 64 2.3.1.b. Scotopic ERG implicit time ....................................................... 71 2.3.1.c. Rod-isolated a-wave response ................................................... 74 2.3.1.d. Scotopic ERG responses to blue light flashes ........................... 76 2.3.1 .e. Naka-Rushton fitting of the first limb of the scotopic intensity-response curves ............................................................ 77 2.3.1.f. Rod flicker ERG response .......................................................... 81 2.3. 1 . g. Oscillatory potentials .................................................................. 83 2.3.2. Photopic ERG responses 2.3.2.a. Photopic a- and b-wave amplitudes ........................................... 85 2.3.2.b. Photopic ERG implicit time ....................................................... 91 2.3.2.c. Cone flicker ERG response ........................................................ 94 2.3.2.d. Photopic negative response ........................................................ 97 2.3.2.c. Long flash ERG response .......................................................... 98 2.4. Discussion ........................................................................................................ 101 CHAPTER 3 PHARMACOLOGICAL DISSECTION OF THE PDE6A ELECTRORETINOGRAM 3.1. Introduction ...................................................................................................... 106 3.2. Materials and methods 3.2.1. Animals ................................................................................................... 107 3.2.2. Pharmacological agents .......................................................................... 107 3.2.3. Intravitreal injection ................................................................................ 108 ‘ 3.2.4. Electroretinographic recording 3.2.4.a. Anesthesia and recording electrode placement ........................ 108 viii 3.2.5. 3.2.4.b. Electroretinographic recording ................................................ 109 3.2.4.c. Electroretinographic test protocols 3.2.4.c (i). Scotopic and photopic short flash electroretinography ................................................ 109 3.2.4.c (ii). Flicker electroretinography .................................... 110 3.2.4.c (iii). Long flash electroretinography .............................. 110 Data analysis ............................................................................................ 110 3.3. Results 3.3.1. Results afier the intravitreal injection of APB 3.3.1.a. Short flash scotopic and photopic ERG intensity~series responses from normal control dogs ........................................ 111 3.3.1.b. Cone flicker responses ............................................................. 115 3.3.1.c. Long flash ERG responses ....................................................... 116 3.3.2. Results afier the intravitreal injection of FDA 3.3.2.a. ERG photopic intensity-series response .................................. 120 3.3.2.b. Cone flicker response ............................................................... 122 3.3.2.c. Long flash ERG response ........................................................ 124 3.3.3. Results after the intravitreal injection of tetrodotoxin 3.3.3.a. Photopic ERG response ........................................................... 127 3.3.3.b. Cone flicker response ............................................................... 127 3.4. Discussion .......................................................................................................... 130 CHAPTER 4 DETAILED HISTOPATHOLOGICAL CHARACTERIZATION OF THE PDE6A DOG PHENOTYPE 4.1. Introduction ...................................................................................................... 136 4.2. Materials and methods 4.2.1. Morphological analyses by light microscopy 4.2.1.a. Tissue collection and processing ............................................. 137 4.2.1.b. Data collection ......................................................................... 140 4.2.1.c. Data analysis ............................................................................ 142 4.2.2. Morphological analyses by transmission electron microscopy 4.2.2.a. Tissue collection and processing ............................................. 142 4.2.2.b. Data analysis ............................................................................ 143 4.2.3. Immunohistochemical analyses of paraffin-embedded sections 4.2.3.a. Tissue collection and processing ............................................. 143 4.2.3.b. Data analysis ............................................................................. 146 4.2.4. Immunohistochemical analyses of frozen sections 4.2.4.a. Tissue collection ...................................................................... 147 4.2.4.b. Data analysis ............................................................................. 149 4.2.5. Terminal Deoxynucleotidyl Transferase-mediated dUTP Nick End Labeling (TUNEL) staining 4.2.5.a. Tissue collection and processing .............................................. 150 4.2.5.b Data analysis ............................................................................. 150 4.3. Results ix 4.3.1. Morphological assessment of the retina by light and electron microscopy 4.3.1.a. Retinal sections at 3 to 10 days of age ...................................... 151 4.3.l.b. Retinal sections at 2 to 3 weeks of age .................................... 153 4.3.1.c. Retinal sections at 4 to 5 weeks of age .................................... 162 4.3.1 .d. Retinal sections at 7 to 12 weeks of age .................................. 170 4.3.1.c. Retinal sections at 16 weeks of age ......................................... 177 4.3.1.f. Retinal sections at 24 weeks of age .......................................... 188 4.3.1. g. Retinal sections at age greater than 60 weeks ........................... 193 4.3.2. Immunohistochemical analyses of paraffin-embedded and frozen sections 4.3.2.a. Immunohistochemistry using rhodopsin ................................... 197 4.3.2.b. Immunohistochemistry using cone arrestin ............................. 201 4.3.2.0. Immunohistochemistry using red/ green opsin ......................... 203 4.3.2.d. Immunohistochemistry using PKCa ....................................... 205 4.3.2.e. Immunohistochemistry using calbindin .................................... 207 4.3.2.f. Immunohistochemistry using Hu C/D ...................................... 208 4.3.2. g. Immunohistochemistry using calretinin ................................... 209 4.3.2.b. Immunohistochemistry using GFAP ........................................ 211 4.4. Discussion ........................................................................................................ 213 CHAPTER 5 INVESTIGATION OF THE TISSUE EXPRESSION OF THE PDE6A GENE 5.1. Introduction ...................................................................................................... 221 5.2. Materials and Methods 5.2.1. 5.2.2. 5.2.3. 5.2.4. 5.2.5. 5.2.6. Tissue collection ..................................................................................... 222 Isolation of total RNA 5.2.2.a. Preparation of instruments and RNA handling ......................... 223 5.2.2.b. Isolation of total RNA using RNeasy Mini Kit ....................... 224 5.2.2.c. Visualization of total RNA for integrity using a formaldehyde gel 5.2.2.c.(i). Preparation of materials ......................................... 225 5.2.2.c.(ii). Formaldehyde (FA) gel electrophoresis ................. 225 5.2.2.c.(iii). Analysis of total RNA by FA gel electrophoresis ........................................... 225 Spectrophotometry of RNA ..................................................................... 226 Reverse Transcription-Polymerase Chain Reaction 5.2.4.a. Synthesis of cDNA .................................................................. 226 5.2.4.b. Polymerase Chain Reaction (PCR) for PDE6A gene expression ................................................................................ 227 Sequencing of PCR products 5.2.5.a. DNA preparation ....................................................................... 229 5.2.5.b. DNA sequencing ....................................................................... 229 Rapid Amplification of cDNA Ends (RACE) to determine full- length sequence of kidney transcript 5.2.6.a. Dephosphorylation of RNA ..................................................... 230 5.2.6.b. Removal of the mRNA cap structure ........................................ 230 5.2.6.0. Ligation of the RNA oligo to de-capped mRNA ..................... 231 5.2.6.d. Reverse transcription of mRNA .............................................. 231 5.2.6.e. Amplification of cDNA ends ................................................... 232 5.2.7. Cloning of PCR products 5.2.7.a. Cloning reaction ........................................................................ 234 5.2.7.b. Transformation of competent cells .......................................... 234 5.2.7.c. Analysis transformed clones .................................................... 235 5.2.8. Northern hybridization 5.2.8.a. Preparation of FA gel for electrophoresis ................................. 235 5.2.8.b. FA gel electrophoresis 5.2.8.b.(i). Preparation of RNA samples and RNA size marker ..................................................................... 236 5.2.8.b.(ii). FA gel electrophoresis ........................................... 236 5.2.8.c. Preparation for an upward capillary transfer 5.2.8.c.(i). Preparation of FA gel ............................................. 237 5.2.8.c.(ii). Preparation of nylon membrane, wicked papers and blotting papers ...................................... 237 5.2.8.c.(iii). Assembly of the capillary transfer system ............. 237 5.2.8.c.(iv). Fixation of the transferred RNA to the membrane ................................................................ 239 5.2.8.d. Northern hybridization 5.2.8.d.(i). Preparation of the DNA probe ............................... 240 5.2.8.d.(ii). Prehybridization ..................................................... 241 5.2.8.d.(iii). Hybridization ......................................................... 241 5.2.8.d.(iv). Washing of the membrane ..................................... 242 5.2.8.c. Exposure of the nylon membrane to phosphor-imager ............ 242 5.3. Results 5.3.1. Expression of PDE6A mRNA in canine tissues 5.3.1.a. Investigation of PDE6A expression by RT-PCR ..................... 242 5.3.1 .b. Investigation of kidney transcript of the normal control .......... 249 5.3.1.c. Investigation of PDE6A expression by northern blot .............. 255 5.4. Discussion ........................................................................................................ 257 CHAPTER 6 FINAL DISCUSSION & CONCLUSIONS ................................................................... 262 CHAPTER 7 FUTURE STUDIES 7.1. Investigation of retinal PDE activity and cGMP levels 7.1.1. Background and Hypothesis ................................................................... 268 7.1.2. Methods 7.1 .2.a. Investigation of PDE activity of the retina 7.1.2.a.(i) I-IPLC analysis ......................................................... 269 . 7.1.2.a.(ii) Western blot analysis ............................................... 269 7.1.2.b. Investigation of cGMP level of the retina ................................ 270 xi 7.2. Assessment of canine cone-specific electroretinography 7.2.1. Background and Hypothesis .................................................................... 270 7.2.2. Methods .................................................................................................. 271 7.2.2.a. ERG assessment of S cone function ........................................ 272 7.2.2.b. ERG assessment of M cone function ....................................... 272 7.3. Investigation of the distribution of canine cone photoreceptors and the pattern of cell loss 7.3.1. Background and Hypothesis .................................................................... 272 7.3.2. Methods .................................................................................................. 273 APPENDIX ..................................................................................................................... 276 BIBLIOGRAPHY ............................................................................................................ 286 xii Table 1.1. Table 4.1. Table 4.2. LIST OF TABLES Summary of the different types of progressive retinal atrophy (PRA) in dogs and cats ......................................................................... 41-42 A list of the primary antibodies, their working dilution and source that are used in paraffin-embedded sections ............................................ 146 A list of the primary antibodies, their working dilution and source that are used in frozen sections ................................................................. 149 xiii LIST OF FIGURES Figure 1.1. Human ocular embryology ........................................................................... 2 Figure 1.2. Cross section of canine retina ....................................................................... 7 Figure 1.3. (A) Schematic drawing of structural elements of rod and cone and (B) A Differential Interference Contrast image of canine central retina from a flat mount preparation focused at the level of the inner segments (magnification 100X) ........................................................... 9 Figure 1.4. Histological section showing canine photoreceptors at 3 (A) and 10 (B) days of age ...................................................................................... 11 Figure 1.5. A simple diagram of the organization of the retina ..................................... 14 Figure 1.6. A schematic diagram demonstrating the central projection of the retina to subcortical regions of the brain as to the visual cortex ................ 22 Figure 1.7. A simplified diagram of the rod phototransduction cascade and visual cycle ................................................................................................. 26 Figure 1.8. Diagram of electrical components (P1, P11 and P111) recorded in the retina as described by Granit ................................................................ 30 Figure 1.9. Canine clinical ERG recordings as recommended by the ERG committee of the ECVO ............................................................................. 32 Figure 1.10. Components of canine scotopic full-field intensity-series ERG recorded after 1 hour of dark adaptation .................................................... 35 Figure 1.11. A canine long flash ERG recorded after 10 minutes of light adaptation ................................................................................................... 37 Figure 1.12. F undus photography of a normal control (A) and a Cargian Welsh corgi (B) with progressive retinal atrophy at 18 weeks of age ................... 43 Figure 1.13. A diagram demonstrating sequences of the PDE6A transcript and amino acid of the normal (A) and mutant (B) alleles ................................. 49 xiv Figure 2.1. Figure 2.2. Figure 2.3. Figure 2.4. Figure 2.5. Figure 2.6. Figure 2.7. Figure 2.8. Figure 2.9. Figure 2.10. Figure 2.11. Representative scotopic ERG recordings from a normal control (A) and the PDE6A mutant dog (B) at 4 weeks of age ............................... 66 Representative scotopic ERG recordings from a normal control (A) and the PDE6A mutant dog (B) at 6 weeks of age .............................. 67 Mean scotopic a-wave amplitudes with standard errors (1x +/- SEM) plotted against stimulus intensity on a log-log scale for normal control and PDE6A mutant puppies ............................................... 68 Mean scotopic b-wave amplitudes with standard errors (1x +/- SEM) plotted against stimulus intensity on a log-log scale from normal control and PDE 6A mutant puppies ............................................... 69 Mean scotopic intensity threshold with standard errors (1x +/- SEM) using 5 uV and 10uV criterion for a- and b-wave amplitude from normal control and PDE6A mutant dogs at all ages .......................... 70 Mean scotopic a-wave implicit times with standard errors (1x +/- SEM) plotted against stimulus intensity on a log-log scale from normal control and PDE6A mutant dogs at 2 & 3 (A),4 & 5 (B), 6 & 7 (C), 9 & 12 (D) and 16 & 20 (E) weeks of age .................................... 72 Mean scotopic b-wave implicit times with standard errors (1x +/- SEM) plotted against stimulus intensity on a log-log scale from normal control and PDE6A mutant dogs at 2 & 3 (A), 4 & 5 (B), 6 & 7 (C), 9 & 12 (D) and 16 & 20 (E) weeks of age .................................... 73 Normalization (for a-wave amplitude) of the rod-isolated a-wave derived from a subtraction of intensity-matched photopic and scotopic ERGs in a normal control and the PDE6A mutant dog at 3 weeks of age ............................................................................................ 75 Representative recordings of rod-mediated ERG response to dim blue flashes of light from dark-adapted normal controls and PDE6A mutant dogs at 4 and 7 weeks of age .............................................. 76 Scotopic b-wave intensity-response function obtained from a normal control and the PDE6A mutant dog at 3 weeks of age .................... 78 A comparison of the mean and standard errors (1x + SEM) of the Naka-Rushton variables; Vmax (A), k value (B) and n value (C) of the normal controls and the PDE6A mutant dogs at all age groups ......................................................................................................... 8O XV Figure 2.12. A comparison of representative rod flicker responses (-l.6 log cds/mz, 5H2) from a normal control (A) and the PDE6A mutant dog (B) at different ages ............................................................................. 82 Figure 2.13. Representative scotopic oscillatory potential (0.85 log cds/mz) from normal control and PDE6A mutant dogs at 5 weeks of age (band pass at 73 to 500 Hz) ........................................................................ 83 Figure 2.14. Mean scotopic oscillatory potential amplitudes with standard errors (1x + SEM) of normal controls and the PDE6A mutant dogs plotted against age .............................................................................. 84 Figure 2.15. Representative photopic ERG recordings from a normal control (A & C) and the PDE6A mutant dog (B & D) at 4 (A & B) and 6 (C & D) weeks of age .................................................................................. 87 Figure 2.16. Mean photopic a-wave amplitudes with standard errors (1x +/— SEM) plotted against stimulus intensity on a log-log scale from normal control and PDE6A mutant dogs at 2 & 3 (A), 4 & 5 (B), 6 & 7 (C), 9 & 12 (D) and 16 & 20 (E) weeks ofage .................................... 88 Figure 2.17. Mean photopic b-wave amplitudes with standard errors (1x +/- SEM) plotted against stimulus intensity on a log-log scale from normal control and PDE6A mutant dogs at 2 & 3 (A), 4 & 5 (B), 6 & 7 (C), 9 & 12 (D) and 16 & 20 (E) weeks of age .................................... 89 Figure 2.18. Mean intensity at threshold from photopic recordings with standard errors (1x +/- SEM) using a 5 uV criterion for a- and b- wave amplitudes from normal control and PDE6A mutant groups at all ages tested .......................................................................................... 90 Figure 2.19. Mean photopic a—wave implicit times with standard errors (1x +/- SEM) plotted against stimulus intensity on a log-log scale from normal control and PDE6A mutant dogs at 2 & 3 (A), 4 & 5 (B), 6 & 7 (C), 9 & 12 (D) and 16 & 20 (E) weeks of age .................................... 92 Figure 2.20. Mean photopic b-wave implicit times with standard errors (1x +/- SEM) plotted against stimulus intensity on a log-log scale from normal control and PDE6A mutant dogs at 2 & 3 (A), 4 & 5 (B), 6 & 7 (C), 9 & 12 (D) and 16 & 20 (E) weeks of age .................................... 93 Figure 2.21. A comparison of representative cone flicker responses (0.39 log cds/mz, 33 Hz) from a normal control and the PDE6A mutant dog at 9 weeks of age ......................................................................................... 95 xvi Figure 2.22. Figure 2.23. Figure 2.24. Figure 2.25. Figure 3.1. Figure 3.2. Figure 3.3. Figure 3.4. Figure 3.5. Figure 3.6. Figure 3.7. Mean and standard errors (1x + SEM) of cone flicker amplitudes (A) and cone flicker implicit times (B) of normal controls and PDE6A mutant dogs (0.39 log cds/mz, 33 Hz) at all age groups ................ 96 Representative photopic ERG responses (1.36 log cds/mz) from a normal control and the PDE6A mutant dog at different ages demonstrating the photopic negative response ........................................... 97 A representative long flash ERG from a normal control dog of 60 weeks of age ............................................................................................... 99 Representative long flash ERG recordings from normal controls (A) and PDE6A mutant dogs (B) with age (week) . .................................. 100 Representative scotopic (A) and photopic (B) intensity-series ERGs from a normal control at 60 weeks of age performed before and at 1.5 hours following an intravitreal injection of APB .................... 113 Representative scotopic intensity—series ERGs from a normal control at 4 weeks of age (A), the PDE6A mutant dog at 4 weeks of age (B) and scotopic ERG from the PDE6A mutant dog at 6 weeks of age (C) performed before and at 1.5 hours following an intravitreal injection of APB .................................................................... 1 14 Representative cone flicker response (0.39 log cds/mz, 33Hz) of a normal control (A) and the PDE6A mutant dog (B) at 6 weeks of age. performed before and at 2 hours following an intravitreal injection of APB ........................................................................................ 115 Representative long flash ERG response from normal controls (A) and the PDE6A mutant dogs (B) at 4 weeks of age performed injection of APB ....................................................................................... 117 Representative long flash ERG response from a normal control (A) and the PDE6A mutant dog (B) at 6 weeks of age performed before and 2 hours following an intravitreal injection of APB ................ 118 Subtraction of post APB long flash ERG from pre APB administration long flash ERG from a normal control (A) and the PDE6A mutant dog (B) at 6 weeks of age ................................................ 119 Representative photopic ERGs at -0001, 0.39 and 1.36 log cds/m2 from a normal control (A) and the PDE6A mutant dog (B) at 7 weeks of age performed before and at 2 hours following an intravitreal injection of FDA .................................................................... 121 xvii Figure 3.8. Representative cone flicker response (0.39 log cds/mz, 33Hz) of a normal control (A) and the PDE6A mutant dog (B) at 7 weeks of age before and 2 hours following an intravitreal injection of PDA ......... 123 Figure 3.9. Representative long flash ERG responses from a normal control (A) and the PDE6A mutant dog (B) at 7 weeks of age performed before and at 1, 1.5, 2 and 2.5 hours following an intravitreal injection of PDA ....................................................................................... 125 Figure 3.10. Subtraction of post PDA long flash ERG from pre PDA long flash ERG from a normal control (A) and the PDE6A mutant dog (B) at 7 weeks of age . ......................................................................................... 126 Figure 3.11. Representative photopic ERGs at 0.85 log cds/m2 from a normal control (A) and the PDE6A mutant dog (B) at 10 weeks of age performed before and at 1.5 hours following an intravitreal injection of TTX ....................................................................................... 128 Figure 3.12. Representative cone flicker response (0.39 log cds/mz, 33Hz) of a normal control (A) and the PDE6A mutant dog (B) at 10 weeks of age before and at 1.5 hour following an intravitreal injection of TTX .......................................................................................................... 129 Figure 4.1. Diagram of the right eyecup of a dog illustrating planes of section and regions of the retina where the thickness of individual retinal layers is measured, and the number of outer nuclear layer (ONL) rows and photoreceptor nuclei per unit length are counted ....................... 139 Figure 4.2. Diagram of the right eyecup of a dog illustrating the regions of the retina where detailed morphological analysis is performed ..................... 140 Figure 4.3. Diagram of a cross section of retina showing a measurement of the number of rows of the ONL and the number of rod and cone nuclei per 100-micrometer length of the retina ........................................ 141 Figure 4.4. Representative photomicrographs of retinal morphology of a normal control (A & C) and the PDE6A mutant dog (B & D) at 4 (A & B) and 10 (C & D) days ofage ........................................................ 152 Figure 4.5. Representative photomicrographs of retinal morphology of a normal control (A & C) and the PDE6A mutant dog (B & D) at 2 (A & B) and 3 (C & D) weeks of age ....................................................... 155 Figure 4.6. Mean retinal thickness of each of the different retinal layers from superior to inferior ora ciliaris retina (vertical section; see Figure xviii 4.1 in materials and methods) from normal control (A), PDE6A carrier (B) and PDE6A mutant dogs (C) at 3 weeks of age . .................... 156 Figure 4.7. Mean retinal thickness of each of the different retinal layers from nasal to temporal region (horizontal section; see Figure 4.1 in materials and methods) from normal control (A) and PDE6A mutant dogs (B) at 3 weeks of age ........................................................... 157 Figure 4.8. Photomicrographs of retinal morphology of the ONL of a normal control (A) at 23 days of age, and PDE6A mutants dogs (B & C) at 24 (B) and 27 (C) days of age and a photomicrograph of the anti-caspase 3 immunohistochemistry in the PDE6A mutant retina at 24 (D) days of age ................................................................................ 159 Figure 4.9. Ultra structural sections of the ONL of a normal control (A) and the PDE6A mutant dog (B) at 23 days of age .......................................... 160 Figure 4.10. Ultra structural appearance of photoreceptor nuclei of normal control (A) and PDE6A mutant dogs (B, C & D) at 27 days of age ................................................................................................................... 161 Figure 4.11. Representative photomicrographs of retinal morphology of normal control (A & C) and the PDE6A mutant dogs (B & D) at 4 (A & B) and 5 (C & D) weeks of age ....................................................... 161 Figure 4.12. Photomicrographs of retinal morphology of the ONL of a normal control (A) at 38 days of age, a PDE6A mutant dog (B) at 42 days of age and caspase 3 immunohistochemistry in a PDE6A mutant retina at 42 days of age (C) ...................................................................... 165 Figure 4.13. Photomicrographs of TUNEL labeling of retina from normal controls (A, C & E) and the PDE6A mutant dogs (B, D, F & G) at 25 day (A & B), 28 day (C & D), 5 weeks (E & F) and 10 weeks (G) of age ................................................................................................... 166 Figure 4.14. Mean retinal thickness of each of the different retinal layers from superior to inferior ora ciliaris retina (vertical section; see Figure 4.1 in materials and methods) from normal control (A), PDE6A carrier (B) and PDE6A mutant dogs (C) at 5 weeks of age ..................... 167 Figure 4.15. Mean retinal thickness of each of the different retinal layers from ,nasal to temporal region (horizontal section; see Figure 4.1 in materials and methods) from normal control (A) and the PDE6A mutant dogs (B) at 5 weeks of age ........................................................... 168 xix Figure 4.16. Figure 4.17. Figure 4.18. Figure 4.19. Figure 4.20. Figure 4.21. Figure 4.22. Figure 4.23. Figure 4.24. Figure 4.25. Figure 4.26. Ultra structural sections of the photoreceptor segments of a normal control (A) and the PDE6A mutant retina (B) at 5 weeks ofage ........................................................................................................ 169 Representative photomicrographs of retinal morphology of a normal control (A & C) and the PDE6A mutant dog (B & D) at 7 (A & B) and 12 (C & D) weeks of age ..................................................... 172 Ultra structural sections of the photoreceptor layer of the PDE6A mutant retina at 9 weeks of age ................................................................ 173 Ultra structural sections of the ONL of a normal control (A) and the PDE6A mutant retina (B) at 9 weeks of age ....................................... 174 Mean retinal thickness of each of the different retinal layers from superior to inferior ora ciliaris retina (vertical section; see Figure 4.1 in materials and methods) from normal control (A), PDE6A carrier (B) and PDE6A mutant dogs (C) at 9 weeks of age ..................... 175 Mean retinal thickness of each of the different retinal layers from nasal to temporal region (horizontal section; see Figure 4.1 in materials and methods) from normal control (A) and PDE6A mutant dogs (B) at 9 weeks of age ........................................................... 176 Representative photomicrographs of retinal morphology of a normal control (A) and the PDE 6A mutant dog (B) at 16 weeks of age ............................................................................................................ 178 Ultra structural sections of the outer retina of a normal control (A & D) and the PDE6A mutant dog (B, C & E) at 16 weeks of age ........... 179 Mean retinal thickness of each of the different retinal layers from superior to inferior ora ciliaris retina (vertical section; see Figure 4.1 in materials and methods) from normal control (A), PDE6A carrier (B) and PDE6A mutant dogs (C) at 16 weeks of age ................... 180 Mean retinal thickness of each of the different retinal layers from nasal to temporal region (horizontal section; see Figure 4.1 in materials and methods) from normal control (A) and PDE6A mutant dogs (B) at 16 weeks of age ......................................................... 181 Scatter plot showing the number of nuclei rows in the ONL (A), number of rod (B) and number of cone (C) nuclei per unit length (100 pm) of the retina (of region #3) plotted against age for normal control (blue), carrier (green) and mutant (orange) dogs .............. 182 XX Figure 4.27. Figure 4.28. Figure 4.29. Figure 4.30. Figure 4.31. Figure 4.32. Figure 4.33. Figure 4.34. Figure 4.35. Figure 4.36. Figure 4.37. Mean number of nuclei rows in the ONL of retinal regions (#1 to #8) in a vertical section through the optic nerve head at 5 (A), 9 (B), 16 (C) and 60 (D) weeks of age ........................................................ 183 Mean number of nuclei rows in the ONL of retinal regions (#9 to #12) in a horizontal section temporal from the optic nerve head at 5 (A), 9 (B), 16 (C) and 60 (D) weeks of age ........................................... 184 Mean number of rod nuclei per unit length (100 run) of retinal regions (#1 to #8) in a vertical section through the optic nerve head at 5 (A), 9 (B), 16 (C) and 60 (D) weeks of age .............................. 185 Mean number of rod nuclei per unit length of retinal regions (#9 to #12) in a horizontal section temporal from the optic nerve head at 5 (A), 9 (B), 16 (C) and 60 (D) weeks of age ....................................... 186 Mean number of cone nuclei per unit length (100 pm) of all retinal regions (#1 to #8) in a vertical section through the optic nerve head (A & B) and horizontal section temporal through the optic nerve head (C) at 5 (A) and 16 (B & C) weeks of age .................... 187 Thickness of the inner segments of normal control and the PDE6A mutant dog for different retinal regions (see Figure 4.2 in materials and methods) at 24 weeks of age .............................................. 189 Thickness of the ONL of normal control and the PDE6A mutant dog for different retinal regions (see Figure 4.2 in materials and methods) at 24 weeks of age .................................................................... 190 Number of nuclei rows in the ONL at different retinal regions (see Figure 4.2 in materials and methods) in normal control and the PDE6A mutant dog at 24 weeks of age .................................................... 191 Number of photoreceptor nuclei per unit length (100 pm) of retina at different retinal regions (see Figure 4.2 in materials and methods) in normal control and the PDE6A mutant dog at 24 weeks of age ............................................................................................. 192 Representative photomicrographs of retinal morphology of a normal control (A) and the PDE6A mutant dog (B, C & D) at 100 weeks of age ............................................................................................. 194 Mean retinal thickness of each of the different retinal layers from superior to inferior ora ciliaris retina (vertical section; see Figure 4.1 in materials and methods) from normal control (A) and PDE6A mutant dogs (B) at greater than 60 weeks of age ........................ 195 xxi Figure 4.38. Figure 4.39. Figure 4.40. Figure 4.41. Figure 4.42. Figure 4.43. Figure 4.44. Figure 4.45. Figure 4.46. Figure 4.47. Mean retinal thickness of each of the different retinal layers from nasal to temporal region (horizontal section; see Figure 4.1 in materials and methods) from a normal control (A) and PDE6A mutant dogs (B) at greater than 60 weeks of age . .................................... 196 Immunohistochemical expression of anti-rhodopsin (a gifl from Dr. P. Hargrave, University of Florida) in retinal sections of normal control (A, C & E) and the PDE6A mutant dogs (B, D & F) at 3 (A & B), 5 (C & D) and 9 (E & F) weeks ofage .......................... 198 Immunohistochemical expression of anti-rhodopsin (Lab Vision) in retinal sections of normal control (A & C) and the PDE6A mutant dogs (B, D, E and F) at 4 (A & B), 8 (C & D), 60 (E) and 210 (F) weeks of age ................................................................................ 200 Immunohistochemical expression of anti-cone arrestin (a gift from Dr. S. Craft, University of Southern California) in retinal sections of normal control (A & C) and the PDE6A mutant dogs (B&D) at9(A&B) and 210(C&D) weeks ofage ............................. 202 Immunohistochemical expression of anti-red/green opsin fluorescence in retinal sections of normal control (A & E) and the PDE6A mutant dogs (B, C, D & F) at 5 (A & E), 7 (B), 16 (C & F), and 60 (D) weeks of age ..................................................................... 204 Immunohistochemical expression of anti-protein kinase C alpha (PKCor) in retinal sections of normal control (A, C & E) and the PDE6A mutant dogs (B, D & F) at 7 (A & B), 9 (C & D) and 60 (E & F) weeks of age ................................................................................ 206 Immunohistochemical expression of anti-calbindin fluorescence in retinal sections of normal control (A & C) and the PDE6A mutant dogs (B & D) at 10 (A & B) and 60 (B & D) weeks of age ......... 207 Immunohistochemical expression of anti-Hu C/D fluorescence in retinal sections of normal control (A) and the PDE 6A mutant dogs (B & C) at 5 (A), 7 (B) and 60 (C) weeks of age ...................................... 208 Immunohistochemical expression of anti-calretinin fluorescence in retinal sections of normal control (A, C & E) and the PDE6A mutant dogs (B, D & F) at 5 (A & B), 10 (C & D) and 60 (E & F) weeks of age .............................................................................................. 210 Immunohistochemical expression of anti-GFAP in retinal sections of normal control (A, C & E) and the PDE6A mutant dogs (B, D, &F) at3(A&B),S(C&D) and 60 (E&F)weeks ofage .................... 212 xxii Figure 5.1. Figure 5.2. Figure 5.3. Figure 5.4. Figure 5.5. Figure 5.6. Figure 5.7. Figure 5.8. Figure 5.9. Upward capillary transfer apparatus ......................................................... 239 Diagram illustrating PDE6A fragments amplified from RT— PCR with various designed primer sets ............................................................ 244 RT-PCR analysis of the PDE6A gene expression in various tissues ....................................................................................................... 246 RT-PCR analysis of the PDE6A gene expression in various tissues using gene specific primers (PDEA1/6835; Figure 5.1) ............... 248 Nucleotide sequence of the assembled kidney PDE6A full-length transcript, the shorter product missing exon 16 from the PDE6A mutant kidney and predicted translated amino acid sequence ......... 250-252 Comparison of a portion of the two fragments of mutant canine kidney PDE6A transcript and amino acid sequence in the region of the mutation responsible for PRA in the PDE6A mutant dog .............. 253 Diagram demonstrating different PDE6A domains from the retina (A) and PDE6A fragment from kidney (B) .............................................. 254 Total RNAs separated by gel electrophoresis using 1.5% formaldehyde prior to blotting on a nylon membrane .............................. 258 Northern blot probed with full-length canine retinal PDE6A cDNA probe .............................................................................................. 256 xxiii Chapter 1 Introduction 1.1. Ocular embryology The components of the canine eye are derived from basically 3 embryonic tissues: neuroectoderrn, surface ectoderm and mesenchyme (from neural crest cells) (Moore & Persaud, 1998). The eye starts to develop early in gestation, with ocular structures starting to form at the embryonic plate (neural plate) stage. At the site of the developing eye a flattened area develops on both sides of neural groove in the forebrain region. The neural groove sinks into the mesoderrn, and detaches from the overlying surface ectoderm to form the neural tube from which the primitive central nervous system develops. At gestation day 13, before the anterior end of the neural tube closes, the optic grooves (optic sulci) are formed in dogs (Figure 1.1A) (Aguirre et al., 1972). A localized area of neuroectoderrn grows outwards towards the surface ectoderm to form the primary optic vesicle (Figure 1.1B) at gestation day 15 in dogs. The optic vesicle remains connected to the forebrain by the Optic stalk. c' forebrain , surface ectoderm .. , surface ectoderm optic vesicle-a neuroectoderrn invagrnating optic stalk lens placode p neuroectodenn vesrcle surface ectoderm surface ectoderm optic cup D. optic vesicle neurosensory retina Retinal pigment \.epitl're|ium optic stalk lens vesi lens vesicle optic fissure Figure 1.1. Human ocular embryology. Note that afier optic sulci are formed (A), the surface ectoderm cells overlying the optic vesicles thickens to form the early lens placode (B). The lens placode invaginates to form the lens vesicle as the optic vesicle is forming the optic cup (C & D). Lens vesicle is positioned within the optic cup (E). (Moore & Persaud, 1998) The optic vesicle induces the overlying surface ectoderm to thicken into lens placode at gestation day 17 (Figure 1.1C), on gestation day 19 as the optic vesicle invaginates and folds onto itself creating the optic cup, the lens placode also invaginates to form the lens vesicle (Figure 1.1D & HE). A separation of the lens vesicle from the surface ectoderm later in development leads to the formation of the anterior chamber. An accompanying migration of mesenchyme between the surface ectoderm and the optic cup forms cornea endothelium and stroma. Surface ectoderm overlying the optic cup forms the corneal epithelium, while the neuroectodenn cells located at the anterior aspect of the optic cup become the epithelium of the iris, which is continuous with the epithelium of the ciliary body. At gestation day 25 where the optic cup meets the optic stalk there is an opening on the ventral surface, the optic fissure, which allows vasculature access to the inside of the developing globe (Flower et al., 1985). The inner (non-pigmented) layer and the outer (pigmented) layer of the posterior aspect of the bi-layered optic cup become the neurosensory retina and retinal pigment epithelium (RPE), respectively. At the time of lens placode induction (gestation day 17), the retinal primordium consists of an outer (nuclear) zone and inner (anuclear) zone, which then form outer and inner neuroblastic layers, which are separated by the fiber layer of Chievitz at gestation day 32. Retinal primordium at this stage consists of the nerve fiber layer, inner neuroblastic layer, proliferative zone, outer neuroblastic layer and outer limiting membrane. Cellular differentiation of the retina, after the opening of the eyelids (postnatal day 10-14), results in 3 layers of neurons. The outer layer consists of photoreceptor cells (rods and cones), the first-order neurons. The middle layer consists of the second-order neurons, mainly bipolar, horizontal, amacrine and also the cell bodies of glial cells known as Muller cells. The inner layer consists of ganglion cells (the third- order neurons) and displaced amacrine cells. 1.2. Ocular anatomy The globe and extraocular muscles are contained within a conical cavity, called the orbit. The orbit provides protection to the eyeball and is accessed by various blood vessels and nerves involved in the function of the eye. The wall of the globe comprises of 3 layers: the fibrous tunic (sclera and cornea), the vascular tunic (choroid, ciliary body and iris), and the nervous tunic (retina). The fibrous tunic is composed of two parts; sclera and cornea. It is responsible for maintaining the shape of the eye, protecting it from the external environment, transmitting and refracting of light rays via the cornea and providing a location for insertion of the extraocular muscles. The sclera consists of a dense network of collagen and elastic fibers and encloses the posterior three-fourths of the globe. The cornea is transparent and located in the anterior portion of the globe. Its transparency is due to a highly ordered arrangement of collagen fibrils and lack of pigment, vessels, and myelinated nerve fibers. The vascular tunic (uveal tract) is the middle layer of the eye and is interposed between the retina and the sclera. It is composed of choroid, ciliary body and iris. The choroid is a pigmented vascular layer that makes up the posterior portion of the uveal tract. The tapetum lucidum, a specialized reflective layer of the choroid, is present in the superior fundus of most domestic species including the dog. It is thought to increase the sensitivity of the retina to low light levels by reflecting light back through the overlying photoreceptor layer (Elliott & Futterman, 1963). The ciliary body is the middle segment of the vascular tunic positioned between the iris and choroid and is highly vascular. It is responsible for aqueous production and outflow. The zonular ligament, which originates from the ciliary body attaches to the lens capsule to suspend the lens. Ora ciliaris retina (ora ciliaris retina) is the boundary where the ciliary body ends posteriorly at the adjacent retina. The iris is the anterior most segment of the vascular tunic, connecting with the ciliary body at the periphery. It acts as a diaphragm to regulate the amount of light that enters through the pupil via two muscles; the sphincter muscle innervated by the parasympathetic nervous system, and the dilator muscle innervated by sympathetic and some parasympathetic nerves. The retina is the innermost layer of the posterior segment of the globe. It develops from the two walls of the optic cup. The outer wall, a single layer of cells, forms the retinal pigment epithelium (RPE) whereas the invaginated inner wall of the optic cup adjacent to the vitreous chamber develops into the neurosensory retina. Macula is a small area near the center of the retina of the human eye, where it is histologically defined as having two of more layers of ganglion cells. Near its center is the fovea, a small cone-rich region, which is also present in primates, birds and reptiles. The fovea is free from rods. Instead of a fovea many domestic species have an area centralis, an area of higher cone density (Henkind, 1966). Nutrient supply to the inner and outer layers of the retina originates from retinal and choriodal vessels, respectively. The vascular pattern of the retina varies between species (Simoens et al., 1988). The dog has a holoangiotic retina in which there is a relatively uniform vascularization of the entire retina (Engerrnan et al., 1966). Histologically 10 layers of the retina are recognized (Figure 1.2.). The 10 layers from proximal to distal are RPE, photoreceptor layer (PRL), outer limiting membrane (OLM), outer nuclear layer (ONL), outer plexifonn layer (OPL), inner nuclear layer (INL), inner plexiforrn layer (IPL), ganglion cell layer (GCL), nerve fiber layer (NFL), and inner limiting membrane (ILM). I " u . Choroid - J .. . - g _ u .9 - A | (tapetal |ucrdum) - ~ - - - - v ’1 I ”:9. x. A: .1. r . r p i I ‘11.: -_,, firm, '2'??- . . f I." r. - ,up’hgpl, 2.; "3,?" . ‘ ' '1 ‘ i” - . f 5 3 . l . ‘3' f ) v‘ I i " < * . - . - IPL .‘ E ' i“, \ “s’g 3' w Vt \ r- vi” ’ . ‘ “’2‘ . ‘ >~’ GCL+NFL ' ‘ “ ' """" . ' ’———— ILM Figure 1.2. Cross section of canine retina. A cross section of canine retina shows the 10 layers recognized histologically. Key: RPE = retinal pigment epithelium, PRL = photoreceptor layer, OLM = outer limiting membrane, ONL = outer nuclear layer, OPL = outer plexiforrn layer, INL = inner nuclear layer, IPL = inner plexiform layer, GCL = ganglion cell layer, NFL = nerve fiber layer, and ILM = inner limiting membrane. 1.2.1. Retinal pigment epithelium The RPE is a single layer of epithelial cells located between the choroidal vasculature and the neurosensory retina. Cells within the RPE are pigmented because they contain melanosomes except for those overlying the tapetal lucidum. The apical surface of the RPE has cytoplasmic apical processes which engulf the outermost portion of photoreceptor outer segments. They act to support the photoreceptors as well as phagocytize photoreceptor outer-segment discs. Disc shedding from the outer segments and phagocytosis by the RPE is controlled by several factors, such as duration of the light/dark cycle (Besharse et al., 1977) and melatonin levels (Besharse & Dunis, 1983). The RPE is involved in the visual cycle whereby ll-cis retinal is generated and supplied to the photoreceptors. In addition, the RPE serves as part of the blood-retinal barrier which limits the diffusion of large molecular-weight molecules from choroidal vessels into the retina (Steuer et al., 2004). 1.2.2. Photoreceptor layer There are two main types of photoreceptors (rod and cone), both of which consist of four functional segments: the outer segment, the inner segment, the cell body/soma and the receptor terminus (Figure 1.3.). The photoreceptor layer consists of the outer and inner segments of photoreceptors, while their nuclei and termini are positioned in the ONL and OPL, respectively. Rods are responsible for night time vision and cones are for day time vision, as well as higher visual acuity and color vision. :— disc <2 C: =_)—— cytoplasm => Outer segment Outer segment cilium mitochondria Inner Inner segment segment nucleus Synaptic Synaptic terminus . terminus Rod IS Figure 1.3. A. A schematic drawing of structural elements of rod and cone. Note that the structure of rods and cones is similar, except for size and shape. Additionally, cone outer segment discs are continuous with the cell membrane, whereas rod outer segment discs are separated from the cell membrane. B. A Differential Interference Contrast image of canine central retina from a flat mountpreparation focused at the level of the inner segments (magnification 100X). Cone inner segment diameter is approximately 6 times that of rod inner segments (as shown in B). The outer segments of rods are long and uniform in width while those of cones are cone-shaped. The outer segments consist of a stack of disc shaped membrane. In rods, the discs are separated from the plasma membrane whereas in cones, discs are continuous with the membrane (Steinberg et al., 1980). Rod and cone outer segments are connected to the inner segment by a cilium. The protein kinesin is closely associated with connecting cilia of photoreceptors. It is located at the axoneme that extends to the outer segment tip of cones (Eckmiller & Toman, 1998) whereas it is surrounding the basal body of the cilium, outer segment axoneme and part of inner segment in rods (Beech et al., 1996; Eckmiller & Toman, 1998). Not only is this protein involved in the translocation of materials between outer and inner segments (Muresan et al., 1997), but it is also involved in the formation of new photoreceptor disc membranes (Williams et al., 1992). During postnatal development in the mouse, membranous discs are formed by the random fusion of small vesicles with the apical swelling of the connecting cilium into a partial stack in order. A partial stack of discs is progressively rearranged during development to form a regular arrangement of outer segments in the mature retina (Obata & Usukura, 1992). The inner segments of rods and cones are responsible for metabolic function, protein synthesis and homeostasis (Baldridge et al., 1998). They contain cell organelles; centrioles, mitochondria, free ribosomes, smooth and rough endoplasmic reticulum, microtubules and small vesicles. Cone inner segments contain more number of mitochondria than rods, compared per unit volume of outer segment (Hoang et al., 2002). 10 t w-a ,,. '—T.' .--'-r r 11.! bfikw~ ‘ '1' 1- .jrm-fiu": ,':..._;’.f.‘:~’~ :1 *, 1*. 3&‘fg1gm. i" " ' ‘ . “&- Sign ’ “ " “we‘d-”ww- .4.“ 9334'“ inner ‘ , .. “vi—r“ WW segments Inner - ' . . segments PR nuclei Figure 1.4. Histological section showing canine photoreceptors at 3 (A) and 10 (B) days of age. Note the inner segments have developed by 3 days of age. By 10 days of age, they have elongated. The outer limiting membrane is present. At this age, photoreceptor nuclei are oval in shape. Bar = 25 um. 1.2.3. Outer limiting membrane The inner segments of photoreceptors are separated from the soma by the OLM (Figure 1.2 & 1.4). This thin membrane is composed of zonular adherens, which firmly attach the inner segments of photoreceptors to Miiller cells and Miiller cells to each other to provide structural support. Gap junctions within the OLM are composed of aggregates of particles of variable size and shape (Whiteley & Young, 1986). The pore radius of the zonulae adherens of the OLM is between 30 and 36 A°, and thus helps to prevent the penetration of protein-rich fluid from the PRL to the inner retina when the outer blood retinal barrier is compromised from disease or injury (Buntmilam et al., 1985). 1.2.4. Outer nuclear layer Photoreceptor cell bodies (soma) are located in the outer nuclear layer. Mature rod nuclei are round in shape, small and more heterochromatic than nuclei of cones. Cone nuclei are large, euchromatic and located close to the OLM. The density of photoreceptors is not uniform across the retina (Curcio, 1986). The density of cones in the dog is reported to be greater in the central retina compared to the periphery (Koch & Rubin, 1972). In humans, cone density declines rapidly towards the periphery from a peak at the fovea while rod density is the highest at a distance of about 3-5mm from the fovea, then its density decreases toward the periphery (Jonas et al., 1992). 1.2.5. Outer plexiforrn layer Photoreceptor synaptic termini lie at the outer aspect of the OPL. Two morphologic categories of synaptic termini are recognized. The rod terminus is called a spherule and is small and round. The cone terminus is called a pedicle and is large and flat. Rod spherules have one or two invaginations whereas cone pedicles have numerous invaginations. Photoreceptor termini are filled with synaptic vesicles. Synaptic ribbons (synaptic lamella) are dense laminar structures present within the photoreceptor termini at the site of synaptic contact. Proteins that have a function in synaptic vesicle docking and fusion as well as endocytosis are present in ribbon synapses. This is an indication of synaptic membrane traffic in ribbon synapses (Ullrich & Sudhof, 1994). Glutamate, a 12 neurotransmitter, is released at ribbon synapses by the calcium-dependent exocytosis of synaptic vesicles (Morgans, 2000). The typical appearance of invaginated synaptic processes in the photoreceptor termini are called triads and consists of a pair of horizontal cell processes flanking a bipolar cell dendrite (Sikora et al., 2005). Occasionally, synaptic units consist of two horizontal cells and two bipolar cells. This arrangement is called a tetrad (Migdale et al., 2003). Rods and cones are primarily interconnected by horizontal cells, the neurotransmitter glutamate, is released from photoreceptor termini to stimulate wide-field and narrow-field horizontal cells (Smith, 1995). Cone synaptic termini make contact with both a superficial, non-invaginated synapse to flat bipolar cells (Hopkins & Boycott, 1995) and an invaginating synapse within the triad to invaginating bipolar cells (Boycott & Hopkins, 1991). 13 Figure 1.5. A simple diagram of the organization of the retina. Key: RPE = retinal pigment epithelium, MiiC = Miiller cell, HzC = horizontal cell, BC = bipolar cell, AC = amacrine cell, GC = ganglion cell, NFL = nerve fiber layer, and ILM = inner limiting membrane. (Source: www.webvision.med.utah.edu) 1.2.6. Inner nuclear layer The inner nuclear layer is composed of the cell bodies of amacrine cells (AC), bipolar cells (BC), Miiller cells (MiiC), horizontal cells (HzC) and interplexiforrn cells. The average percentages of AC, BC, MiiC and HzC differ among species. For example, in the mouse retina there are 41% of AC, 40% of BC, 16% of MiiC and 3% of HzC (Jeon et al., 1998). 1.2.6.a. Amacrine cells Amacrine cell bodies are located in the inner region of the INL. Narrowofield, bistratified amacrine cells (AII amacrine cells) are the most numerous amacrine cells in mammals and are known as rod-mediated depolarizing cells. Their axons terminate at the sublamina b of the IPL close to the GCL. Not only do the All amacrine cell transmits the signal from rod bipolar cells via gap junction to ganglion cells, the All amacrine cells also couple to themselves to improve signal/noise ratio in their network (Vardi & Smith, 1996). The A17 amacrine cells have diffuse axonal processes terminating at the sublamina b of the IPL. Reciprocal synapses of the A17 amacrine cells to rod bipolar cells suggest their role in the rod pathway (Hartveit, 1999). In addition to All and A17 amacrine cells, other types of amacrine cells such as A2 and A8 amacrine cells are involved in cone pathways. The amacrine cells express ionotropic (NMDA, AMPA and kainate) and metabotropic (mGluRl, mGluR2, mGluR4 and mGluR7) glutamate receptors to uptake glutamate from the synaptic cleft (Duarte et al., 1998). Activation of these glutamate receptors stimulates the release of gamma aminobutyric acid (GABA) (Gleason et al., 1994) and acetylcholine (Linn et al., 1991) from amacrine cells. Calretinin is present in the All amacrine cells (Gabriel & Witkovsky, 1998) and can be used as a marker for these cells. 15 1.2.6.b. Bipolar cells Bipolar cells are the second-order neuron in the retina connecting the photoreceptors to the ganglion cells. Their dendrites synapse with photoreceptors and horizontal cells in the OPL, and axons synapse with amacrine and ganglion cells in the IPL. Each rod bipolar cell in the monkey connects to about 20 rods at 2—4 mm eccentricity and 60 rods at 6-7 mm eccentricity (Grunert & Martin, 1991). The connections have a sign-inverting synapse (ON-bipolar cells). Rod bipolar cell termini in the mammalian retina contact to ganglion cells via amacrine cells. There is no direct synapse between rod bipolar cells and ganglion cells. In some instances, rod bipolar cells contact the narrow-field, bistratified (AII) amacrine cells, which in turn make gap junctions with cone bipolar cells in the sublamina b of the IPL (Strettoi et al., 1994). Alternative rod pathways in rodents have been shown where rods bypass the rod bipolar cells and directly contact cone OFF-bipolar cells (Soucy et al., 1998; Hack et al., 1999). All cone bipolar cells synapse with amacrine and ganglion cells. Two classes of cone bipolar cells based on responsiveness to light stimulation are recognized. They are depolarizing (ON or invaginating bipolar cells) and hyperpolarizing (OFF or flat bipolar cells). The synapses between cones and ON-bipolar cells and cones and OFF-bipolar cells are sign-inverting and a sign—preserving, respectively. Cone bipolar cells substantially outnumber rod bipolar cells all across retina in rabbits (Strettoi & Masland, 1995). 16 l.2.6.e. Mfiller cells During development of the retina the retinal microglial cells originate from hemopoietic cells. These cells invade the developing retina via blood vessels. They play an important role in host defense against invading microorganisms, in immunoregulation, and tissue repair (Chen et al., 2002). Miiller cells are the principal glial cells of the retina. Their nuclei are angular in shape with dense chromatin and are located toward the outer region of the INL. Miiller cell processes extend radially from the OLM to the ILM. The classic type of Miiller cell in mammals has diffuse and abundant descending processes. Numerous voltage-gated channels and neurotransmitter receptors are expressed in Miiller cells and these modulate neuronal activity by regulating the extracellular concentration of neuroactive substances, including potassium (K+), glutamate, GABA and hydrogen (H+) (Newman & Reichenbach, 1996). In the OLM, Miiller cell processes tightly ensheath photoreceptor termini (Derouiche, 1996). Miiller cell processes at the end feet have the ability to phagocytose foreign substances and enwrap the axons of the nerve fiber layer to enhance electrical transmission along the ganglion cell axons. Morphological and cellular changes develop in Muller cell in response to retinal damage reflect their role in neuroprotection (Derouiche, 1996; Garcia & Vecino, 2003). Horizontal cells 1.2.6.d. Horizontal cells Horizontal cell nuclei are located at the outer region of the INL with their processes extending horizontally through the OPL to couple with photoreceptors. 17 Horizontal cell dendrites are arranged on either side of the photoreceptor ribbon synapses along with bipolar cell dendrites at the center of the triad. This organization helps to include responses from the surrounding region of retina. Two types of horizontal cell are recognized in domestic species. Type A horizontal cells have no axons and synapse with all types of cones. Type B horizontal cells have axons and synapse with both rods and cones. The type A horizontal cell is strongly reactive to anti-calbindin—D antibody compared to type B horizontal cell (Lyser et al., 1994; Gabriel & Witkovsky, 1998). Calbindin can be used as a marker for type A horizontal cells. l.2.6.e. Interplexiforrn cells In the INL, interplexiform cells are positioned among amacrine cells and have processes that ramify in the OPL and IPL. They provide a feedback mechanism between the inner and outer retina. Most interplexiform cells are dopaminergic and well developed in Teleost fish, rodents, rabbit and primates (Nguyenlegros, 1991). In other species, these cells release either GABA or other neuroactive substances, such as glycine, somatostatin or serotonin. In the goldfish the synthesizing enzyme for epinephrine was identified in the interplexiform cells, and this transmitter influences horizontal cells (Baldridge & Ball, 1993; Savy et al., 1995). Dopaminergic interplexiform cells, along with amacrine cells are involved in the generation of the oscilatory potentials of the electroretinogram (ERG) (Citron et al., 1985). 18 1.2.7. Inner plexiform layer The IPL is a multilaminated region containing different types of synapses between bipolar, amacrine, interplexiform and ganglion cell processes. A synapse between presynaptic cells (bipolar cells) and two postsynaptic cells; either a ganglion cell or amacrine cell process or two amacrine cells is known as a dyad. In the cat, OFF- bipolars and ON-bipolar cell axon termini are positioned in sublamina a (outer region) and sublamina b (inner region), respectively. They correspondingly synapse with ganglion cell subtype a and b. Rod bipolar axons synapse with A17 and All amacrine cells in sublamina b (Kolb, 1979; Derouiche, 1996; Garcia & Vecino, 2003). In monkey and human retina, the IPL is divided into five layers. Amacrine processes are distributed in three bands corresponding to different levels of the peptides, dopamine, and GABA, whereas bipolar cell processes are distributed in four broadly overlapping bands (Koontz & Hendrickson, 1987). Some horizontal cells in the bovine retina have additional thick processes descending to the IPL, where they are postsynaptic at the dyads (Chun & Wassle, 1993). 1.2.8. Ganglion cell layer This layer contains cell bodies of most of the ganglion cells, displaced amacrine cells and some astroglial cells as well as blood vessels. Morphologically, ganglion cells can be divided into three types; alpha, beta and gamma. The alpha ganglion cells have the largest cell bodies, uni-stratified dendrites and axons occupying two strata in the outer half of the 19 IPL (Wassle et al., 1981b). The beta ganglion cells have more branching dendrites than the alpha cells. The gamma ganglion cells have the smallest cell bodies, widely extended thin and winding dendrites, and are concentrated in the visual streak (Saito, 1983). In the cat retina, 55% of all ganglion cells are beta, 41% are gamma, and 4% are alpha cells (Wassle et al., 1981a). Ganglion cells receive inputs from neighboring photoreceptors in a circumscribed area of the retina (their receptive field) and transmit these inputs as a train of action potentials. Two classes of ganglion cells, based on differential illumination of their receptive fields, are ON-center and OFF-center ganglion cells. The ON-center ganglion cells are depolarized when light is directed to the center of their receptive field, whereas OFF-center ganglion cells are hyperpolarized (Purves et al., 2004). Displaced amacrine cells are present in the GCL in many species, including the dog (Marroni et al., 1995). These cells form synapses with other cell processes in the IPL (Wong & Hughes, 1987) and can be labeled for either GABA or glycine (Koontz et al., 1993). The proportion of displaced amacrine cells to ganglion cells varies among species. This ratio decreases during grth of the fish’s eye (Mack et al., 2004). Five to 20% and more than 40% of the neurons in the GCL are displaced amacrine cells in the cat (Koontz et al., 1993) and hamster retina (Linden & Esberard, 1987), respectively. 20 1.2.9. Nerve fiber layer Axons of ganglion cells, inner fragments of Miiller cell processes and retinal blood vessels are present in the NFL. Ganglion cells have axons that converge to form the optic nerve, which projects to the lateral geniculate nucleus (LGN) of the thalamus. In the NFL, the axons are unmyelinated but as they converge on the optic nerve head, they gain a myelin sheath. The myelination of the fibers at the optic nerve head gives the canine optic nerve head its characteristic slightly raised appearance. Astroglial cells have a simultaneous contact with ganglion cell axons and blood vessels (Runggerbrandle et al., 1993). The majority of astroglial processes are aligned in parallel with the ganglion cell axons in the central region of the retina and are radially arranged in the periphery (Karschin et al., 1986). 1.2.10. Inner limiting membrane The ILM is the innermost layer of the retina. Astrocytes and Miiller cells contribute to the formation of this membrane. In the rabbit the ILM is a thin basement membrane throughout all areas of the retina, whereas the ILM of the cynomolgus monkey is a thick basement membrane in the peripapillary region and a thin basement membrane in the region of the fovea (Matsumoto et al., 1984). Like humans, the ILM in cynomolgus monkeys thickens with age (Matsumoto etal., 1984). 21 1.3. The central visual pathway Visual information from the retina is conducted from all ganglion cells axons to the optic nerve where ganglion cell axons are bundled and myelinated. At the optic chiasm, the axons from the nasal portion of each retina cross to the contralateral side of the brain whereas axons from the temporal portion of the retina remain on the ipsilateral side of the brain. The degree of axon decussation varies between species. In the dog 78% of axons cross over to the contralateral side of the brain (Lee et al., 1999). From the optic chiasm, axons from the left portion of each retina project in the left optic tract and vice versa for axons from the right half. All axons project to three major subcortical regions; the lateral geniculate nucleus (LGN), the pretectum and the superior colliculus (Figure 1.6). , Optic tract Optic nerve Optic chiasm Hypothalamus Lateral geniculate ‘ -. Pretectum nucleus ‘. g Optic radiatio 4-5 yrs (Dekomien et al., 2000) Persian cat pra ? 6 wks (Rah et al., 2005) Abyssinian cat rdy ? 8-12 wks (Curtis et al., 1987) LATE ONSET Poodle; * 3-5 yrs (Koskinen et al., 1985) Miniature,Toy 1’er & Standard American prcd * 3-5 yrs (MacMillan & Lipton, 1978) cocker spaniel Portugese prcd "' 3-6 yrs X water dog Labrador prcd * 4-8 yrs (Kommonen & Karhunen, 1990) retriever English cocker prcd "‘ 4-6 yrs X spaniel Dachshund; pra ? 5-7 mths (Curtis & Barnett, 1993) miniature longhaired Table 1.1. Summary of the different types of progressive retinal atrophy (PRA) in dogs and cats. Key: PDE6A = alpha subunit of phosphodieasterase, PDE6B = beta subunit of phosphodieasterase, erd = early retinal degeneration, NR = no report, PDC = phosducin, pd = photoreceptor dysplasia, pra = progressive retina atrophy, prcd = progressive rod cone degeneration, rdy = retinal dystrophy, rcd = rod-cone dysplasia, rd = rod dysplasia, UN = un-classified, X-pra = X-linked progressive retina atrophy. 41 BREED DISEASE GENE ONSET OF REFERENCES FUNDUS ABNORMALITY LATE ONSET Tibetan terrier pra ? 1-1.5 yrs (Barnett & Curtis, 1978) Akita pra ? 1.5-2 yrs (Toole & Roberts, 1984) Samoyed pra ? 2-4 yrs (Dice, 1980) Irish pra ? NR (Gould et al., 1997) wolfhound English setter pra ? > 7yrs (Bjerkas, 1990) Tibetan pra ? 3-4 yrs (Bjerkas & Narfstrorn, 1994) spaniel Papillon pra ? > 3 yrs (Narfstrom & Wrigstad, 1999) Mastiff pra Rod opsin ? (Kijas et al., 2003) Abyssinian cat pra ‘? 1.5-2 yrs (Narfstrom, 1983) Siberian husky X-pra RPGR 1.5-2 yrs (Acland et al., 1994) Mixed breed X-pra RPGR ? (Zhang et a1, 2002) Table 1.1 (continued) Star (*) indicates the gene for prcd locus reportedly been identified although this information has not been published of the time of writing. Question mark (?) indicated the gene responsible for PRA in the particular breed has not been indicated. X indicated no direct scientific reference for that particular breed. Note that PRA is also reported in other dog breeds; American Eskimo dog, Australian cattle dog, Australian shepherd, Basenji, Chesapeake bay retriever, Doberman pinscher, Entlebucher mountain dog, German shorthaired pointer, Newfoundland, Nova Scotia duck tolling retriever, Border collie and Shetland Sheepdog (www.0ptigen.com). 42 Similar to RP, PRA is a bilateral condition with both eyes affected to a similar extent. The earliest clinical sign in PRA-affected dogs is often impaired vision in dim light. As the retina undergoes degeneration, the pupillary light reflex is affected, and becomes more sluggish with progression of the disease. Nystagmus is reported in Abyssinian cat with dominant PRA (Curtis et al., 1987). Ophthalmoscopically, most PRA forms show a progressive development of tapetal hyperreflectivity and attenuation of retinal blood vessels as the retina undergoes degeneration (Figure 1.12). Depigrnentation mixed with patchy areas of increased pigmentation develops in the nontapetal fimdus with advanced disease. Atrophy of the optic nerve head also develops. Secondary cataracts are common in PRA-affected dogs, particularly those with late-onset disease (Priester, 1974). Electroretinography is a useful tool to detect functional changes in the retina of the PRA-affected animals prior to development of ophthalmoscopic changes. Figure 1.12. Fundus photography of a normal control dog (A) and a Cargian Welsh corgi with progressive retinal atrophy at 18 weeks of age (B). Note that mutant fundus (B) has tapetal hyperreflectivity, attenuation of retinal blood vessels and optic nerve head atrophy. 43 Several genes associated with PRA had been identified (Lin et al., 2002), however more genetic investigation is still required. Two mutations in the gene encoding the beta subunit of PDE (PDE6B) protein are reported as a cause of PRA in two breeds of dog; Irish setter (Suber et al., 1993) and Sloughi (Dekomien et al., 2000). Mutations in this gene are also reported in humans with arRP (Danciger et al., 1995). PRA in the Irish setter is known as rod-cone dysplasia type 1 (rcdl) and is due to a nonsense mutation at codon 807 of the PDE6B (Suber et al., 1993). The PDE6B gene in the rcdl dog has a transition of guanine to adenine at nucleotide 2420 that is predicted to cause a premature termination of the PDE6B protein by 49 amino acid residues. The truncated PDE6B protein, if translated, would be lacking the membrane binding site and part of the catalytic domain. Mutant dogs have a deficiency in PDE catalytic activity and subsequently develop on accumulation of cGMP (Suber et al., 1993). A significant reduction in the level of PDE6B mRNA is present in the developing rcdl retina prior to retinal degeneration (Farber et al., 1992). Rod flicker ERG responses could not be detected at 24 days of age whereas cone flicker responses were present but abnormal (Aguirre & Rubin, 1975). The rch Irish setter has disorganization of rod outer segments with fewer rod nuclei compared to cone nuclei by 6 weeksof age. These changes reflect a severe loss of rods at an early age, with simultaneous a slower degeneration of cones. Loss of day time vision was present by as early as one year of age. PRA in the Sloughi was found to be due to an 8-bp insertion in exon 21 of PDE6B (Dekomien et al., 2000). It seems likely that the form of PRA in the Sloughi will also be rod-cone dysplasia, although there are no histological or ERG studies to' 44 characterize the disease. Mice with PDE6B defects have a variability in phenotype that correlates to the predicted effect of the mutation (Hart et al., 2005). A dominantly inherited form of PRA has been described in the Mastiff and Bull Mastiff breeds (Kijas et al., 2003) (Kijas et al., 2002). Dominant PRA in the Mastiffs is caused by a mutation in the rod opsin gene, a common cause of RP. The affected dogs have an abnormal ERG by 12-18 months of age. The ophthalmoscopic signs of this form of PRA are not typical. The more severely affected parts of the retina are those that are predicted to receive more light exposure. Therefore, the central retina is more severely affected, with hyperreflectivity being seen around and lateral to the optic nerve head. Hyperreflectivity of the peripheral retina does not develop until later in the disease. At the time of writing the genes that cause PRA in cats have not been reported. However, Rhodopsin, ROMl, PDE6G (Gould & Sargan, 2002) and phosducin (Gorin et al., 1995) were excluded as candidate genes for retinal dystrophy in the rdy Abyssinian cats. The rdy Abyssinian cats with an autosomal dominant trait (Curtis et al., 1987) develop ocular abnormalities at a young age. Severe degeneration of outer segments of photoreceptors occurs in rdy kittens by 22 days of age with impairment of development of inner segments. Persian cats with PRA develop vascular attenuation as early as 5 weeks of age, followed by marked tapetal hyperreflectivity at 16 weeks of age. At this time, retinal vascularization is barely visible and only one or two rows of photoreceptor nuclei remain. Abnormality of RPE such as cytoplasmic vacuolation and cell swelling also occurs in cats with the PRA in this breed (Rah et al., 2005). 45 In the Miniature schnauzer affected with photoreceptor dysplasia (pd), a missense mutation was detected in codon 82 of phosducin (Zhang et al., 1998). It was predicted that the mutation would create a non-conservative substitution (Arginine to Glycine) in the translated protein, with the altered amino acid being located close to the amino acid that directly interacts with the Bat-subunits of transducin. This mutation is deleterious to retinal function and causes by a decrease in ERG response and a retardation of photoreceptor development by 24 days of age (Parshall et al., 1991). However, some dogs with the mutantation are heterozygous for the mutant allele, and some are homozygous for the wild-type allele. Thus, the disease alleles at more than one PRA causing locus may be segregating in the Miniature schnauzer breed. Currently two forms of X-linked PRA in dogs are recognized and they are due to separate mutations within a similar region of the RP GTPase regulator (RPGR) gene. Despite the mutations being in the same region of the same gene, the phenotypes differ. The disease, X-linked PRA type 1 (flPRAI) that occurs in the Siberian husky and Samoyed breeds, is less severe than disease the X-linked PRA type 2 (HPRAZ) that was identified in a line of crossbred dogs (Zhang et al., 2002). The mutations in the two canine diseases involve the gene called open reading frame 15. The mutations are in the same region of the human gene that has been described as being a mutation “hot-spot” for several different X-linked retinitis pigrnentosa (V ervoort et al., 2000). Dogs with flPRAI have ERG abnormalities are present from 6 months of age and there is a rod-led retinal degeneration leading to an “end-stage” retina by 4 years of age. XLPRAZ-affected dogs have abnormal photoreceptor development with ERG abnormalities from 6 weeks 46 of age. A progressive retinal degeneration follows, resulting in an “end-stage” retina by approximately 2 years of age. A late-onset PRA or progressive rod-cone degeneration (prcd) has been reported in several breeds of dogs (see table 1.1) and a phenotypically similar disease is seen in the Abyssinian cat with a recessive mode of inheritance. Opsin (Ray et al., 1999), rhodopsin, rds/peripherin (Ray et al., 1996), phosducin (Lin et al., 1998), and PDE6B (Acland et al., 1998) were excluded as breeds responsible for prcd. The prcd locus was mapped to canine chromosome 9. The mutation is reported to have been identified but not published at the time of writing and is in previously undescribed gene (G. Acland personal communication to S. Petersen-Jones, 2005). Histopathological and ERG studies have shown that prcd dogs have a normal development and functional maturation of the retina followed by rod-led photoreceptor degeneration. PRA in Cardigan Welsh corgis was first reported in the veterinary literature in 1972 (Keep, 1972). It is an early-onset form of PRA that leads to night blindness detectable from as early as 7 weeks of age. Loss of daytime vision occurs more slowly and the age of which the dogs are totally blinded varies between individuals. PRA in Cardigan Welsh corgis is autosomal recessive and due to a one-base pair deletion of an adenine at codon 616, nucleotide 1939-1940 (numbering according to Kylma and others (Kylrna et al., 1997) in the alpha subunit of rod cGMP-PDE (PDE6A). This results in a frame shift with a run of 28 codons encoding for altered amino acids and 47 followed by a premature termination codon (Figure 1.138). This is predicted to result in truncation of the protein by 218 amino acids, if the mRNA is translated (Petersen-Jones et al., 1999). The translated protein would be missing part of the catalytic domain (Baehr et al., 1991) and the C-terminal cysteine responsible for membrane binding (Ong et al., 1989). It is therefore most likely a functional null mutation. 48 A. Emu Emn15 901 W W M WAC m RGTNNL YQMK SQN PLA sumaccrcmcmmm KLHGSSILERHHLEFGK IOSIWWWWW QHEHAIHMMDIAIIAT Exont7 1101WWWWQW DLALYRKKRTMFQKIVD B. Emu Enon15 901 W W moo elem RGTNNLYQMKSQTHWP 951mmmmmmm SSMGPPSWKDTTWSSAK 50016 1001 W W CTW WC! CLAW RCCEMRA 1051 mm mm WC museum-ca W Emnfl 1101 W W W W new Figure 1.13. A diagram demonstrating sequences of the PDE6A transcript and amino acid of the normal (A) and mutant (B) alleles. The mutation is an adenine deletion (A in red) that causes a flame shift and premature termination stop (TGA in green). Altered amino acids after the mutation site are colored pink. 49 A DNA—based test using a mismatch PCR-restriction enzyme digestion was developed (Petersen-Jones & Entz, 2002) to ascertain the genotype of dogs for the presence of the codon 616 adenine l—bp deletion in the PDE6A gene responsible for PRA in Cardigan Welsh corgi. 1.8. Laboratory animal models for inherited retinal dystrophies Studies of mouse and rat models of retinal dystrOphies has not only provided candidate genes for the investigation of ocular conditions in humans, but has also offered the opportunity to investigate the effect of gene dysfunction in more detail. Animals with retinal dystrophies that were homologous to human retinal dystrophies have also become useful in the development of drug or gene therapy for these retinal conditions. For example, expression of functional bovine PDE6B delivered via a recombinant adeno- associated virus (rAAV) vector in rd] mice which have a nonsense mutation in the gene encoding the PDE6B protein (Pittler & Baehr, 1991) have increased the number of photoreceptors and caused a two-fold increase of light sensitivity in the mice (Jomary et al., 1997). The RCS (The Royal College of Surgeons) rat has a retinal dystrophy that is a classic model of recessively inherited retinal degeneration. In these rats, the RPE fails to phagocytose shed outer segments, leading to photoreceptor cell death (Katai et al., 1999a). The defect is due to the mutation in the gene encoding the receptor tyrosine kinase (Mertk) (D'Cruz et al., 2000) that is expressed in phagocytic monocytes. This 50 protein is co-localized with outer segment material during phagocytosis by the RPE cells. Several attempts have been made to rescue photoreceptors in this model. Delivery of rat Mertk to cultured RCS RPE cells by means of a recombinant adenovirus results in infected RCS RPE cells ingested exogenous outer segments to the same extent as wild- type RPE cells (Feng et al., 2002). Subretinal transplantation of brain-derived precursor cells promote photoreceptor survival in the RCS rat at very young age (Wojciechowski et a1,2002) In addition to studies of spontaneous gene mutations causing retinal dystrophies in mice, the laboratory mouse offers the opportunity for genetic manipulation. Transgenic mice or gene knockout mice may be created to mimick genetically caused human diseases or to over express a target gene. These mice may be used to study the role of a specific gene product on retinal function in vivo. For example, the Pr023His rhodopsin mutant mouse with a missense mutation (P23H) in the rod opsin gene was used as an animal to study rod degeneration in adRP patients (Olsson et al., 1992). The rds mouse carrying a mutation in the peripherin gene, which encodes a protein that is located at the periphery of the outer segment disc membrane of photoreceptors, was not only used to study the mechanism of retinal degeneration, but also for gene therapy trials (Ali et al., 20001 In addition to creating animals carrying gene mutations in gene encoding phototransduction proteins, several mouse models have been produced to investigate the role of proteins in the visual cycle. For example: rpe65 knockout mice that lack RPE65 51 protein, which is essential for formation of ll-cis retinal (Redmond et al., 1998). This model can be used to study the mutations in the human RPE65 gene that causes to Leber congenital amaurosis (Allikmets, 2004), a disease that causes of childhood blindness characterized by a severe retinal dystrophy at a young age. Although the mouse is the most commonly used model species for the study of retinal degeneration, the small eyes of the mouse means they are not ideal for modeling certain procedures that would need to be performed on human eyes for drug or gene therapy. The larger eyes of dogs and cats are more suitable for such manipulations. The dog model for arRP due to the PDE6B mutation has provided an opportunity to study the cellular mechanisms of the disease as described above. Briard dogs, an animal model of Leber congenital amaurosis in humans due to RPE65 mutation, have added to understanding the impact of the genetic defects on retinal cell death, and as a model for the use of gene therapy (Narfstrom et al., 2003). 52 Chapter 2 Detailed electroretinographic characterization of the PDE 6A dog phenotype 2.1. Introduction The electroretinogram (ERG) has been used as a measurement of retinal function in the study of many retinal diseases. Various procedures, based on light intensity, duration and color of light stimulus, or light or dark adaptation condition, can be used to identify and characterize the contributions to the ERG components from different neurons in the retinal pathways. Alterations of the shape of the ERG waveform are often used to qualitatively indicate reduced contributions of some pathways to the ERG waveforms. However, quantitative measurement of the amplitude and the time course (implicit time) of each ERG components can provide us with a measure of retinal function and how it is altered by the disease process. The ERG is a summation of the electrical responses recorded from the entire retina, meaning that quantitative measurement of isolated ERG components can be used to assess the function of specific retinal cells. For instance: variables of maximum rod response and rod sensitivity obtained by fitting the leading edge of the isolated rod a- wave in response to intensity flashes (Hood & Birch, 1996) have been used to assess rod photoreceptor function in patients with retinitis pigmentosa (RP) (Hood & Birch, 1994). Additionally, fitting the scotopic b-wave amplitude to a non-linear regression model 53 allowing assessment of maximal rod response and rod sensitivity has also been used to investigate rod function in RP patients (Birch & Fish, 1987). The purpose of this study was to use the ERG to characterize retinal dysfunction in the PDE6A mutant dog. 2.2. Materials and methods 2.2.1. Animals Homozygous PDE6A mutant, heterozygous PDE6A carrier, and homozygous PDE6A normal dogs were used in this experiment. Genotyping for the PDE6A mutation was performed as previously described (Petersen-Jones & Entz, 2002). All dogs were maintained at the Vivarium of the College of Veterinrary Medicine, Michigan State University under 12 hours light/dark cycles. 2.2.2. Electroretinographic recording 2.2.2.a Anesthesia Animals were anesthetized under a dim red light. Two anesthesia protocols were used based on the age of the animals: less than 9 weeks of age; induction and maintenance with halothane delivered in oxygen, at 9 weeks of age and higher; premedication with acepromazine maleate (0.1-0.3 mg/kg) intramuscularly, followed by 54 induction of anesthesia with intravenous thiopental sodium (Pentothal®; 6-12 mg/kg). Anesthesia was maintained with halothane delivered in oxygen. A pulse-oximeter (Vet/Ox 4400, Heska Corporation, Fort Collins, CO) was used to record pulse rate and oxygen saturation for the duration of the procedure. Body temperature was maintained using a heat pad. Pulse rate, oxygen saturation and body temperature were recorded every 5 minutes during the ERG recording. 2.2.2.b. Recording electrode placement and animal positioning Dogs were positioned in sternal recumbency. The left eye was used for ERG recording; the right eye was taped close. The left pupil was maximally dilated by applying 1% tropicamide (Mydriacyl®, Alcon Laboratories, Honolulu, HI) and 10% phenylephrine hydrochloride (AK-Dilate®, Akorn Inc, Buffalo Grove, IL). The globe was positioned in primary gaze with stay sutures of 4-0 silk (Ethicon, Inc, Piscataway, NJ) placed in the conjunctiva adjacent to the limbus. A drop of 2.5% hydroxypropyl methylcellulose solution (Goniosol®, Iolab Pharmaceutical Inc, Claremont, CA) was applied to keep the cornea moist. Burian-Allen bipolar contact lenses (Hansen Ophthalmic Development Laboratory, Coralville, IA) were used. These have a loop silver electrode that contacts the cornea and are referenced to a spectrum that contacts the palpebral conjunctiva and is coated in a silver conductive paint. The ground electrode consisted of needle electrode and was placed subcutaneously at the back of cervical region. 55 2.2.2.c. Electroretinographic recording Full-field short flash ERGs were recorded using the UTAS-E 3000 electrophysiology unit (LKC Technologies Inc; Gaithersburg, MD). The band pass was set at l to 500 Hz; gain setting varied from 2x103 to 4x104. The time base was set to record 20 msec pre-stimulus. Inter-stimulus intervals were set and ERG responses averaged based on flash intensities. Flash stimuli were delivered to the tested eye via a spherical bowl (Spafford et al., 1993) painted with reflective white paint. ERG responses were amplified and stored for further analysis. 2.2.3. Electroretinographic test protocols 2.2.3.a. Intensity-series electroretinography Intensity-series ERGs were recorded from four homozygous PDE6A mutant, five heterozygous PDE6A carriers, and four homozygous PDE6A normal dogs at 2, 3, 4, 5, 6, 7, 9, 12, 16, 20 and 52 weeks of age. 2.2.3.a.(i) Scotopic intensity-series electroretinography After dogs were dark-adapted for 60 minutes, scotopic ERG responses from a series of 16 white flash stimuli (-3.18, -2.98, -2.79, -2.6, -2.0, -1.6, -1.19, -0.79, -0.39, 56 -0.001, 0.39, 0.85, 1.36, 1.9, 2.38, and 2.82 log cds/mz) were recorded. Inter-stimulus intervals were increased from one second at low intensities to 360 seconds at the highest intensity to avoid light adapting the rods. Preliminary studies showed that these inter- stimulus intervals were adequate to present rod adaptation (data not shown). 2.2.3.a.(ii) Photopic intensity-series electroretinography Dogs were light-adapted to a rod saturating white background white light of 30 cd/m2 for 10 minutes after the dark-adapted ERG had been recorded. Photopic ERG responses were recorded from a series of 10 white flashes (~0.39, —O.22, —0.001, 0.16, 0.39, 0.85, 1.36, 1.9, 2.38, and 2.82 log cds/mz), superimposed on the same background white light. Inter—stimulus intervals were one second from -0.39 to 1.36 log eds/m2 and 5, 10, and 15 seconds for 1.9, 2.38, and 2.82 log cds/mz, respectively. 2.2.3.b. Flicker electroretinography Flicker ERGs were recorded from four homozygous mutant, five heterozygous carriers, and four homozygous normal dogs at 2, 3, 4, 5, 6, 7, 9, 12 and 16 weeks of age. Recording electrode placement and animal positioning were described in 2.2.2.b. The band pass was set at l to 500 Hz; gain setting varied from 4x103 to 4x104. 57 2.2.3.b.(i). Rod flicker ERG While the dog was dark-adapted, rod flicker ERG responses were recorded in response to white flashes of light -1.6 log cds/m2 in intensity at 5H2, and 15 tracings averaged. 2.2.3.b.(ii). Cone flicker ERG Cone flicker ERG was recorded while the dog was still dark-adapted using white flash stimulus at 0.39 log eds/m2 intensity at 33 Hz, and 15 tracings averaged. 2.2.3.c. Electroretinography using blue flashes Rod-mediated ERGs were recorded from dark-adapted retina using blue flashes (wavelength 400-560 nm) obtained with 3 #47A Wratten filter (Kodak, Rochester, NY). See 2.2.2.c. for ERG setting. 2.2.3.d. Long flash electroretinography Full-field long flash ERGs were recorded using a customized Ganzfeld stimulator unit connected to the UTAS-E 3000 electrophysiology unit (LKC Technologies Inc; Gaithersburg, MD). The flash duration was set at 150 msec. The light source was a 12-V 50-W tungsten-halogen lamp giving a maximum white light stimulus of 180 cds/mz. 58 Constant background illumination inside the Ganzfeld bowl was generated from a 12-V light bulb. A diffuser in front of the bulb was adjusted to give a homogeneous background white light of 42cd/m2 (Sieving, 1993). Responses were amplified with a bandwidth of 0.3 to 500 Hz, and 30 responses averaged. All ERG waveforms were averaged, stored and displayed by LKC software for further analysis. 2.2.4. Data analysis 2.2.4.a. The a- and b-wave amplitude and implicit time The a- and b-wave amplitude (microvolt; uV) and implicit time (millisecond; msec) were measured for each averaged response. The a-wave amplitude was measured from the onset of light stimulus to the trough of the first negative wave; b-wave amplitude from the trough of the first negative wave to the peak of the first positive wave. A-wave implicit time was time measured from the onset of the light stimulus to the time when the maximal a-wave trough occurred and b-wave implicit time from the onset of the light stimulus to the time when the peak b-wave was present. Means (+/- SEM) of scotopic and photopic ERG amplitudes and implicit time were calculated and plotted as a function of light stimulus. 59 2.2.4.b. Flicker amplitude and implicit time Flicker amplitude (uV) and implicit time (msec) were measured for the entire recording period (250msec) and averaged. Amplitude was measured from trough to peak of each wave; implicit time was duration of time measured from time at the trough to time at the peak of each wave. 2.2.4.c. Naka-Rushton function The first limb of the scotopic b-wave amplitude as a function of log stimulus was fitted by a non-linear regression with the 3-parameter Hill equation as follow: V (I) = (Vmax * I") / (1n + k") (1) In this equation, V, retinal responsiveness, is the b-wave amplitude (uV) to a stimulus of luminance I, Vmax is the maximum response amplitude, I is the stimulus luminance, k is the semi-saturation constant or luminance required to elicit a response equal to one-half the amplitude of Vmax, which is considered to represent retinal sensitivity, and n is proportional to the slope of the graph of equation 1 at the point where the stimulus luminance is taken to be k, and is considered an indicator of retinal homogeneity. The b-wave response curve was fit to equation 1 using SigrnaPlot, version 5.0 (Systat Software, Inc, Richmond, CA), and independent variables (Vmax, k and n) were derived. 60 From 2.2.4.a. to 2.2.4.c. the experiments have a repeated measures structure. Data was statistically analyzed using Proc Mixed, SAS version 9.1 (SAS Institute Inc., Cary, NC). Analysis of residuals were obtained and the experiment variables (amplitude, implicit time, Vmax, k value and n value) were transformed to a logarithmic scale to obtain approximately normally distributed residuals, and the value of the confidence interval (CI) was calculated. A set of models with the same fixed effects, but different covariance structures was compared using the Bayesian Information Criterion (BIC). The model with the smaller value (best fit) was selected. A different covariance structure for the repeated measures was allowed for each variable. Comparisons among fixed effects (age, genotype and their interaction) were performed based on the selected model. Interaction between age and genotype was tested at each flash intensity used. For any situations where statistical significance was found, difference among genotypes was tested at each given age. Data were considered significant at a level of significance less than 0.05 (P<0.05). 2.2.4.d. Criterion threshold of means ERG amplitudes Criterion threshold of means of ERG scotopic and photopic amplitudes were assigned based on data from normal control animals; 10 uV was selected for b-wave scotopic amplitude, 5 uV for a- and b-wave photopic and a-wave scotopic amplitudes. A calculation of flash intensity-at-criterion threshold was performed by linear interpolation. 61 On every occasion that the highest value of ERG amplitude was below the given threshold, the data was labeled as censored. Censored values were then replaced by the maximum light intensity (2.82 log cds/mz). A non-parametric ANOVA was computed using the Nparlway procedure of SAS (SAS version 9.1, SAS Institute Inc., Cary, NC). An exact Kruskal-Wallis test was used to analyze the difference among genotypes at each age. For ages where a significant difference among genotypes was found, a parametric Tukey-Kramer test was applied to detect differences between each genotype. Data were considered significant at a level of significance of less than 0.05 (P< 0. 05). 2.2.4.c. Rod-isolated responses Rod—isolated responses were derived by subtraction of intensity-matched photopic (cone only) ERG responses from scotopic (rod plus cone) ERG responses (Hood & Birch, 1990). The a-wave of the rod-isolated responses at 1.36 log cds/m2 were normalized for amplitude and the leading edge of the a-wave compared between PDE6A mutant and normal control dogs from 2 to 6 weeks of age. 2.2.4.f. Oscillatory potentials Oscillatory potentials are high-frequency ERG components located on ascending limb and peak of b-wave. Scotopic OPs were isolated from scotopic ERGs recorded from -0.001, 0.39, 0.85 log eds/m2 flash stimuli by electronically applying a band pass filter at frequencies of 73 to 500 Hz. OP amplitude was measured from the peak of a positive 62 wave to the preceding negative trough. Amplitudes of five OPs were summed in each dog, averaged within the genotype, and then compared between two homozygous PDE 6A mutant and two homozygous PDE6A normal dogs at 3, 5, 7, 9, and 12 weeks of age. 2.2.4.g. Photopic Negative Response Photopic Negative Response is a negative waveform that occurs after the photopic b—wave. PhNR at -0.001, 0.16, 0.39, 0.85, 1.36 log eds/m2 was compared between homozygous PDE6A mutant and homozygous PDE6A normal dogs at 5, 7, 12 and 50 weeks of age. 2.3. Results There were no significant differences (P>0.05) between the ERGs recorded from PDE6A carriers and the normal controls for any of the parameters examined in this study. 2.3.1. Scotopic ERG responses The scotopic ERG waveforms were analyzed in the following ways: 0 a— and b-wave amplitudes and implicit times 0 amplitude of a- and b-wave at selected criterion threshold (5 uV and 10 BV for a- and b-wave amplitude, respectively) 63 o the slope of the leading edge of the derived rod-isolated a-wave o the response in dark-adapted dogs to blue flashes of light 0 modeling of the first limb of the b-wave intensity-response curve by Naka- Rushton fit to derive maximal rod response and photoreceptor sensitivity 0 rod flicker responses 0 oscillatory potentials 2.3.1.a. Scotopic ERG amplitude (Figures 2.1, 2.2, 2.3, 2.4 & 2.5) Scotopic a- and b-waves could be recorded in normal controls and the PDE6A mutant dogs after the opening of the eyelids (approximately 2 weeks of age). With age, the mean a— and b-wave amplitudes of normal controls progressively increased and reached a peak at about 6 weeks of age (Figures 2.2A, 2.3 & 2.4) while the mean scotopic intensity threshold of a- and b-wave amplitudes continuously declined from 2 to 6 weeks of age, after which they slightly increased and then plateaued until 20 weeks of age (Figure 2.5). The mean scotopic a- and b-wave amplitudes from the mutant dogs (Figures 2.3 & 2.4) reached their peaks at approximately 3 weeks of age, at which time they were significantly smaller (P<0.05) compared to those of normal controls. Loss of a-wave response and OPs continued with age in the mutant dog (Figures 2.18 & 2.2B). The mean scotopic intensity threshold of the a-wave was significantly higher (P<0. 05) by 1.5 to 2.0 64 log units at all ages investigated compared to that of the normal controls (Figure 2.5). After 5 weeks of age, it did not reach the SuV-criterion threshold. Although the mean b-wave amplitude decreased from 3 weeks of age in the mutant dogs, a temporary increase was observed at 6 weeks of age followed by a constant decline (Figure 2.4). The ratio of b-wave amplitude to a- wave amplitude (b/a ratio) was relatively constant in normal controls, whereas it increased in mutant dogs with increasing age and stayed at abnormally high levels throughout the period ERGs could be recorded. With age, development of the b-wave intensity threshold of the mutant dogs paralleled that of normal controls until 5-6 weeks of age, after which there was a sharp increase in threshold from 7 to 9 weeks of age. The mean b-wave threshold values were significantly higher (P< 0.05) by about 2.5 log units than those of the normal controls at all ages investigated (Figure 2.5). It is of interest that the scotopic and photopic waveforms of mutant dogs were similar suggesting a response primarily from cones. 65 -3.18 -2.98 -2.79 -2.6 -2.10 -1.6 4.19 -0.79 —O.39 -0.001 0.39 0.85 1.36 1.9 2.38 2.82 7777????» -20 O 20 4O 80 80 100 120 -20 0 20 40 60 80 100 120 Time (msec) Time (msec) Figure 2.1. Representative scotopic ERG recordings from a normal control (A) and the PDE6A mutant dog (B) at 4 weeks of age. Light intensities (cds/mz) are indicated in the figure. The onset of flash is at 0 msec. A) In the normal control, scotopic ERG amplitude increases with flash intensity whereas ERG implicit time shortens. The b-wave threshold is at -2.79 log eds/m2. The a-wave threshold is at approximately -0.39 log eds/m2. Both a- and b-wave amplitude increases with increasing intensity. Four small oscillatory potential wavelets are present on the b-wave, in particular at high light intensities. B) The b-wave threshold of the PDE6A mutant dog is elevated (-2.10 log cds/mz) compared to that of the normal control. The a-wave is much reduced at this age and the b-wave response is small and peaks at an intensity of 0.001 log eds/m2 then decreases with further increases in light intensity. This is suggestive of a predominantly cone response. B-wave implicit time normally gets shorter with higher light intensities, whereas in the mutant dog it tends to stay the same at first then increases. ERGs of this mutant dog can not be recorded at very high light intensities (2.38 and 2.82 log eds/m2). Size bars indicate amplitude in microvolt. 66 3.18 m m 2.98 FA 2.79 A 2 6 2.82 W 2.38 r V r -20 0 20 4O 60 80 100 12) -20 0 20 40 60 80 100 120 Time (msec) Time (msec) Figure 2.2. Representative scotopic ERG recordings from a normal control (A) and the PDE6A mutant dog (B) at 6 weeks of age. Light intensities (eds/m2) are indicated on the figure. The onset of flash is at 0 msec. A) Compared to responses at 4 weeks of age (Figure 2.1) the b-wave threshold of the normal control is decreased (-3.18 log eds/m2) while a- and b-wave amplitudes are increased. Oscillatory potentials are more prominent. B) Compared to the normal control, intensity threshold of the PDE6A mutant dog is markedly higher (-0.79 log cds/mz). A small a-wave is present only at very bright flash intensities while the b-wave amplitude is markedly reduced and decreases with increasing flash intensity after 0.85 cds/mz. The b-wave implicit time in the normal control decreases with increasing stimulus intensity whereas in the mutant dog the implicit time increases with the brighter intensities. Size bars indicate amplitude in microvolt. 67 r l i T 100i 9 i 3 l O l“ 3 l =roe E t a j: ------------------------ 8" i -‘ r 1e i 4' Z. l 0,11 -- -_.__-_ _ -4 -3 -2 -l O l 2 3 4 Logintensity(cdslm2) —O—-2wk —*-—3wk ~t~~4wk -—>€—5wk +6wk +7wk -—+--9wk 12wk *~——16wk +20% ---+---2wk -------3wk - A~~4wk ----x--~-5wk -~--x---~6wk “-0'm7wk ~-+---9wk -------- 12wk ------16wk ~~O~~20wk Figure 2.3. Mean scotopic a—wave amplitudes with standard errors (1x +/- SEM) plotted against stimulus intensity on a log-log scale for normal control and PDE6A mutant puppies. The selected a-wave scotopic criterion threshold is 5 uV (dashed black line). Solid lines and dot lines represent values from normal controls and the mutant dogs, respectively. Various colors represent the mean scotopic a-wave amplitude at different ages. (11 = 4 for each group of dogs) Note a significant difference (P< 0. 05) of the mean a- wave amplitude is found between the normal controls and the PDE6A mutant dogs at all age groups investigated. The PDE6A mutant photoreceptors do not respond to light at low intensities, and after 3 weeks of age their mean amplitudes decrease at very bright intensities. Increased scotopic a—wave criterion threshold in the PDE6A mutant reflects a marked reduction of photoreceptor sensitivity. Key: [iv = microvolt, wk = week. 68 Log amplitude (uV) -4 -3 -2 -l 0 l 2 3 4 Log intensity (eds/m2) —+—2wk +3wk -*-—— 4wk —-X—5wk +6wk -O— 7wk —+-— 9wk 12wk 16wk ——O-- 20wk ~--+~-2wk ---~'----3wk ~ ~A~~-4wk ""Xm-ka WX6wk m‘m'7wk “-+'“9wk """""" 12wk ~--~-l6wk ““0"“20wk Figure 2.4. Mean scotopic b-wave amplitudes with standard errors (1x +/- SEM) plotted against stimulus intensity on a log—log scale fi'om normal control and PDE6A mutant puppies. The selected b-wave scotopic criterion threshold is 10 uV (dashed black line). Solid lines and dot lines represent values from normal controls and the mutant dogs, respectively. Various colors represent the mean scotopic b-wave amplitude at different ages. (n = 4 for each group of dogs). Note a significant difference (P< 0. 05) of the mean b-wave amplitude is found between the normal controls and the PDE 6A mutant dogs at all age groups investigated. The mean scotopic b-wave amplitudes in both normal and PDE6A mutant dogs develop to peak level at 6 weeks of age. In the normal control after this age there is a slight decrease to adult levels. In the PDE6A mutant dogs, there is a progressive decrease in amplitudes with age. At 6 weeks of age the 10 uV-amplitude criterion threshold is approximately 2.5 log units higher in the mutant dogs than in the controls, after that the b-wave amplitudes in the mutant dogs decline to below the threshold criterion level. Key: pV = rrricrovolt, wk = week. 69 9w H." 191w Log intensity (eds/m?) I .9 or r'o H-l l-H m m m m m 1'1 -3. r: -3-5 1 T fl I l T l I l l l 0 2 4 6 8 10 12 14 16 18 20 22 Age (week) ==I PDE6A +I+ — PDE6A -/- Figure 2.5. Mean scotopic intensity threshold with standard errors (1x +/- SEM) using SuV and 10uV criterion for a- and b-wave amplitude from normal control and PDE6A mutant dogs at all ages. (n = 4 for each group of dog) Solid lines represent a-wave intensity threshold, and dot lines represent b-wave intensity threshold. Note that significantly higher (P< 0. 05) mean a- and b-wave intensity thresholds are observed in the PDE6A mutant group at all ages examined. Key: * and 3 indicate statistical differences of the a- and b-wave intensity threshold, respectively, at P<0.05. 70 2.3.1.b. Scotopic ERG implicit time (Figures 2.1, 2.2, 2.6 & 2.7) The mean a- and b-wave implicit times of normal control dogs decrease with increasing light intensities. Development of the implicit times is related to age. In normal controls, a- and b-wave implicit times shortened over the period of retinal maturation to 6 or 7 weeks of age and then stayed similar for the rest of the study period. A delay of both implicit times was observed in the ERGs recorded from mutant dogs (examples are shown in Figure 2.18 & 2.28). A significant delay (P<0.05) of the mean a-wave implicit time was found at 2, 3, 7, 16 and 20 weeks of age at 0.39 log eds/m2 at lower intensities, whereas it was significantly delayed at 7 weeks of age compared to normal control at higher intensities (Figure 2.6). For the mean b-wave implicit time, it was significantly delayed (P<0.05) in the mutant dogs at 4, 5, 7, 9, 12, 16 and 20 weeks of age at low light intensities, as well as at intensities higher than 0.85 log cds/m2 (Figure 2.7). 71 roo —. 100 . Log imptcit time (msec) 8 Log implicit time (msec) 8 g -4 -3 -2 - l 0 l 2 3 4 -4 -3 -2 -l 0 l 2 3 4 Log iniemity (cdslrrr?) Log imeneity (edslm?) +2wk -—‘—-3wk -- t- --4wk —X—5wk ---§---2wk “'I'~3wk " A-“4wk ""Xm-ka 100 ' LA ¢jp 0 Log implant time (msec) 5 Log implicit time (msec) 2'5 l r r r T l r r r -4 -3 -2 -l 0 l 2 3 4 -4 -3 -2 -l 0 l 2 3 4 Log irtensity (edslm?) Log irkemity (cddm?) +6wk +7wk —-+—-9wk 12wk ----x----6wk ---o--~7wk ----+-~-9wk -------- 12wk E. 100 W —-—— — — —— 1 E g. . "5 10 3; R‘i ’6 l a i ‘3 .§ " 1 o l 3 .1 4-3-2-101234 Log idemity (eds/m?) - — 16wk ——9— 20wk -~~--~-16wk ---+~-20wk Figure 2.6. Mean scotopic a-wave implicit times with standard errors (1x +/- SEM) plotted against stimulus intensity on a log-log scale from normal control and PDE6A mutant dogs at 2 & 3 (A), 4 & 5(8), 6 & 7 (C), 9 & 12 (D) and 16 & 20 (E) weeks of age. Solid lines and dot lines represent values from normal controls and the mutant dogs, respectively. Various colors represent the mean scotopic a-wave implicit time at different ages. (11 = 4 for each group of dogs). Note a significant delay (P<0. 05) of the mean scotopic a-wave implicit time of the PDE6A mutant group is found at light intensity between 1 to 2 log eds/m2 in most of age_groups. Key: msec = millisecond, wk = week. 72 E i g "We-2i. g , M a: ‘ f .- '6 2a 10 ‘3 10 g E o o 3 l T T T T Y I 1 3 l T T T T I l -4 -3 -2 -l 0 l 2 3 4 -4 -3 -2 -l 0 l 2 3 4 Log irlensity (eds/m?) Log itlensity (eds/m?) + 2wk -——I— 3wk —-—--—e— 4wk -—X—-5wk M...2wk --l--3wk ‘ ~4wk ""X""5Wk C. D If“) ‘1’ ------- NIX) T — r — i 1 i v 1m ‘L v 1m 2» s 1 = g ’5 i '0 j a lo 1 l s : E " § s? 1 Y I 7 r r r r 1 4-3-2-10l234 4-3-2-101234 Loo idemity (eds/m?) Log imemfly (Wm?) —ai-a'--—-6wk —O—7wk ——+——9er —~—12wk ,_ X 6wk ---O~-'7wk ----+----9wk ------- 12wk E. 1W ' g “m M g fihfit’r"h i m g , o 3 I l r r f T . T T a -4 -3 -2 -l 0 l 2 3 4 Logimensilymdslrn?) ___.— 46er —o—-20wk .-- 16wk ~-+-'-20wk Figure 2.7. Mean scotopic b—wave implicit times with standard errors (1x +/- SEM) plotted against stimulus intensity on a log-log scale from normal control and PDE6A mutant dogs at 2 & 3 (A), 4 & 5(8), 6 & 7 (C), 9 & 12 (D) and 16 & 20 (E) weeks of age. Solid lines and dot lines represent values from normal controls and the mutant dogs, respectively. Various colors represent the mean scotopic b-wave implicit time at different ages. (1) = 4 for each group of dogs). Note that from statistical data, the mean scotopic b- wave implicit times between the two groups are comparable at 2 to 3 weeks of age (A). A significantly delayed implicit time (P< 0. 05) is observed at ages greater than 4 weeks, and at intensities higher than 1 log eds/m2 in particular. Key: msec = millisecond, wk = week. 73 2.3.1.c. Rod-isolated a-wave response In an attempt to detect the presence of a rod-mediated a-wave in the PDE6A mutant dog, a subtraction of cone-mediated responses from mixed rod-cone responses was performed. To allow a comparison of a-wave slope, subtracted waveforms were normalized for a-wave amplitude (Figure 2.8). The subtraction showed that the rod- mediated a-wave responses of the PDE6A mutant dogs were either non-recordable or delayed. At 3 weeks of age a very low amplitude negative waveform of delayed implicit time (for an a—wave) was present after the subtraction and preceded a positive waveform (b-wave) also of increased implicit time compared to the normal control. With such low amplitude responses it is difficult to be certain that this actually represents a true rod- driven a-wave. Furthermore the subtraction of light-adapted cone responses from dark- adapted cone responses may have affected the appearance of the final waveform because it is known that dark-adapted cone response differ from those of light-adapted cones. 74 20« M i f \ A 10 ,, "i ‘ > - g a) 0m ,. / 5“ ___‘fi __ '0 l' ' ' 3 2 ‘1 E .101 ll \ ' < '20" '. I 1.6 — -30.! 2] 14 l V ' q .401 . a a e 4 . - - . . 1.2 a o 20 40 so so 100 120 140 150 130 200 220 240 Time (msec) .1.2~.Tfi, .,-L 0510152025303540455055 Time (msec) =2: PDE6A +/+ — PDE6A -/. Figure 2.8. Normalization (for a-wave amplitude) of the rod-isolated a-wave derived from a subtraction of intensity-matched photopic and scotopic ERGs in a normal control and the PDE6A mutant dog at 3 weeks of age. The onset of flash is at 0 msec. Inset illustrates actual scotopic ERG responses from the same normal control and the PDE6A mutant dog from which the normalized waves are derived. Note that rod-isolated ERG responses in the PDE6A mutant dog are much reduced compared to the normal control (inset). The time to peak of the normalized “a-wave” of the PDE6A mutant dog is considerably delayed at 50 milliseconds, compared to 15 milliseconds in a normal control. Key: uV = microvolt, msec = millisecond. 75 2.3.1.d. Scotopic ERG responses to blue light flashes Dim blue flashes were used to preferentially stimulate rod photoreceptors of dark— adapted dogs. In normal controls the scotopic ERG response to blue flashes developed at 2 weeks of age (data not shown). The response grew in amplitude with age, to peak at 6 and 7 weeks of age for a- and b-wave amplitude, respectively. The blue flash ERG of PDE6A mutant dogs in contrast was of very low amplitude. An a-wave could not be recorded at any age. A b-wave of very low amplitude could only be detected at 7 weeks of age (Figure 2.9). 20 11V 0 20 40 6O 80 100 120 140 160 180 200 220 240 Time (msec) === PDE6A +/+ — PDE6A -/- Figure 2.9. Representative recordings of rod-mediated ERG response to dim blue flashes of light from dark-adapted normal controls and PDE6A mutant dogs at 4 and 7 weeks of age. The onset of flash is at 0 msec. Note the amplitude of the waveform from the normal control dog increases with age. ERG responses are barely detectable in the PDE6A mutant dog suggesting very reduced or absent rod responses. Key: uV = microvolt, msec = millisecond, wk = week. 76 2.3.l.e. Naka-Rushton fitting of the first limb of the scotopic intensity-response CUTVCS A plot of b-wave amplitude against stimulus intensity typically results in a two- limbed curve. The first limb is considered to primarily represent rod responses and the second limb consists of a saturated rod response plus cone response. To analyze rod responses the first limb of the response curve is fitted using the Naka-Rushton formula. The Naka-Rushton equation (non-linear regression) successfully fitted the first limb (portion) of the normal canine scotopic b-wave intensity-response curve (Figure 2.10). The correlation coefficient (Burstedt et al., 2000), which is a measure of the covariation in the magnitude of two variables, was used to describe how well the predicted value from the model (predicted by Naka-Rushton equation) fits with the actual data (scotopic b-wave amplitude from the first limb). R2 is a number between 0 and 1.0. As the strength of the relationship between the predicted values and actual values increases, the R2 value is close to 1.0. The majority of the first—limb scotopic b-wave of the normal control dogs had R2 value at 0.97 to 0.99 except for 0.93 at 2 weeks of age. The PDE6A mutant dogs also had R2 value at 0.93 at 2 weeks of age. Even though the value increased to 0.97 at 4 to 5 weeks of age, it decreased afterward to approximately 0.88 at older ages indicating a poorer fit between the predicted value and the actual data. 77 120 _ l()0 i o l Normal control I; O :1 O E 80 r B o E g 60 ~ ° Q) 56 E ______________ mam ......... 40 q ”.1 ”x PDEGA mutant ..X7 I ‘/2 Vmax (1) x' Vmax (2) ' 20‘J .qu . ‘/2 Vmax (2) ,1" : X*%‘ W i 0 I ' ' ‘ ‘ ' k(1) k(2) -4 -3 -2 -l 0 l 2 3 4 Log intensity (eds/m2) Figure 2.10. Scotopic b-wave intensity-response function obtained from a normal control (diamond symbol) and the PDE6A mutant dog (square symbol) at 3 weeks of age. The superimposed curve represents the predicted rod b-wave amplitude from normal control (dot curve) and the PDE 6A mutant dog (continuous curve) that are fitted from the Naka- Rushton model. Note that the PDE6A mutant dog has a low Vmax and high k value compared to normal controls, which indicates low rod response and low rod sensitivity. Key: 1 = normal control, 2 = the mutant dog, Vmax = maximal rod response, k = log intensity at half maximal rod response. A progressive increase of the mean Vrnax was observed from 2 to 6 weeks of age in both normal control and the PDE6A mutant groups although it was considerably slower for the PDE6A mutant group (Figure 2.11A). The mean Vmax reached the peak at 6 weeks of age at 102.93 +/- 9.09 BV and 19.7 +/- 7.42 1.1V for normal controls and the PDE6A mutant dogs, respectively. Following 6 weeks of age, it was maintained in the normal controls but progressively declined in the mutant dogs. A significant difference 78 (P<0.05) of the mean Vmax for the PDE6A mutant groups was found at all ages investigated except at 2 weeks of age. The k variable corresponds to the luminance required to elicit a b-wave amplitude equal to one-half the amplitude of Vmax, and it is considered to represent rod sensitivity. In the normal control, the mean k value rapidly decreased from 2 (3.87 +/- 2.11) to 4 (0.06 +/- 0.03) weeks of age whereas it slowly declined in the PDE6A mutant dogs (1.36 +/- 0.25 to 0.85 +/- 0.39). After that, the mean k value continued to decrease in both groups of dogs however, that of the PDE6A mutant dog was significantly elevated (P<0.05) compared to normal controls (Figure 2.118). The n variable represents retinal homogeneity and is thought to be superior when it is close to 1.0 in normal retina. The mean n value was significantly larger in the PDE6A mutant dogs at all ages except for at 4 weeks of age (Figure 2.11C). 79 2968:: u >2 .36v& 8 moist? :88 2: mo 85856 338265 8:8me ... unav— 6068 8mm #68 8 98% E82: ~06QO 2: E Awedvn: 2:80ch banfiEwB 2m mug—amt? :oEmzm $me :88 =« 35 202 Row mo 98% some 88 v n 5 .mqsew own =m 8 mwou ESE: VeMQm 2: can 205:8 38.5: 2: mo ADV 03? a can Am: 029 v— .A ”mo—para.» :ofimaméxmz of mo 32mm + x: 88.5 28:me Ba :88 05 .8 83388 < A 2.. chewE 2858... NNQNEEEESm e e N o r F F Li _ p _ _ p L r o a- (omen. IIII 1+ (omen. .IilHu SDIBA u 2093 oo< :35 mod. NN cm 2 a. v. N. o. w o v N o NN cm 2 o. I N_ o. w c v N o if r L :9 h b p p l? r —1 . p p p p p F _ L L p O lv—1 F—o H O 00 (N1) xBUJA (zur/spo) anieA )j H H r .2. CN— l" \O V) V }——i T r ev— 80 2.3.1.f. Rod flicker ERG response Rod flicker response in normal dogs was detectable at 3 weeks of age, and had an adult-like appearance by 4 weeks of age. Its peak amplitude and implicit time was found at 6 weeks of age in the normal controls. In comparison, a rod flicker response could not be recorded in the PDE 6A mutant dogs (Figure 2.12). 81 .0663 u :3 8:88:58 n 82: £08.88: n >1 93M .woc 882: “.6QO a Soc 8898: 8on 8 82 65 082 8:85 8on .8 88¢ 888?: :8 988m .8? Subaru 8 av we: 88:: VbMQR 2: 28 2V 8:86 388: m 82.: 82m .~E\m8 we. QTV 880:8: .685 8: 03888682 .8 88888 < .N_.~ 8:me A895 oEP 8095 95» ooom owe—con. 3282 of. com o2 o coon om: com—own. coo. own cow onm o q._+_____1_ .J..n..._. {E 12% >18 x26 rig; ism i? {325% x>>N 82 2.3.1 . g. Oscillatory potentials Four major OPs are present in the ERG of normal dogs with the greatest peak at the 0P3 (Figure 2.13). In normal controls, OPs obtained from 0.85 log eds/m2 flash stimulus were first observed at 3 weeks of age with a mean summed value of 22.95 +/- 6.65 uV, and then peaked at 9 weeks of age (131.4 +/- 16.8 11V). Very small values of summed OPs were measured in the PDE6A mutant dogs at all ages investigated compared to normal controls. There was no significant change in OPs recorded from mutant dogs from 3 to 12 weeks of age (Figure 2.14). 10pV O 10 20 30 40 50 6O 70 80 Time (msec) u:=a PDEGA +l+ _ PDE6A -/- Figure 2.13. Representative scotopic oscillatory potential (0.85 log cds/mz) from normal control and PDE6A mutant dogs at 5 weeks of age (band pass at 73 to 500 Hz). The light onset is at 0 msec. Note that the scotopic ERG response of the mutant dog does not contain a typical canine OP feature such as that seen in the normal control. Key: 11V = microvolt, msec = millisecond. 83 160 j .40 l r 3 1204 T 8 3 100 i r 5% 80* 8 a 60‘ e 51 4°“ T . 20- 0 LL______-__h_____i_ 3 5 ‘ 7 9 12 Age (week) =2 PDE6A +/+ — PDE6A -I- Figure 2.14. Mean scotopic oscillatory potential amplitudes with standard errors (1x + SEM) of normal controls and the PDE6A mutant dogs plotted against age. (11 = 2 for each group of dogs). Note that the PDE6A mutant dogs have diminutive OP amplitudes at all ages investigated. Key: uV = microvolt. 2.3.2. Photopic ERG responses The photopic ERG waveforms were analyzed in the following ways: 0 a- and b-wave amplitudes and implicit times 0 amplitude of a- and b-wave at selected criterion threshold (5 11V) 0 cone flicker responses 0 photopic negative responses 0 long flash ERG responses 84 2.3.2.a. Photopic ERG amplitudes (Figures 2.15, 2.16, 2.17 & 2.18) The mean photopic ERG amplitudes of normal controls increased with increasing flash intensity up to an intensity of about 1.9 log cds/mz. The amplitudes, particularly of the b-wave tended to decrease slightly at the brightest of intensity. In the PDE6A mutant dogs the means of both a- and b-wave peaked at 3 weeks of age, after which the mean a- wave amplitudes progressively decreased and were significantly smaller (P<0.05) compared to those of normal controls. Following a comparable photopic a-wave intensity threshold at 2 weeks of age between the PDE6A mutant group and normal controls, the mean intensity threshold of the PDE6A mutant dogs was significantly higher (P<0.05) than the normal controls (Figure 2.18). Although after 3 weeks of age, the mean a-wave photopic amplitudes of the mutant dogs decreased with age, those of the b-wave were relatively well maintained (Figure 2.17). By 6 weeks of age (Figures 2.15D & 2.16C), the mean a—wave amplitude of the PDE6A mutants did not reach the threshold criterion, while the threshold of the normal controls remained constant (Figure 2.18). A significant difference (P<0.05) between the mean photopic a-wave amplitude of the two groups of dogs was found from 6 weeks of age onward at the higher light intensities investigated (Figure 2.16C to E). The mean b-wave amplitudes were comparable until 7 weeks of age then decreased afterward particularly at the higher intensities (Figure 2.17). The b-wave intensity threshold response was also comparable between the two groups throughout the 85 investigation (Figure 2.18). The mutant dogs had a significantly smaller b-wave amplitude (P<0.05) for the brightest stimulus intensities at 9, 12 and 16 weeks of age. 86 -0.39 -039 M 0.22 M -022 M; L_ A _. 000‘ -o.oo1—"'r-" \v L, -20 o 20 4o 60 80 100 120 .20 o 20 40 so so 100 120 Time (msec) Time (msec) C. D. .039 __ ‘* -O.39 '8‘ A '0.22 MW '0,22 “7 A -0.001M— -o.001 M ’0.39 M A L_——- -0.39 M 0.39 Av“; 1.9 1.36 m 2.38 W 1.9 W F I I I r I I T I T j I I -20 0 20 40 60 80 100 120 .20 0 20 40 80 80 100 120 Time (msec) Time (msec) Figure 2.15. Representative photopic ERG recordings from a normal control (A & C) and the PDE6A mutant dog (8 & D) at 4 (A & B) and 6 (C & D) weeks of age. Light intensities are indicated in the figure. The onset of flash is at 0 msec. A & B) In the normal control at 4 weeks of age, photopic ERG responses consist of a- and b-wave. A reduction in b-wave amplitude occurs at very bright flashes such as 2.38 and 2.82 log eds/m2. This phenomenon is known as the photopic hill. In the PDE6A mutant dog in contrast, the a-wave is considerably smaller while b-wave is similar in amplitude to normal control. C & D) At 6 weeks of age, a small a-wave is only recordable in the PDE6A mutant dog at 0.16 and 1.36 log eds/m2. The b-wave is still present at all light intensities, but is reduced in amplitude in comparison to that of the normal control. Size bars indicate amplitude in microvolt. 87 100 {Air — ———--’— ——-— —— 1(1) 1’3 1 A 3: i A it 2 § i g I. ‘E, f v i o a Q E 1 E o o 3 3 +» J 0'1 V‘ V‘ V 0.] T T r T 1 2 l 0 l 2 3 4 -2 -l 0 l 2 3 4 Log insanity (cdslm?) Log intensity (eds/m2) + 2wk + 3wk —t—— 4wk -—)(— ka ---+---2wk “"I-m3wk A '4wk '°'°X""ka . D 100 irg - —— —— '-— — 100 37—“ — 1 1‘ ’6‘ ’6 .. 3 . . E. E 10 o o 3. E . °~ “ii a i 5 * 3 - § 4 - 0" V f I 0.] 1 fit 2 l O l 2 3 4 -2 l 0 l 2 3 4 Log intensity (cdelm?) Log imemity (eds/m3) —-*-—6wk --0--7wk _,_ ~vx~~6wk -.-.--.M --—-+-»—-%E -------- 313‘; E. 100 - -—- — — ~ é o 3 g a. E O D .3 0.1 r 2 l 0 l 2 3 4 Logimemitfiodslm?) ———- 16wk +20wk ---’ 16wk ~-+'-‘20wk Figure 2.l6. Mean photopic a-wave amplitudes with standard errors (1x +/- SEM) plotted against stimulus intensity on a log-log scale from normal control and PDE6A mutant dogsat2&3(A),4&5(B),6&7(C),9&12(D)and l6&20(E)weeksofage.The selected a-wave photopic criterion threshold is 5 uV (dashed black line). Solid lines and dot lines represent values from normal controls and the mutant dogs, respectively. Various colors represent the mean photopic a-wave amplitudes at different ages. (11 = 4 for each group of dogs) Note that at flash intensities higher than 0.5 log eds/m2, a significant difference (P< 0. 05) in the mean photopic a-wave amplitudes between the two groups of dog is observed at all ages for nearly all intensities. After 5 weeks of age the a- wave amplitudes of the PDE6A mutant dogs do not reach the selected criterion threshold. 88 100 — i 100 — o o 3 8 5 a E ; E ° , s o " 01 - = 'J 0.1 f T . T -2 -l 0 l 2 3 4 -2 -l 0 1 2 3 4 Log idensity (edslm?) Loo imamily (eds/m?) +2wk —|—3wk —t—4wk --X—5wk ---+---2wk ~ -~~3wk A~~4wk ----x----5wk C. D. e A it s : ~ g ' E 101:L o 0 IP 3 3 : E 3 g g 1 ‘5 a 3 5' 6 § ‘ 9 0.1 T -2 l 0 l 2 3 4 Logimensitnedslm?) —+——9wk 12wk ----+----9wk -------- 12wk i I 3 a O. E a § 2 -l 0 l 2 3 4 Logirlersitficdslm?) W‘— 16wk —9——20wk --'---—l6wk ---+~--20wk Figure 2.17. Mean photopic b-wave amplitudes with standard errors (1x +/- SEM) plotted against stimulus intensity on a log-log scale from normal control and PDE6A mutant dogsat2&3 (A),4&5 (B),6&7(C),9& 12(D)and l6&20(E)weeksofage. The selected b-wave photopic criterion threshold is 5 uV (dashed black line). Solid lines and dot lines represent values from normal controls and the mutant dogs, respectively. Various colors represent the mean photopic b—wave amplitudes at different ages. (n = 4 for each group of dogs) The mean photopic b-wave amplitudes are similar between control and PDE6A mutant dogs except for the brighter intensities from 9 weeks of age onwards when the amplitudes are significantly smaller (P<0. 05) in mutant dogs. 89 N N 01 co 1. _J.___J o i——¢——i i—HH—i 1.5 7 i—-¢—-i Log intensity (eds/m2) w I f ...... 1 { HH NH 914—: HH m ~11 r r f T l r T fir T T l 0 2 4 6 8 1O 12 14 16 18 20 22 Age (week) :2: PDE6A +/+ — PDE6A -/- Figure 2.18. Mean intensity at threshold from photopic recordings with standard errors (1x +/- SEM) using a SuV criterion for a- and b-wave amplitudes from normal control and PDE6A mutant groups at all ages tested. (n = 4 for each group of dog) Solid lines represent a-wave intensity threshold, and dot lines represent b-wave intensity threshold. Note that there is a significant difference (P<0. 05) in mean a-wave intensity threshold in the PDE6A mutant group at 3 weeks of age and older whereas there is no significance difference (P>0.05) in b-wave intensity threshold between the two groups. Key: * indicates statistical difference of the a-wave intensity threshold at P< 0.05. 90 2.3.2.b. Photopic ERG implicit time (Figures 2.19 & 2.20) The mean photopic a- and b-wave implicit times tended to decrease with increasing light intensity in normal dogs. There was a more marked reduction in a-wave implicit time than b-wave implicit time. The a- and b-wave implicit times of the mutant dogs were similar to controls. For the older age groups tested, the a-wave of the mutant dogs was not always recordable in response to brighter flashes and in some instances when it was still recordable had a longer implicit time at the brighter intensities. 91 mo “A. ___j 100 $3. 2 g ' If F ' E “ x- . .g ,0 I '3. 10 1; IN 2% :z a a :F E . .g .. 3 i g . o i _. r _J l T T i l 1 T T I T I -4 -3 -2 -l 0 l 2 3 4 -4 -3 -2 -1 0 l 2 3 4 Log idensity (eds/m?) Log imensity (cdslm’) + 2wk + 3wk + 4wk -—)(—- ka ---+---2wk ------~-3wk ~~~A---4wk ----><----5wk C. D 100 i: "-—'—"— “| 100 2 1 g 4. g 4 ' . . ., i g .. . .- 'f 10 . . i 101: "4’. g : i ’ .6 2* ‘ ‘ : a .0 g «» i .g 4» o o , 3 i 3 l l T . - r 1 4‘ l I T T -4 -3 -2 -l 0 l 2 3 4 -4 -3 -2 -1 0 1 2 3 4 Log idensity (cdslm’) Log imemity (cdsImZ) +6wk —O—7wk —+—9wk 12wk '-'-X-~--6wk WOW-7wk ---+~-9wk -------- 12wk E. 100 4: -- - ~— 4» -r i i H i v ' o 10': f: i I i ‘ i I ! Log impicit time (msec) ] T fl T l -4 -3 -2 -l 0 1 2 3 4 Log intensity (eds/m?) —-——16wk —--o—-20wk -l6wk ---+~~-20wk Figure 2.19. Mean photopic a-wave implicit times with standard errors (1x +/- SEM) plotted against stimulus intensity on a log-log scale from normal control and PDE6A mutant dogs at 2 & 3 (A), 4 & 5 (B), 6 & 7 (C), 9 &12 (D) and 16 & 20 (E) weeks of age. Solid lines and dot lines represent values from normal controls and the mutant dogs, respectively. Various colors represent the mean photopic a-wave implicit times at different ages. (n = 4 for each group of dogs) Note a similar pattern of a decrease in the mean photopic a-wave implicit time with increasing light intensity in both groups of dogs. From 9 to 20 weeks of age the a-wave implicit time of the mutant dogs is significantly increased compared to controls (P< 0. 05) and not recordable at some of the brightest intensities. 92 MIX) 1 — 1am I A 3: g: 3E . , . g, 100 0 ' £100 ‘5; g 1 M l 3 5E M '5 i '3 3' .o-' i '6 5;; l0 3. , E1 ‘0 1f g , l E: 2: -‘ 1 T T . r - 4 "I 1 . 7 T . -4 -3 -2 -l 0 1 2 3 4 -4 -3 -2 -l 0 l 2 3 4 Log irtensity (eds/m?) Log irtensity (eds/m2) -—+-— 3wk ——1— —-—— 4wk --)(— ka ---—-~-~3wk -~~A---'4wk WXka D. 5 1000 g. g ‘ :2: .00 1* V _ _ :1 3: _. 5 a f ’6 .. g , a 101 E l E II D a 4. 3 J 3 1 ‘ 4-3-2-101234 4-3-2-101234 Log intensity (eds/m?) Log intensity (eds/m?) ——3K—6wk —-O—-7wk -—+—9wk 12wk Wx6wk ....-..-7wk -~+---~9wk """" lZWk E. 1000 7 ‘ ———"**‘i ’2? 5t ’ g 100 3* g 1: ' *M-ar“ ’6 T3 10 ' .E i a 4 3 l f r T l -4 -3 -2 -l 0 l 2 3 4 Log "tensity (cdslm?) ———~-——- 16wk —-—o—-— 20wk -----~--16wk -~+---20wk Figure 2.20. Mean photopic b-wave implicit times with standard errors (1x +/- SEM) plotted against stimulus intensity on a log-log scale from normal control and PDE6A mutant dogs at 2 & 3 (A), 4 & 5 (B), 6 & 7 (C), 9 & 12 (D) and 16 & 20 (E) weeks of age. Solid lines and dot lines represent values from normal controls and the mutant dogs, respectively. Various colors represent the mean photopic b-wave implicit times at different ages. (11 = 4 for each group of dogs) Note that the mean b-wave implicit time tends to become significantly delayed (P<0. 05) from 5 weeks of age at higher light intensity and in older puppies also at lower intensities. 93 2.3.2.c. Cone flicker ERG response (Figures 2.21 & 2.22) The cone flicker response could be detected from 3 weeks of age and at that age was similar between normal controls and mutant dogs. In normal controls the amplitude increased with retinal maturation to peak at 6 weeks of age. This was followed by a gradual decrease with age. In the PDE6A mutant dogs the cone flicker amplitude followed a similar pattern to the normal controls, however it was significantly smaller (P<0.05) from 5 weeks of age onward (Figure 2.22A) although at 20 weeks of age the difference was not significant. By 52 weeks of age the cone flicker response could not be detected in the PDE6A mutant dogs (data not shown). A similar pattern of the mean cone flicker implicit time development was also observed; it slightly increased with age prior to 5 weeks of age in normal controls and mutant dogs and then progressively decreased until 16 weeks of age. Except at 3 weeks of age, the mean cone flicker implicit time was smaller in the PDE6A mutant dogs compared to the normal controls, but these differences were not significant (Figure 2.228). 94 IOpV L l l l l l l 4 l 7 T O 20 4O 6O 8O 100120140160180200 220 240 Time (msec) == PDE6A +l+ _ PDE6A -I- Figure 2.21. A comparison of representative cone flicker responses (0.39 log cds/mz, 33 Hz) from a normal control and the PDE6A mutant dog at 9 weeks of age. Spiking bar indicates onset of flicker flashes within the recording time (250 msec). A bi-phasic appearance of cone flicker response is seen in the normal control. Note that the PDE6A mutant dog has reduced flicker amplitude and slight delayed flicker implicit time compared to the control. Key: pV = microvolt, msec = millisecond. 95 30a 111 204 Amplitude (0V) 0 2 4 6 810121416182022 Age(week) 38~ 36* 34* 32* 30~ 284 26‘ I I 24* Implicit time (msec) 22 ‘ 20 O 2 4 6 810121416182022 Age(week) ====| PDE6A +l+ — PDE6A -/- Figure 2.22. Mean and standard errors (1x + SEM) of cone flicker amplitudes (A) and cone flicker implicit times (B) of normal controls and PDE6A mutant dogs (0.39 log eds/m2, 33 Hz) at all age groups. (n = 4 for each group of dog) Note there is a significantly smaller (P< 0. 05) photopic flicker amplitude in the PDE6A mutant group in comparison to normal controls for 5 to 16 weeks of age. Note that the differences in implicit times are not statistically significant. Key: uV = microvolt, msec = millisecond. 96 2.3.2.d. Photopic negative response (Figure 2.23) A PhNR was present at 2 weeks of age in normal controls and increased with age. In the normal controls it matured at 5 to 6 weeks of age (~ 15 11V) (Figure 2.23) and was maintained until the last time point examined (52 weeks of age) (data not shown). The PhNR in the PDE6A mutant dogs was smaller than in controls. It also increased with age from 2 weeks and matured at 5 to 7 weeks of age. It then progressively decreased. By 52 weeks of age, it was non-recordable (data not shown). / \ , WW war»; .M 24WK 10 0V 0 20 4O 6O 80 100 120 140 160 180 200 220 240 Time (msec) ====I PDE6A +I+ — PDE6A -/- Figure 2.23. Representative photopic ERG responses (1.36 log cds/mz) from a normal control and the PDE6A mutant dog at different ages demonstrating the photopic negative response. The light onset is at O msec. Note that the PhNR, a response measured from the baseline to the negative trough after the peak of the b-wave is smaller in the PDE6A mutant dog when compared to that of the normal control. Key: uV = microvolt, msec = millisecond, wk = week. 97 2.3.2.e. Long flash ERG response (Figure 2.24 & 2.25) The long flash ERG response in dogs following 10 minutes of white light adaptation consists at light onset of an a-wave, followed by a b-wave and a post negativity of the b-wave (PhNR). While the light remained on, there was usually a slow increase towards the baseline. Approximately 20 to 25 milliseconds after the light offset, a small positive wave appeared, which was then followed by a larger negative wave (d- wave) (Figure 2.24). In normal controls, long flash ERG responses increased with age over the period of retinal maturation, whereas they diminished in the PDE6A mutant dogs (Figure 2.25A & 2.25B). The b/d amplitude ratio of the normal control increased from 4 to 7 weeks of age and was then fairly constant. The d-wave recorded from mutant dogs at 5 to 6 weeks of age appeared to have one of two forms. In 3 of 7 mutant dogs tested the amplitude was decreased while in 4 of the 7 there was very much increased d—wave amplitude (Figure 2.25B shows one example of each response seen). By 28 weeks of age in the mutant dog, the d-wave had vanished while b-wave was still present but very small (data not shown). By 52 weeks of age, long flash ERG responses were not recordable in the PDE6A mutant dogs (data not shown). With the exception of the puppies with large d-wave amplitudes at 5 to 6 weeks of age, the b/d amplitude ratio in mutant dogs was high compared to that of normal controls at all ages tested. 98 1 11’ ”1 ”Wk WWMW " .f _ \ w pr¢»-¢“7 R$~Jf ”‘0‘\Wfl/‘”M 20uV T T W7 Y j r -20 30 80 130 180 230 280 330 380 430 480 Time (msec) Figure 2.24. A representative long flash ERG from a normal control dog of 60 weeks of age. ERG-Jet lens is used as a corneal recording electrode. Bar indicates flash duration, which is 150 msec (top tracing) and 300 msec (bottom tracing). At light onset, a-wave (the first negative potential) is seen preceding the b—wave (the first positive potential). Following the post b-wave negativity there is a slight increase towards the baseline. The off-response is shown at light offset, and it comprises of a small positive potential immediately followed by a larger negative response. Key: uV = microvolt, msec = millisecond. 99 183 u x3 6:88:28 n 82.: £882.: 0 >1 ”mov— doom 8: mm 238.“ $5 .meEOoE A283 8&ch .mwoc 850 E 038508 3:868: E 8:883? 0333: owcfl be.» m mwov 2:8 5 .0? Co 8.83 o «a wow E83: VBMQK 2: 5m :32? 08 "288033 95 :2: 202 .wov ESE: EMQQ 05 E c2155 2m 88088 2: 882:» omm £3, 88082 0538 was .9 mm. wine—gov 9597. 35:8 Echo: one .385 ow: cone—6 swam 82865 Sm .0858? 95:88 .585 :o:<-§t:m mi? 8282 8a $55 of SE 202 .383 owm 5:5 Amy 38 E83: meQ& 98 2V 20:80 RES: Soc mmEEooE 0mm :8: mac. o>388moc8m .mm.m oBmE AeomEv oEc. A82; mE; COM OWN com 02 co. on O can omN com of OS Om o _ L P h L r P _ L L >10; >1o—_ 2.4. Discussion The ERG study showed that the PDE6A mutant dogs have a severe lack of rod response from an early postnatal age. There was a marked increase in scotopic threshold, reduced scotopic amplitudes, undetectable a-wave responses to blue flashes and an absence of rod flicker responses. Similar abnormal changes in the ERGs were recorded from red] dogs at very young age (Buyukmihci et al., 1980), the rcd2 dogs (Wolf et al., 1978) and the erd dogs (Acland & Aguirre, 1987), all of which have an early-onset retinal degeneration. Cone responses of the PDE6A mutant dogs are initially normal but show a progressive decline with age. Several ways were used in this study to investigate whether any rod function developed in mutant puppies. An attempt was made to assess rod function by analysis of the leading edge of the rod-mediated a-wave at high flash intensity (a-wave modeling) as derived fiom a subtraction of intensity-matched cone-only response from rod plus cone response (Lamb & Pugh, Jr., 19923). A-wave modeling was not possible in the mutant dog because scotopic ERG amplitudes were very small and hardly met the minimum amplitude criterion of 9uV suggested for the analysis of the a-wave leading edge in humans. Fitting very low rod-mediated response to an equation of the a-wave leading edge may considerably lose the fitting quality, which could lead to data misinterpretation (Lamb & Pugh, Jr., 1992b; Tzekov et al., 2001). Therefore a-wave modeling using the data from mutant dogs was not performed. One of the alternative methods that we used in this study to assess rod function was to qualitatively evaluate the subtracted rod a-wave 101 response using a normalization (Hood & Birch, 1996) to standardize and compare the slope of the a-wave leading edge between the normal and mutant dogs. Using this technique a delayed negative amplitude response was derived from the rod-only mutant dog ERG, which was very rapidly lost in the first few weeks of age, suggesting a lack of rod function. It is not clear whether this waveform was truly the result of residual rod photoransduction in the young mutant puppy. Application of the Naka-Rushton equation to fit the first limb of the scotopic b- wave intensity-response curve is suggested as a method to assess rod function. The first limb of the scotopic b-wave plotted against flash intensity from the PDE6A mutant dogs yielded poor fitting to the model, markedly reduced Vmax values, increased k and n values. It seems likely that the response in the mutant dog was primarily from cones and it was the initial portion of the cone b-wave intensity-response curve that was fitted to the equation. All of our investigations have shown severely reduced or absence rod responses in the PDE6A mutant puppies from an early age. It should be noted that the scotopic ERG waveforms recorded from the mutant dog at 6 weeks of age were very similar to those of the photopic waveforms suggesting that the residual responses may be due primarily or completely to cone-only function. Although cone responses were initially similar between mutant dogs and controls, a significant decrease in the mean photopic a-wave amplitude from the PDE6A mutant dogs was observed afier a few weeks of age. This ERG finding is similar to that reported in the rcdl dogs in the postnatal period 102 (Buyukmihci et al., 1980), which showed that cone responses were also lost and that this was subsequently to rod degeneration. Significantly diminished cone sensitivity is shown as a shift to the right of the photopic a-wave intensity-response plot as well as significantly increased mean photopic a-wave threshold in contrast to the mean a-wave threshold of normal controls. Photopic hill is a phenomenon that as the flash intensity increases, the photopic b- wave reaches peak amplitude and then decreases as the flash intensity increases. This results from the reduction of the ON-bipolar response and a delay in the OFF-bipolar response at high light intensities (Ueno et al., 2004). Compared to normal controls, the photopic hill from the PDE6A mutant dogs was shifted to the left as seen on the intensity- response plot suggesting that cone response was reduced. Besides a change of the photopic hill, a reduction of the mean photopic b-wave amplitude occurred in the mutant dogs at 9 weeks of age onward indicative of a progressive loss of cone function. Interestingly the photopic a-wave of mutant dogs started to decrease in amplitude in response to the brighter flashes and in some instances the ERG became unrecordable. The photopic a-wave has major contributions from the inner retina and there may have been some alteration of inner retinal responses in the mutant dog retina in response to brighter light stimulation. Long flash ERG was performed to investigate any differential effect in ON- and OFF-pathways. It was shown that canine off-response consists of a transient small positive-going potential preceding a larger negative-going potential, which was then 103 followed by a slow off-component toward the baseline. Features of canine long flash waveform had previously been described (Shirao et al., 1997). A negative d-wave is also seen in amphibians (Dick & Miller, 1985a) and some mammals such as cats (Frishman & Steinberg, 1990), rabbits (Dick et al., 1985) and rats (Lei, 2003). Conversely, a positive d-wave is present in humans, primates and chickens. An ERG with a negative d-wave was described as the E-type (excitatory) retina and that with a positive d-wave the Hype (inhibitory) retina by Granit (1935). Granit (1935) suggested that the difference between these two types of retina might depend on the number of cones. Evers and Gouras (1986) suggested that it may depend on the type of cones, with S-cone responses in monkeys having an E-type response and M- and L-type cones an I-type d-wave. Granit’s theory is supported by evidence in humans with cone-rod dystrophy, once the photopic ERG amplitudes were reduced by 90%, the d-wave became negative. We showed that the d-wave of normal controls increased in amplitude to about 9 weeks of age, whereas in the mutant dogs, the d-wave was small at all ages investigated except for a subpopulation of mutant dogs at 5 to 6 weeks of age. Size of the d-wave is proportional to the b-wave in both normal controls and mutant dogs although the b/d amplitude ratio in mutant dogs was higher. The abnormally large d-wave transiently recordable from some mutant dogs at 5 to 6 weeks of age may represent some differential effect of the disease on the ON- and OFF- pathways and requires investigation to elucidate it further. A dissection of the components of the on- and off-responses to identify their neuron of origin forms part of chapter 3. In addition to changes of d-wave, CPS and PhNR are also influenced by photoreceptor loss. 104 The results of our ERG studies in the PDE6A mutant dogs showed that rod function is reduced or absent from an early stage in retinal maturation, and is followed by a slow deterioration in cone function. Reduced signal transmission from photoreceptors to the second- and the third-order neurons reflects reduced responses from rod and cone pathways in the mutant dogs. A long flash study using different light wavelength to differentially stimulate the two types of cones could be used to detect any differential effect of the disease on the two cone types of the dog. 105 Chapter 3 Pharmacological dissection of the PDE6A electroretinogram 3.1. Introduction As described in Chapter 1, intravitreal drugs that block intraretinal pathways have been used to investigate the origin of components of the electroretinogram (ERG). Conventional ERG investigations (Chapter 2) showed the PDE 6A mutant dogs have very reduced ERG waveforms. To further investigate this, we used drugs to dissect the canine ERG. It was necessary to establish the effect of the drugs on the ERG of normal dog before they were applied to the ERG of the PDE6A mutant dog. The glutamate analog 2-amino-4-phosphonobutyric acid (APB) selectively blocks the influence of photoreceptors on ON-bipolar cells. Thus APB blocks rod and cone transmission to ON-bipolar cells, leaving the cone connected to OFF -bipolar cells intact. Application of APB in primates eliminates the scotopic and photopic OPs indicating that the drug has an effect on the third-order neurons as well (Jamison et al., 2001). The glutamate antagonist Cis-2,3-piperidine-dicarboxylic acid (PDA) blocks transmission through OFF-bipolar cells, horizontal cells and amacrine cells without diminishing the light response of ON-bipolar cells (Sieving et al., 1994). 106 Tetrodotoxin (TTX), a voltage-gated sodium channel blocker, prevents action potentials in ganglion cells, some types of amacrine cells (Bloomfield, 1996) and interplexiform cells (Bui & Fortune, 2004b). TTX has been used to block the PhNR and mimic the effect on the ERG of glaucoma or optic neuropathy in a primate model (Rangaswamy et al., 20043). 3.2. Materials and methods 3.2.1 . Animals Homozygous PDE6A mutant dogs and normal controls at 4, 7, l6, and 60 weeks of age were used in this experiment. 3.2.2. Pharmacological agents 3.2.2.a. 2-arnino-4-phosphonobutyric acid (APB) (Sigma Aldrich; St. Louis, MO) 3.2.2.b. Q's-2,3-piperidine-dicarboxylic acid (PDA) (Sigma Aldrich; St. Louis, MO) 3.2.2.c. Tetrodotoxin (TTX) (Sigma Aldrich; St. Louis, MO) 107 3.2.3. Intravitreal injection The maximum volume injected for all agents was 60 ul in puppies and 150 pl in 16 and 60 week-old dogs. To calculate the amount of agent that had to be injected to achieve the desired final vitreal concentration, the volume of the vitreous body of euthanized dogs had been previously measured by aspirating it into a syringe. The target vitreal concentrations of APB, PDA and TTX were selected based on the results of studies in other species and were 3mM, 7mM and 6uM, respectively. APB was dissolved in 0.9% sterile saline, heated at 60°C for 3 hours and filtered through a sterile 0.22pm filter (Millex-GS, Millipore Corp, Bedford, MA). PDA and TTX were dissolved in 0.9% sterile saline and filtered through a sterile 0.22pm filter (Millex-GS, Millipore Corp, Bedford, MA). Agents were injected into the vitreous through the pars plana approximately 4-7 mm posterior to the dorsal corneal limbus using a Hamilton syringe fitted with a 30-gauge needle. The needle was directed towards the region of the optic nerve head to avoid contacting the lens. 3.2.4. Electroretinographic recording 3.2.4.a. Anesthesia and recording electrode placement These were as described previously (see Chapter 2; section 2.2.2.a and 2.2.2.b.) 108 3.2.4.b. Electroretinographic recording A baseline ERG was recorded immediately before the intravitreal injection and at several different times after the injection to detect when the maximum response had developed. A Burian—Allen bipolar contact lens was used as the recording electrode. A needle electrode placed subcutaneously at the back of cervical region was used as a ground electrode. The UTAS-E 3000 electrophysiology unit (LKC Technologies Inc; Gaithersburg, MD) was used to record scotopic and photopic short flash ERG and flicker responses. The band pass was set at l to 500 Hz; gain setting varied from 2x103 to 4x104. The time base was set to record 20 msec pre-stimulus. Inter-stimulus intervals were set and ERG responses averaged based on flash intensities as described in chapter 2. Full-field long flash ERGs were recorded using a customized Ganzfeld stimulator unit connected to the UTAS-E 3000 electrophysiology unit and the duration of flash was set at 150 msec. Responses were amplified with a bandpass of 0.3 to 500 Hz, and up to 30 responses averaged. All ERG waveforms were averaged, and then stored for further analysis using LKC software. 3.2.4.c. Electroretinographic test protocols 3.2.4.c (i). Scotopic and photopic short flash electroretinography 109 For scotopic ERGs dogs were dark-adapted for 60 minutes. A series of white flash stimuli ranging in intensity from -2.6 to 1.9 log cds/m2 were used. For photopic response, the dogs were light-adapted for 10 minutes to a background white light of 30 cd/mz. Then a series of white light stimuli ranging in intensity from -0.001 to 1.36 log cds/m2 were superimposed on the same background light. (See Chapter 2 for more details) 3.2.4.c (ii). Flicker electroretinography Rod and cone flicker ERGs were recorded using a white light of —1 .6 log eds/m2 at 5 Hz and 0.39 log eds/m2 at 33 Hz, respectively, on the dark-adapted eye. 3.2.4.c (iii). Long flash electroretinography Dogs were light-adapted for 10 minutes to a background light of 42 cd/mz, followed by a white flash stimulus of 2.25 log eds/m2 of 150 msec duration superimposed on the same background light. (See chapter 2 for more details) 3.2.5. Data analysis ERG was qualitatively compared between age-matched homozygous PDE6A mutant dogs and normal controls before and afier the intravitreal injections. 110 3.3. Results 3.3.1. Results after the intravitreal injection of APB (Figures 3.1 to 3.6) 3.3.1.a. Short flash scotopic and photopic ERG intensity-series responses from normal control dogs In studies using a normal dog as a control, intensity-series ERGs performed before and after the intravitreal injection of APB revealed that the peak effect of the APB had developed at between 1.5 and 2 hours post injection. The action of the drug to block ON-bipolar cells almost completely eliminates the b-wave. At dim lights, the scotopic b- waves were suppressed. At brighter intensities, the scotopic a-waves were slightly increased in amplitude most likely by removal of the imposition of the b-wave on the a- wave (Figure 3.1A). Following the a-wave there was a positive-going potential that returned towards the baseline. The UPS were suppressed by the action of APB. The effect of the APB on the photopic intensity-series ERGs was similar to the effect of the APB on the scotopic intensity-series ERGs. A-wave amplitudes were slightly increased; and the b-waves and OPs were suppressed (Figure 3.1 B). In the normal control and the mutant puppies at 4 weeks of age (Figure 3.2A & 3.28), APB suppressed the b-wave leaving the a-wave. By 6 weeks of age, in the mutant puppy the a—wave was not recordable and the b-wave was reduced in amplitude. lll However, the effect of APB was still evident and eliminated the residual b-wave (Figure 3.2C). 112 0 ‘1 0 20 30 40 50 60 70 80 Time (msec) I I l r . V j I '. 0 10 20 3O 40 50 60 7O 80 Time (msec) ....................... Pre —— P031 Figure 3.1. Representative scotopic (A) and photopic (B) intensity-series ERGs from a normal control at 60 weeks of age performed before and at 1.5 hours following an intravitreal injection of APB. Light intensities are -1.l9, -0.79, -0.39, -0.001, 0.39, 0.85, 1.36, 1.9, 2.38, and 2.82 log cds/m2 (note that the highest two intensities are not used in mutant dog; see Figure 2). Light onset is at 0 msec. Note that the effect of APB is similar between scotopic and photopic ERG. A-wave amplitude is slightly increased at the brightest intensity (solid arrow) while the b-wave is suppressed. 113 ZOuV 0 10 20 30 40 50 60 70 80 Time (msec) B. ‘ ‘.,-..,»::2.\;nll.o":‘:“ll‘r:: !‘-..as..v-._'""h’.~:..t:. \-‘ SpV 0 10 20 3O 4O 50 60 70 80 Time (msec) C. SuV O 10 20 3O 40 SO 60 70 8O Time(msec) .................--- Pre —— Post Figure 3.2. Representative scotopic intensity-series ERGs from a normal control at 4 weeks of age (A), the PDE6A mutant dog at 4 weeks of age (B) and scotopic ERG from the PDE6A mutant dog at 6 weeks of age (C) performed before and at 1.5 hours following an intravitreal injection of APB. Light intensities used for A & B are -0.001, - 0.39, 0.39 and 1.9 log eds/m2, and for C is 1.9 log cds/mz. Light onset is at O msec. As in normal adult dogs, APB suppresses the b-wave of both control and mutant puppies. Key: pV = microvolt, msec = millisecond. 114 3.3.1 .b. Cone flicker responses APB markedly reduced the amplitude of the cone flicker response from the normal control dog and also resulted in a delay in response, although the peak to peak time was unaffected (Figure 3.3A). Cone flicker response post APB was not recordable in the mutant dog (Figure 3.3B). O 20 40 60 80 IOO 120 140 H30 180 200 220 240 2 1.1V O 20 40 60 80 100 120 140 160 180 200 220 240 Time (msec) «mum-m Pre —— Post Figure 3.3. Representative cone flicker response (0.39 log eds/m2, 33Hz) of a normal control (A) and the PDE6A mutant dog (B) at 6 weeks of age performed before and at 2 hours following an intravitreal injection of APB. Light onset is at O msec. Spike bar indicates onset of flicker flashes within the recording time (240 msec). Note that a biphasic peak of cone flicker response is present (solid arrows). Following the administration of APB, the flicker response is reduced in the control dog and effectively eliminated in the mutant dog. The biphasic peak disappears in both groups of dogs. Key: uV = microvolt, msec = millisecond. 115 3.3.1.c. Long flash ERG responses Long flash ERG responses recorded in a normal control at 4 and 6 weeks of age at 2 hour post injection of APB showed suppression of b-wave and an enlarged and extended a-wave (Figure 3.4A & 3.5A). At light offset there was a small positive d-wave followed by a slow recovery to the baseline. A similar response was recorded from the PDE6A mutant dog at 4 weeks of age (Figure 3.4B). However in the PDE6A mutant dog at 6 weeks of age, the a-wave was not recordable pre-APB injection and elimination of the b-wave by APB did not reveal a PIII component. A small negative d-wave was still recordable prior to APB injection and this was converted to a positive waveform by the action of APB (Figure 3.5B). Subtraction of the long flash ERG waveform pre/post APB injection (Figure 3.6) demonstrated that APB mainly suppresses b- and d-wave. 116 SuV I I I l I I I I l O 50 100 150 200 250 300 350 400 450 500 Time (msec) 2.5 uV I I I T I T I T I I O 50 100 150 200 250 300 350 400 450 500 Time (msec) ....................... Pre —— P051 Figure 3.4. Representative long flash ERG response from normal controls (A) and the PDE6A mutant dogs (B) at 4 weeks of age performed before and at 2 hours following an intravitreal injection of APB. Bar indicates flash duration (150 msec). Note that APB has a similar effect to retinas of a normal control and the PDE6A mutant dog. A-wave is isolated while the b-wave is suppressed (black arrow). Pre drug administration, the off- response consists of a small positive potential followed by a large negative waveform (the d-wave; solid arrow). In both control and mutant dogs, the off-response is converted to a positive waveform (arrowheads). Key: pV = microvolt, msec = millisecond. 117 '.'| . . “Jul 0 v M“, ~‘w : flw’f’f‘" n‘N' SpV j j T 0 50 100 150 200 250 300 350 400 450 500 Time (msec) 2.5 uV I I I I I I I I I I 1 0 50 100 ISO 200 250 300 350 400 450 500 Time (msec) ....................... Pre —— Post Figure 3.5. Representative long flash ERG response from a normal control (A) and the PDE6A mutant dog (B) at 6 weeks of age performed before and 2 hours following an intravitreal injection of APB. Bar indicates flash duration (150 msec). A) Amplitude of a- wave of the normal control is slightly increased by APB and the b-wave is eliminated. The D-wave is altered from a negative response before APB (solid arrow) to a small positive wave (arrowhead) after APB administration. B) In the PDE6A mutant dog, APB eliminates the b-wave. After APB, the d-wave is converted to a positive wave (arrowhead) as in the normal control. Key: pV = microvolt, msec = millisecond. 118 5 pV r 0 50 100 150 200 250 300 350 400 450 500 Time (msec) B. 12 . SHV *I I I I I r O 50 100 150 200 250 300 350 400 450 500 Time (msec) Figure 3.6. Subtraction of post APB long flash ERG from pre APB administration long flash ERG from a normal control (A) and the PDE6A mutant dog (B) at 6 weeks of age. Bar indicates flash duration (150 msec). The subtraction reveals the portion of the long flash ERG that is suppressed by APB. As APB predominantly suppresses ON-bipolar cells, this waveform represents the ON-bipolar response. Note that APB has the same effect to both control and mutant dogs by suppressing the positive on-response (b-wave) and the negative off-response (d-wave). Key: pV = microvolt, msec = millisecond. 119 3.3.2. Results after the intravitreal injection of PDA (Figures 3.7 to 3.10) 3.3.2.a. ERG photopic intensity-series response After 2 hour post injection of PDA, a small reduced a-wave was only observed at 1.36 log cds/m2 in a normal control as well as the PDE6A mutant dog (Figure 3.7). The b- wave was partially suppressed at all intensities. B-wave implicit time was increased, particularly in the PDE6A mutant dog and there was a reduction in the photopic negative response (PhNR). OPs were reduced by the action of PDA and this was more obvious in the control dogs where OPs were more prominent prior to PDA injection (Figure 3.7). 120 10 uV O 50 100 ISO 200 250 Time (msec) \m» HW’W “T” ./\‘ '\ I: 1.36 \\ ”W. I10 pV 0 50 mo 150 200 250 Time (msec) ....................... Pre —— P051 Figure 3.7. Representative photopic ERGs at -0.001, 0.39 and 1.36 log eds/m2 from a normal control (A) and the PDE6A mutant dog (B) at 7 weeks of age performed before and at 2 hours following an intravitreal injection of PDA. Light onset is at 0 msec. Note that in both groups of dogs, PDA has very little effect on the a-wave while it reduces b- wave amplitude and increases b-wave implicit time. B-wave post negativity is reduced (black arrow) and returns to a baseline at the same time shown at before the injection of PDA. OPs are also diminished. Key: uV = microvolt, msec = millisecond. 121 3.3.2.b. Cone flicker response PDA had a similar effect on the cone flicker response recorded from the normal control and PDE6A mutant dogs at 7 weeks of age (Figure 3.8). The amplitude was reduced and the response delayed by 5 milliseconds although the peak to peak interval was unaffected. The biphasic peak of the flicker response was partially removed. 122 10pV L 1 l l l l l l I v 1 1 r r r r v r r I 0 20 4O 6O 80 100 120 140 160 180 200 220 240 Time (msec) 0 20 40 60 80 100 120 140 160 180 200 220 240 Time (msec) ....................... Pre —— POSt Figure 3.8. Representative cone flicker response (0.39 log cds/mz, 33Hz) of a normal control (A) and the PDE6A mutant dog (B) at 7 weeks of age before and 2 hours following an intravitreal injection of PDA. Light onset is at O msec. Spike bar indicates onset of flicker flashes within a recording time (240 msec). Note that following an application of PDA cone flicker response is reduced and delayed. The delay induced by PDA is more pronouced in the normal control than in the mutant dog. In the mutant dog, the pre-PDA flicker response is delayed compared to that of the normal control. Key: pV = microvolt, msec = millisecond. 123 3.3.2.c. Long flash ERG response The maximal effect of PDA on the long flash ERG had developed by 2 to 2.5 hours post injection (Figure 3.9). PDA had a similar effect on the long flash ERG of both the PDE6A mutant dog at 7 weeks of age and an age-matched normal control. The small a-wave present prior to injection was completely suppressed. Compared to the response at pre injection, the b-wave had an increased implicit time and a more rounded shape with a lack of the post b-wave negativity. Instead, there was an elevation of the b-wave descending limb, which lasted for the entire duration of light exposure. At the light offset, the small initial positive component of the d-wave was not detected and the negative component was increased in amplitude. Subtraction of the long flash ERG waveform pre/post PDA injection (Figure 3.10) demonstrated that PDA enhances d- and a-wave, suppresses the post b-wave negativity and the PhNR. This subtracted waveform shows the responses from the OFF-bipolar cells and horizontal cells that are PDA responsive. 124 Pre 1hr Post 2hr Post 2.5hr Post 10 “V T i I I T I I l 0 50 100 150 200 250 300 350 400 450 500 Time (msec) Ma J; \ “My... " \ ,...n. W Pre N \ , W W.” H, W 1"" Post 2hr Post W 2.5hr Post l10uV l I T I I l I I O 50 100 150 200 250 300 350 400 450 500 Time (msec) Figure 3.9. Representative long flash ERG responses from a normal control (A) and the PDE6A mutant dog (B) at 7 weeks of age performed before and at 1, 1.5, 2 and 2.5 hours following an intravitreal injection of PDA. Bar indicates flash duration (150 msec). A) A- wave amplitude of the normal control is eliminated by PDA. B-wave maintains its amplitude but has a delayed implicit time and the degree of the post b-wave negativity is decreased. The negative d-wave response is increased. B)'In the PDE6A mutant dog, the small a-wave is suppressed after the injection of PDA the b-wave response delayed and its post negativity eliminated. The negative component of the d-wave is also enlarged by PDA. Note that the small positive potential at light offset is blocked by the action of FDA. Key: pV = microvolt, msec = millisecond. 125 5uV I I I I 1 O 50 100 150 200 250 300 350 400 450 500 Time (msec) 5 uV I I I I I I If 7 I fl 0 50 100 150 200 250 300 350 400 450 500 Time (msec) Figure 3.10. Subtraction of post PDA long flash ERG from pre PDA long flash ERG from a normal control (A) and the PDE6A mutant dog (B) at 7 weeks of age. Bar indicates flash duration (150 msec). This represents the components of the long flash ERG response that result from OFF-bipolar and horizontal cells. Note that PDA has the same effect to both control and mutant dogs by suppressing the a-wave, the post b-wave negativity, the PhNR and the small positive d-wave at light offset, whereas the b-wave amplitude is fairly maintained and the negative d-wave response is slightly increased. Key: pV = microvolt, msec = millisecond. 126 3.3.3. Results afler the intravitreal injection of tetrodotoxin (Figures 3.11 & 3.12) 3.3.3.a. Photopic ERG response In the normal control (Figure 3.11A), TTX slightly increased the b-wave amplitude and partly suppressed OPs. A slight delay in the b-wave descending limb was noticed with an elimination of the post b-wave negativity. The PhNR was suppressed by approximately 70% compared to the pre injection response. The a—wave was not affected by TTX. In the PDE6A mutant dog (Figure 3.11B) TTX reduced the b-wave amplitude but had a similar effect on the PhNR as shown in the normal control. 3.3.3.b. Cone flicker response The cone flicker amplitude was approximately 25-30% reduced after 1.5 hour post injection of TTX, in both normal control and PDE6A mutant dogs. The biphasic peak of the normal control flicker response was still present even though the amplitude was smaller (Figure 3.12A). The peak of the positive flicker waveform in the PDE6A mutant dog was relatively broader, which obscured the biphasic peak present pre injection (Figure 3.12B). The phase of the flicker responses were not altered by TTX in the normal control or the PDE6A mutant dog. 127 SuV 0 50 100 150 200 250 Time (msec) 5pV 0 50 100 150 200 250 Time (msec) ....................... Pre ' P051 Figure 3.11. Representative photopic ERGs at 0.85 log eds/m2 from a normal control (A) and the PDE6A mutant dog (B) at 10 weeks of age performed before and at 1.5 hours following an intravitreal injection of TTX. Light onset is at O msec. Note that TTX increases the b-wave amplitude of the normal control whereas it suppresses the b-wave amplitude of the PDE6A mutant dog as well as eliminates the CPS. B-wave of a normal control and the PDE6A mutant dog slowly recovers (arrowheads), which then follows by elevated post negativity @lack arrows) Key: pV = microvolt, msec = millisecond. 128 O 20 40 6O 80 100 120 140 160 180 200 220 240 Time (msec) 0 20 40 60 80 100 120 140 160 ISO 200 220 240 Time (msec) ...... ....... Pre —— Post Figure 3.12. Representative cone flicker response (0.39 log cds/mz, 33Hz) of a normal control (A) and the PDE6A mutant dog (B) at 10 weeks of age before and at 1.5 hour following an intravitreal injection of TTX. Light onset is at O msec. Spike bar indicates onset of flicker flashes within a recording time (240 msec). Note that the cone flicker delay is observed from the mutant dog compared to a normal control. A reduction of the flicker response is noticed in both groups of dogs. Key: uV = microvolt, msec = millisecond. 129 3.4. Discussion The effect on the short and long flash ERG of the intravitreal injection of APB, PDA and TTX to block specific retinal neuron pathways in normal and mutant dogs was investigated. The use of these drugs allowed the contributions of difference neurons to the formation of the normal ERG waveform of the dog to be investigated. Blockage of photoreceptor input to ON-bipolar cells by APB virtually eliminated the normal scotopic and photopic b-waves. Thus in the dog, similar to other species, the ON-bipolar cell makes a major contribution to the shape of the b-wave. The action of PDA is to block the OFF-bipolar cells and horizontal cells leaving the ON-bipolar response unaffected. Blocking the OFF-bipolar and horizontal cell pathway with PDA results in a modification of the shape of the photopic b-wave tending to reduce its amplitude and broaden its shape. In the short flash recordings PDA did not eliminate the a-wave, whereas in the long flash ERGs the a—wave was eliminated, while the effect on the b-wave was similar to that seen in the short flash ERG. These results are in contrast to those in the primate photopic ERG (Sieving et al., 1994) where PDA and kynurenic acid were used to block the OFF-bipolar and horizontal cell responses. This resulted in a reduction in a-wave amplitude and an enhancement of the b-wave. Subtraction of post drug injection responses from predrug injection responses showed that the contribution of OFF -bipolar and horizontal cell pathways to the primate photopic ERG is a sustained negative response that lasts the duration of light exposure (Sieving et al., 1994). We performed a similar subtraction and found that the canine OFF-bipolar and horizontal cell responses had an initial brief small negative and then small positive response followed by a much 130 larger sustained negative response that was maintained while the light was on and then a small negative response at lights off followed by a larger positive waveform. This predominantly negative component is similar to that of the primate but appears to be a slower response in the dog so that its peak negativity coincides with the normal descending phase of the b-wave and thus it has the effect of broadening the b-wave and extending it, eliminating the post b-wave negativity, rather than increasing the peak of the b-wave as it does in primates. Similar subtraction to demonstrate the ON-bipolar cell response (the portion of the waveform blocked by APB) showed the major contribution the ON-bipolar cell pathways make to the scotopic and photopic b-wave and also the negative component of the canine d-wave. APB had a similar effect on cone flicker (33Hz) as previously described in primates (Sieving et al., 1994) (Hare & Ton, 2000). The amplitude of response was very reduced and the response slightly delayed although it remained in phase with the stimulus. A similar, but less severe, reduction in cone flicker amplitudes resulted from administration of PDA. These results show the major contribution to the flicker response made by the ON- and OFF- pathways in the inner retina. The alteration to canine cone flicker responses by administration of PDA was similar to that described in primates by the work of Hare and Ton (2002), but differed from the result obtained in primates in an earlier study by Bush and Sieving (Bush & Sieving, 1996) who found that PDA had little effect on the primate cone flicker response. These contradictory results were discussed by Hare and Ton (2002) who suggested that they may have been due to differences in the 131 type of stimulus used, Hare and Ton (2002) used a brief flash from a xenon flash unit (as we used in this study) whereas Bush and Sieving used a square wave stimulus. The use of these drugs allowed investigation of the source of the OFF-response of the long flash ERG in the dog. Intravitreal injection of APB changed the d-wave polarity from predominantly negative to a small positive potential. A similar result has been described in rabbits (Knapp & Schiller, 1984b). A study in macaque monkey using different colored light stimuli to investigate the responses from different cones concluded that the S-cones have a negative off-response whereas the d-wave recorded when medium and long wave cones were stimulated was positive (Evers & Gouras, 1986). Furthermore the administration of APB eliminated the negative d-wave of S-cones but enhanced the positive going d-wave of the M- and L-cones. The effect of intravitreal PDA in the dog was to increase the negativity of the off-response. In primates PDA tends to reduce the d- wave response (push it towards negativity) (Bush & Sieving, 1994). The difference in off-responses in dogs compared to primates can simply be explained by the difference in the amplitudes and timings of responses originating from the APB responsive on- pathway and the PDA responsive off-pathway. The response from ON-bipolar cells was a positive deflection at light onset that was maintained, but slowly drifted down, while the lights remained on and then, in both primates and dogs, when the light stimulus stopped the waveform had a negative deflection. The response from the OFF-bipolar cell pathway is negative at light onset and remains negative with a slow positive drift while the light is on and then has a positive deflection at light offset. The ON-bipolar cell response accounts for the major component of the off response in the dog resulting in an overall 132 negative deflection whereas the OFF-bipolar response probably accounts for the small positive deflection that typically precedes the negative d-wave and then a the initial positive return towards the baseline following the negative deflection of the d-wave. In primates the OFF -bipolar response positive deflection at light offset appears to be faster and is greater in amplitude than the ON-bipolar negative response resulting in an overall positive d-wave. Intravitreal injection of TTX enhanced the b-wave amplitude of the normal control dog and reduced the post b-wave negativity (photopic negative response). This result is the same as obtained from rabbits under bright light stimulation (Dong & Hare, 2000) and in primates independent on light stimulation (Hare & Ton, 2000). Considering the major differences in ERG amplitudes between normal dogs and mutant dogs the administration of APB and PDA to mutant dogs had very similar effects on short flash, long flash and flicker ERG responses to those in the control dogs. This suggests that early in the disease process there are no profound abnormalities in the ON and OFF pathways in the mutant retina. Considering the lack of rod driven responses in mutant puppies the effect of APB on the short flash in the dark adapted mutant dog is most likely to represent the drug’s blockage of cone ON-bipolar cells rather than rod ON- bipolar cells as the latter are unlikely to be contributing much to the overall ERG tracing because of the lack of rod function. 133 The relatively smaller d-wave of the mutant dog compared to b-wave appears to result from changes in the ON—bipolar pathway. Subtraction of the post APB long flash ERG from the pre APB injection ERG showed that the negative deflection at light off from the ON—bipolar pathway was proportionally smaller in amplitude than the positive defection at light on. The OFF-bipolar pathway response revealed by intravitreal PDA from the mutant dog seemed to have waveforms of reduced but similar proportions to the normal control. This finding may indicate an early abnormality in the cone ON-bipolar pathway as a result of the disease process in the retina of the mutant dog. There was a difference in the mutant dog response to TTX compared to the control. In the mutant, TTX acted to decrease the b-wave amplitude, whereas in the control it increased it. The effect on post b-wave negativity was similar between mutant and control, although the mutant dog had a smaller PhNR prior to drug administration than the control. The effect on the mutant dog b-wave is similar tothat described for the rabbit under low light stimulation (Dong & Hare, 2000). TTX was found to eliminate the action potential-dependent inhibition to the glycinergic and GABAergic amacrine cells in rabbit retina (Dong & Hare, 2000) while its effect on rat photopic ERG was attributable to voltage-gated sodium current mainly in amacrine and interplexiform cells (Bui & Fortune, 2004a). According to different results following TTX administration in various species, the effect of TTX may reflect some combination of blocking activity in amacrine cells, ganglion cells and interplexiform cells (Hare & Ton, 2000). Failure of TTX to increase the photopic b-wave in the PDE6A mutant dog and an absence of photopic OPS suggest a loss of action potentials originating in the third-order neurons. A smaller 134 reduction of PhNR in the PDE6A mutant dog compared to the normal control after the intravitreal injection of TTX supports the conclusion that the third-order neurons are affected in the PDE6A mutant dog at young age probably due to altered input from the outer retina. 135 Chapter 4 Detailed histopathological characterization of the PDE6A dog phenotype 4.1. Introduction The PDE6A mutant dogs are night blind at the earliest age they can be tested (approximately 6 to 8 weeks of age). Scotopic ERG responses are dramatically decreased suggesting a lack of rod activity. Results of electroretinographic studies (Chapter 2 & 3) indicate that rod photoreceptors in the PDE6A mutant dogs fail to develop normal function in the early postnatal period. An arrest in rod differentiation followed by a rapid decrease in number of rod photoreceptors occurs in animals with PDE6B mutation (Buyukmihci et al., 1980; Iimenez et al., 1996). Furthermore, there were major pathological changes of second-order neurons in the rd] mouse following rod degeneration (Strettoi et al., 2002). The aims of this part of the study were to document the morphological changes in photoreceptors in the PDE6A mutant dogs over time, investigate the effect of degeneration on the cellular organization of the inner retina, and to document modifications in the retina after photoreceptor cell loss. 136 4.2. Materials and methods Animals: see 2.2.1. (Chapter 2) 4.2.1. Morphological analyses by light microscopy 4.2.1 .a. Tissue collection and processing Dogs at 2, 3, 4, 5, 7, 9, 12, 16, and 60 weeks of age were euthanized by administration of an overdose of pentobarbital sodium (Fatal-Plus; Vortech pharmaceuticals, Dearborn, MI) intravenously. After euthanasia, enucleations were performed. All globes were trimmed of extraocular muscles. Four slits 3mm from and parallel to the limbus were made on the right globe using a #11 scalpel blade to facilitate intraocular penetration of the fixative, and then the globe was submerged in a mixture of 3% glutaraldehyde, 2% paraformaldehyde, and 0.1M Sodiurn-cacodylate buffer (pH 7.2) for two hours at 4°C. The anterior half of the globe, lens and vitreous were then removed. The posterior half of the globe (eyecup) was transferred to the same fixative and left overnight at 4°C . The globe was dehydrated in a graded series of ethanol solutions and infiltrated with Immuno-Bed solution, prepared according to the manufacturer’s instructions 137 (Electron Microscopy Sciences, Ft. Washington, PA). After the eyecup had polymerized overnight at —20°C, a vertical cut was made from the superior ora ciliaris retina through the optic nerve head to the inferior ora ciliaris retina (Fig 4.1). The nasal half of the hemisectioned eyecup was mounted to an EBH-2 block holder (Electron Microscopy Sciences, Ft. Washington, PA). The remaining temporal half of the eyecup was hemi- sectioned in the horizontal plane from the optic nerve head to the temporal ora ciliaris retina, and mounted to an EBH-2 block holder (Electron Microscopy Sciences, Ft. Washington, PA). Sections, each approximately 3 pm thick, were cut at three-micrometer thickness using a glass knife and stained with hematoxylin and eosin (H&E) for light microscopic analysis. 138 Superior ora ciliais retina Temporal I I I I ora ciliaris retina Inferior ora ciliaris retina Figure 4.1. Diagram of the right eyecup of a dog illustrating planes of section and regions of the retina where the thickness of individual retinal layers is measured, and the number of outer nuclear layer (ONL) rows and photoreceptor nuclei per unit length are counted. Key: location 1, 8, and 12 are SOO-micrometers from the ora ciliaris retina, location 4, 5, and 9 are 500-micrometers from the edge of the optic nerve head. To allow investigation of additional retinal regions, globes from homozygous PDE6A mutant and homozygous PDE6A normal dogs at ages from 5 to 7 months were plastic-embedded and sectioned as shown in figure 4.2. Three-micrometer sections were stained with H&E for measurement of retinal thickness and counting of photoreceptor nuclei in the outer nuclear layer. 139 Superior Temporal Nasal Inferior Figure 4.2. Diagram of the right eyecup of a dog illustrating the regions of the retina where detailed morphological analysis is performed. 4.2.1.b. Data collection Images were captured using a Nikon microphot-FXA microscope (Nikon Inc, Garden City, NY). The length of each retinal section was measured using NeuroExplorer software (MicroBrightField Inc, Williston, VT), acquired with NeuroLucida software; version 3 (MicroBrightField Inc, Williston, VT), and then divided by three to allow the same relative retinal region to be examined independent of the size of the globe. The thickness of the following retinal layers were measured at 3 different areas within one microscopic field: retinal pigment epithelium (RPE), photoreceptor outer segment (OS), photoreceptor inner segment (IS), outer nuclear layer (ONL), outer plexiforrn layer (OPL), inner nuclear layer (INL), inner plexiform layer (IPL), ganglion 140 cell layer and nerve fiber layer (GCL & NFL). Measurements were taken at all regions across the retina (Figure 4.1 & 4.2) under 40-time magnification using the Neurolucida software; version 3 (MicroBrightField Inc, Williston, VT). The number of rows of photoreceptor nuclei in the ONL and the number of rod and cone nuclei identified by morphological features of their nuclei (see introduction; Chapter 1) were counted over lOO-micrometer length of the retina at the regions above using the Neurolucida software; version 3 (MicroBrightField Inc, Williston, VT) (Figure 4.3). Figure 4.3. Diagram of a cross section of retina showing a measurement of the number of rows of the ONL and the number of rod and cone nuclei per IOO-micrometer length of the retina. Key: ONL = outer nuclear layer. 141 4.2.1.c. Data analysis The thickness of the eight retinal layers measured and number of cells counted (4.2.1.b.) were mapped across the length of all sections. All measurements derived from the PDE6A mutant dogs were compared with the identical areas from age-matched normal controls, and analyzed using ANOVA. Independent analyses for each region and retinal layer were performed. The fixed effects included in the model were age and disease genotype. For measurement of retinal thickness, a random effect of dog was included to account for repeated measures (triplicates) within dog and age. For measuring the number of cells, no random effects were considered and consequently a linear model of fixed effects was used. No covariance among ages was modeled as the measures at different ages corresponded to independent dogs. No variable transformation was done because the residual analyses revealed fulfillment of model assumptions (normality, heteroskedasticity) (data not shown). Data were deemed significant when P<0.05. All analyses were performed using SAS Proc Mixed software; SAS version 9.1 (SAS Institute Inc, Cary, NC). 4.2.2. Morphological analyses by transmission electron microscopy 4.2.2.a. Tissue collection and processing 142 Dogs at 2, 3, 4, 5, 7, 9 and 16 weeks of age were euthanized by administration of an overdose of pentobarbital sodium (Fatal-Plus; Vortech pharmaceuticals, Dearbom, MI) intravenously. After euthanasia, enucleations were performed. All globes were processed, fixed (as described in 4.2.1.a.), and then sectioned in a vertical plane through the Optic nerve head. The nasal half of the eye was post—fixed in osmium tetroxide, and embedded in resin consisting of Poly/Bed, Araldite, DDSA, and DMP-30 accelerator. Semi-thin sections (0.7 to 1 micrometer thickness) were cut with a glass knife and stained with toluidine blue. Ultra-thin sections (0.06-0.08 micrometer thickness) were cut with a diamond knife and stained with uranyl acetate and lead acetate. Sections from the central tapetal regions (region #3; Figure 4.1.) were examined with a Philips 301 transmission electron microscope. 4.2.2.b. Data analysis Sections from region #3 (Figure 4.1.) from the PDE6A mutant and age-matched normal control eyes were compared. The ultra structural morphologic appearance of photoreceptor outer segments as well as the photoreceptor cell bodies, inner segments, and synaptic termini were assessed. 4.2.3. Immunohistochemical analyses of paraffin-embedded sections 4.2.3.a. Tissue collection and processing 143 Dogs at 2, 3, 4, 5, 7, 9, 12, 16, 60 and 210 weeks of age were euthanized by administration of an overdose of pentobarbital sodium (Fatal-Plus; Vortech pharmaceuticals, Dearbom, MI) intravenously. After euthanasia, enucleations were performed. Four slits were made in the left globe (free of extraocular tissues) at 3 mm posterior to the limbus to facilitate intraocular penetration of the fixative, and then the globe was initially fixed in a mixture of 4% paraformaldehyde in 0.1M phosphate buffered saline (PBS; 0.05M sodium phosphate, l95mM NaCl; pH 7.4).) for two hours at 4°C. The anterior segment of the globe, lens, and vitreous body were removed. The posterior eyecup was further fixed in the same fixative for another 20 hours at 4°C. After that, the eyecup was gently rinsed with 0.1M PBS buffer plus 3% sucrose and cut in a vertical plane through the optic nerve head. The medial half of the eyecup was de- hydrated in ethanol from 65% to 100% concentration, rinsed twice in xylene, and then embedded in paraffin. Paraffin-embedded sections (5pm thickness) were cut, mounted on positively-charged glass slides, and dried at 65°C for 20 minutes. A sample section was stained with H&E to assess the quality of the section prior to immunohistochemical processing. Paraffin-embedded retinal sections were cut at a five-micrometer thickness, air- dried overnight, de-paraffinized in xylene (twice) followed by a gradual rehydration in 100% and 95% ethanol, and finally in distilled water. The sections were incubated in a preheat antigen retrieval buffer (Citrate buffer; DakoCytomation, Carpinteria, CA) for 20 minutes at 97°C. After the section had been cooled to 50°C, it was incubated in 50 mM 144 TRIS-buffered saline (TBS; pH 7.6) for 5 minutes, followed by 10 minutes incubation with a protein-blocking agent (DakoCytomation, Carpinteria, CA) prior to application of the primary antibodies (see Table 4.1. for a list of antibodies used). Goat anti-rabbit and goat anti-mouse secondary antibody from the Labelled Streptavidin-Biotin 2 System, Horseradish Peroxidase (LSAB2 System-HRP; DakoCytomation, Carpinteria, CA) was used to reveal primary antibody-positive immunoreactivity. Immunoreaction was visualized with 3,3’-diaminobenzidine substrate (Liquid DAB substrate chromogen system; DakoCytomation, Carpinteria, CA), and the sections were counterstained with hematoxylin (Gill III formulaTM; Surgipath Medical Industries Inc, Richmond, IL) for 10 minutes. The sections were then washed in distilled water, rinsed in tap water for 5 minutes, blued with 0.04% lithium carbonate, washed again in distilled water, rehydrated in 100% and 95% ethanol then in xylene before being coverslipped. Positive control was included for each set of staining. PRIMARY ANTIBODY CONCENTRATION SOURCE OF PRIMARY ANTIBODY OF PRIMARY ANTIBODY MOUSE ANTI- 1:50 A GIFT: DR PAUL HARGRAVE; U OF RHODOPSIN FLORIDA MOUSE ANTI- 1:2 LAB VISION; FREMONT, CA RHODOPSIN RABBIT ANTI-GFAP 121600 DAKOCYTOMATION;CARPINTERIA, CA MOUSE ANTI-PKC 1:50 BD BIOSCIENCE; ROCKVILLE, MD ALPHA RABBIT ANTI-CONE 112000 A GIFT: DR CHERYL CRAFT; U OF ARRESTIN SOUTHERN CALIFORNIA RABBIT ANTI- 1:100 RESEARCH DIAGNOSTICS; CASPASE 3 FLANDERS, NJ 145 Table 4.1. A list of the primary antibodies, their working dilution and source that are used on paraffin-embedded sections. Key: GFAP = Glial Fibrillary Acidic Protein, PKC = Protein Kinase C. For caspase—3 immunohistochemistry, paraffin-embedded retinas sections (5- micrometer thickness) were de—paraffinized (EZ PrepTM; Ventana Medical System Inc, Tucson, AZ), washed with SSC solutionTM (Ventana Medical System Inc, Tucson, AZ), incubated with reaction bufferTM (Ventana Medical System Inc, Tucson, AZ) at 37°C for 2 minutes. Anti-caspase-3 antibody (1:100) (Casp3actabr; Research Diagnostics Inc, Flanders, NJ) was applied and the slide incubated at 37°C for 10 minutes, biotinylated anti-mouse and anti-rabbit secondary antibody conjugated with alkaline phosphatase- streptavidin (Enhanced alkaline phosphatase red detection kit; Ventana Medical System Inc, Tucson, AZ) was applied to locate and visualize the bound primary antibody. After that, the section was incubated at 37°C for 32 minutes, counterstained with Hematoxylin, . followed with a bluing reagent to change the hue of the hematoxylin to a blue color (Ventana Medical System Inc, Tucson, AZ). To remove the liquid cover slip reagent the section was washed with a detergent and rinsed with warm tap water. Finally the section was rinsed with 100% ethanol, followed by a 1:1 mixture of 50% ethanol and 50% xylene, then 100% xylene, and then coverslipped. Positive control was included for each set of staining. 4.2.3.b. Data analysis 146 Retinal sections of the PDE6A mutant and normal control dogs were examined for differences in the distribution and morphologic appearance of the immunoreactive cells. Images were captured using an Olympus microscope model BX51 or model BX40 (Olympus America Inc, Melville, NY), equipped respectively with the In Sight QE digital camera model 4.2 (Mikron Instruments Inc, San Marcos, CA) or Olympus digital camera model E10 (Olympus America Inc, Melville, NY). Images were optimized by using SPOTTM software v. 4.5 (Westgate Software Inc, Draper, UT). 4.2.4. Immunohistochemical analyses of frozen sections 4.2.4.a. Tissue collection Dogs at 5, 7, 9, 12, 16, and 60 weeks of age were euthanized by administration of an overdose of pentobarbital sodium (Fatal-Plus; Vortech pharmaceuticals, Dearbom, MI) intravenously. After euthanasia, enucleations were performed. The globe was dissected as described in 4.2.3.a, and fixed in a mixture of 4% paraformaldehyde plus 3% sucrose in 0.1M phosphate buffer (pH 7.4) for 15 minutes at 4°C. The anterior segment of the globe, the lens, and vitreous body were then removed. The posterior eyecup returned to the same fixative for another 20 minutes, and then washed three times in PBS. The eyecup was placed in PBS plus 30% sucrose for at least 24 hours, immersed in embedding medium (OCT-compound; Tissue-Tek; Miles Inc, Elkhart, IN), and mounted onto sectioning blocks. Sections (14 um thickness) were cut in a vertical plane through 147 the optic nerve head and thaw-mounted onto Super-FrostTM slides (Fisher Scientific Ltd, Leicestershire, UK). The sections were air-dried and stored at -20°C until use. Sections were thawed at room temperature, ringed with rubber cement, washed three times in PBS, covered with primary antibody solution (see Table 4.2. for a list of antibodies used) (200 pl of antiserum diluted in PBS plus 5% normal goat serum, 0.2% Triton X-100, and 0.01% NaN3), and incubated for 24 hours at 20°C in a humidified chamber. The slides were washed in PBS, covered with secondary antibody solution, and incubated for 1, hour at 20°C in a humidified chamber. Secondary antibodies included goat-anti-rabbit-Alexa488, goat-anti-mouse Alexa488/568 and goat-anti-mouse-lgM Alexa568 (Molecular Probes Inc., Eugene, OR) were diluted to 1:1000 in PBS plus 0.2% X-100. The method of Triton detection streptavidin-immunoperoxidase (DakoCytomation, Carpinteria, CA) was applied tO reveal primary antibody-positive immunoreactivity. Finally, the slides were washed three times in PBS, the rubber cement was removed from the slides, and a coverglass was applied in 4:1 (v/v) glycerol to water. PRIMARY CONCENTRATION SOURCE OF SECONDARY ANTIBODY OF PRIMARY PRIMARY ANTIBODY ANTIBODY ANTIBODY RABBIT ANTI- 1:600 CHEMICON; GOAT-ANTl-RABBIT- RED/GREEN PITTSBURGE, PA ALEXA488 01>er MOUSE ANTI- 1:200 MONOCLONAL GOAT-ANTI-MOUSE- W cm ANTIBODY FACILITY; IGM ALEXA568 ’ U OF OREGON MOUSE ANTI- 1:800 SIGMA ALDRICH; ST. GOAT-ANTI-MOUSE- CALBINDIN LOUIS, MO IGM ALEXA568 148 RABBIT ANTI- 121000 SWANT; GOAT-ANTI-RABBIT- C ALRETININ BELLINZONA, ALEXA488 SWITZERLAND Table 4.2. A list of the primary antibodies, their working dilution and source that are used in frozen sections. Key: Alexa-488 = green and Alexa-568 = red. The immunohistochemical staining utilizing frozen retina was performed in Dr Andy Fischer’s laboratory (Department of Neuroscience, The Ohio State University, Columbus, Ohio). 4.2.4.b. Data analysis Photomicrographs were taken by using a Leica DM5000B microscope equipped with epifluorescence and a 12 megapixel Leica DC500 digital camera (Meyer Instruments, Houston, TX). Images were Optimized for color, brightness and contrast, and double-labeled images overlaid by using PhotoshopTM 6.0. Retinal sections Of the PDE 6A mutant dogs were examined for differences in the distribution and morphology of the immunoreactive cells and compared to the normal control eyes. 4.2.5. Terminal Deoxynucleotidyl Transferase-mediated dUTP Nick End Labeling (TUNEL) staining 149 Direct TUNEL labeling assay was performed using In Situ cell death detection kit, rhodamine (Roche Diagnostics Corporation, Indianapolis, IN) to detect DNA strand breaks in apoptotic cells by using an Optimized terminal transferase (TdT) to label free 3’OH ends in genomic DNA with rhodamine-dUTP. 4.2.5.a. Tissue collection and processing Terminal enucleation was performed on dogs at 3, 4, 5 and 9 weeks of age. See 4.2.4.a. for a detail of tissue processing. Slides were thawed and fixed for 1 hour at room temperature, washed and then permeabilized on ice for 2 minutes. Samples were finally incubated with TUNEL reaction mixture (TdT and flurescein-dUTP solution) at 37°C for 1 hour. 4.2.5.b Data analysis Incorporated rhodamine/fluorescein was visualized by fluorescence microscopy. 4.3. Results NO significant histological differences were observed between the homozygous PDE6A normal and heterozygous carrier dogs. 150 4.3.1. Morphological assessment of the retina by light and electron microscopy 4.3.1.a. Retinal sections at 3 to 10 days of age At 3 to 10 days of age, the morphological features Of the retina of mutant dogs were similar to those of control dogs. The development of retinal layers starts with the inner layers of the retina and then moves progressively outward. The central region of the retina matures prior to the peripheral region. At 3 days of age (Figure 4.4A), the neurosensory retina consisted of two layers; inner and outer neuroblastic layer. The RPE layer was also present at this age. Primitive horizontal cells appeared within the outer neuroblastic layer at 4 days of age (Figure 4.4B) and the OLM was apparent. Separation of the outer neuroblastic layer into the INL, OPL and ONL had occurred by 7 days Of age (Figure 4.4C). The inner segments of photoreceptors had begun to develop in the central retinal region, and by 10 days of age (Figure 4.4C) their length was ranged from 3 to 4.5 pm by 10 days of age (Figure 4.4D). 151 Figure 4.4. Representative photomicrographs of retinal morphology of a normal control (A & C) and the PDE6A mutant dog (B & D) at 4 (A & B) and 10 (C & D) days of age. Bar = 50 pm. A & B) the RPE, IPL, GCL and NFL are formed by 4 days after birth. Retinal nuclei in the outer neuroblastic layer are elongating and photoreceptor inner segments are begining to develop (white arrows). Primitive horizontal cells are present at this age (arrowheads; B). C & D) Retina at 10 days Of age consists of three layers of nuclei; the ONL, the INL and the GCL. Photoreceptor irmer segments have increased in length (white arrow). NO difference is observed between the retina of the normal control and the PDE6A mutant dog at this age. Key: ONBL = outer neuroblastic layer, INBL = inner neuroblastic layer, PRL = photoreceptor layer, ONL = outer nuclear layer, OPL = outer plexiform layer, INL = inner nuclear layer, IPL = inner plexiform layer, GCL = ganglion cell layer, NFL = nerve fiber layer. 152 4.3.l.b. Retinal sections at 2 to 3 weeks Of age All retinal layers had developed by the second week Of life and the photoreceptor inner segments had increased in length (Figure 4.5A & 4.5B). The photoreceptor outer segments had started to develope at this age except for in the far periphery of the retina. Chromatin-dense photoreceptor nuclei were elongated in shape in the normal control retina (Figure 4.5A). A few nuclei with less dense chromatin located at the inner region of the ONL were present in the PDE6A mutant retina (Figure 4.5B). They were spherical in shape with vacuolation Of the cytoplasm. These changes in the nuclei indicated an early stage of degenerative alteration of cells. At 3 weeks of age, photoreceptor inner and outer segments had elongated in the normal control retina (Figure 4.5C). Cone nuclei (large cell bodies with pale chromatin) could be seen located adjacent and internal to the OLM whereas rod nuclei (small, oval cell bodies with very dense chromatin) occupied the rest of the ONL. In the PDE6A mutant retina the photoreceptor outer segments appeared disorganized at this age and surrounded by empty spaces, suggesting a loss of some of photoreceptor outer segments (Figure 4.5D). The mean thickness of the photoreceptor inner segment layer was however comparable with normal control (vertical section; 10.38 +/- 0.78 pm and horizontal section; 8.89 +/- 0.38 pm) and the PDE6A mutant (vertical section; 12.54 +/— 0.54 pm and horizontal section; 10.55 +/- 0.71 pm) (Figure 4.6 & 4.7). Nearly all retinal layers have the greatest thickness at superior region close to the optic disc (region #4) and the least thickness at the far inferior region (region #8). There were no Significant differences 153 in the thickness of each retinal layer (P<0.05) in vertical and horizontal sections between normal control and the PDE6A mutant dog retina at these ages. 154 Figure 4.5. Representative photomicrographs of retinal morphology of a normal control (A & C) and the PDE6A mutant dog (B & D) at 2 (A & B) and 3 (C & D) weeks of age. Bar = 50 pm. A & B) Photoreceptor outer segments of both groups have begun to develop at 2 weeks of age (white arrows) while the inner segments have grown in length. Photoreceptor segments of the PDE6A mutant are of comparable length to those of normal control. Changes Of chromatin pattern present in several photoreceptor nuclei (solid arrows) that are indicative of cell death (apoptosis) in the retinal section of the PDE6A mutant dog. C & D) Photoreceptor cell bodies of the normal retina at 3 weeks of age are arranged in an organized manner, and the photoreceptor inner segment and outer segment are longer compared to those at 2 weeks of age. Spaces in the PRL of the PDE6A mutant retina (black arrows) indicate degenerative outer segments. Apoptotic nuclei indicated by the presence of spherical-shaped photoreceptor nucleus are present (solid arrows) with weak surrounding euchromatin staining and fragmented heterochromatin. Key: PRL = photoreceptor layer, ONL = outer nuclear layer, OPL = outer plexiform layer, INL = inner nuclear layer, IPL = inner plexiform layer, GCL = ganglion cell layer. 155 1234disc5678 Vertical region GCL+NFL lPL INL ‘3 I" 12 3 4disc5 6_7.8 Vertrcalregron 1 2 3 4 disc5 6 7 8 Vertical region Figure 4.6. Mean retinal thickness of each of the different retinal layers from superior to inferior ora ciliaris retina (vertical section; see Figure 4.1 in materials and methods) from normal control (A), PDE6A carrier (B) and PDE6A mutant dogs (C) at 3 weeks of age (n=3). Note that photoreceptor outer segments of all genotypes have developed in particular the central region. All retinal layers are thinner at the superior and inferior regions close to the ora ciliaris retina (#1 & #8). The central retinal thickness tends to be greater than the periphery. There are no obvious differences in thickness of the various retinal layers at this age. Key: RPE = retinal pigment epithelium, OS = outer segment, IS = inner segment, ONL = outer nuclear layer, OPL = outer plexiform layer, INL = inner nuclear layer, IPL = inner plexiform layer, GCL = ganglion cell layer, NFL = nerve fiber layer. 156 250 -_.-__.__ _-._ -_-_ _-._ ___.-_-_-_- -_ - 200 GCL+NF L IPL INL OPL 12 11 10 9 disc Horizontal region 250 ~ — — -- .3----_____._ 0 Z r- 200 i : RPE 12 11 10 9 disc Horizontd region Figure 4.7. Mean retinal thickness of each of the different retinal 'layers from nasal to temporal region (horizontal section; see Figure 4.1 in materials and methods) from normal control (A) and PDE6A mutant dogs (B) at 3 weeks of age (n=3). Note that the thickness Of all retinal layers is least at the far temporal region (#12) and greatest at the region close to the Optic disc (#9). Photoreceptor segments are developing. NO difference in retinal thickness is apparent between normal control (A) and PDE6A mutant dogs at this age (B). Key: RPE = retinal pigment epithelium, OS = outer segment, IS = inner segment, ONL = outer nuclear layer, OPL = outer plexiform layer, INL = inner nuclear layer, IPL = inner plexiform layer, GCL = ganglion cell layer, NFL = nerve fiber layer. 157 The shape of rod nuclei, the appearance Of the chromatin and of the rod cell cytoplasm of mutant dogs at approximately 3 weeks of age appeared to differ from the controls. While photoreceptor nuclei of the normal control compared to the previous week had further elongated (Figure 4.8A, 4.9A & 4.10A), some of those of the PDE6A mutant dog had undergone degeneration. Morphological changes representing several stages of apoptosis (programmed cell death) were seen in the PDE6A mutant retina during this period of time (Figure 4.8B, 4.8C & 4.8D). In the early stages Of apoptosis, nuclei were spherical and swollen with densely fragmented heterochromatin and a pale euchromatin (Figure 4.9B). Fragmented heterochromatin then became clustered adjacent to the nuclear membrane and afterward the cell appeared pyknotic with significant smaller nuclei at the later stages of cell death (Figure 4.10C). Dead cells in the process Of being removed appeared without distinct detail of organelles (Figure 4.10D). A dramatic increase in the number of apoptotic cells occurred from 24 to 27 days of age in the mutant retina although Significant number of TUNEL-positive cells was present in the mutant retina at 28 days of age (figure 4.13D). 158 Figure 4.8. Photomicrographs of retinal morphology of the ONL of a normal control (A) at 23 days of age, and PDE6A mutants dogs (B & C) at 24 (B) and 27 (C) days of age and a photomicrograph of the anti-caspase 3 immunohistochemistry in the PDE6A mutant retina at 24 (D) days of age. Bar = 25 pm. A) In normal control retina, photoreceptor nuclei are elongated in shape. Cone cell bodies (black arrows) with lighter chromatin are located at the outer border of the ONL whereas rod cell bodies with darker chromatin are located throughout the ONL (white arrows). B & C) The PDE6A mutant retinas at 24 and 27 days of age illustrate a number of apoptotic photoreceptor nuclei; spherical, swollen cells with fragmented chromatin and vacuolation (Opened arrows), smaller spherical cells with very dense heterochromatin (solid arrows) and dying cells with fading euchromatic appearance (arrowheads). D) No caspase 3-immunopositive cells are detected in the immunohistochemical section of the PDE6A mutant retina at 24 days, which is comparable to normal control retinal section (data not shown). 159 £4; 33:67.3. ‘43:?» g 6: 3 L , 33%,: rm? ‘3'? gar-2.3- 77%" 3“ W a: 32% is M 53?; § . ,'L , ~ ang‘gfié $83 ‘ ‘8 Q iféfli 5 Figure 4.9. Ultra structural sections of the ONL of a normal control (A) and the PDE6A mutant dog (B) at 23 days of age. Bar = 10 um. Note that photoreceptor nuclei of the normal control dog are oval in shape with a cluster of chromatin (A). In contrast, apoptotic nuclei (arrows) are scattered in the ONL of the PDE6A mutant retina (B). The nuclei appear more spherical in shape with a decrease in staining of euchromatin and irregular nuclear membrane compared to those of normal nuclei surrounded. 160 "'I ‘7‘ '{x-. ‘1", ‘ .- 5‘ t" ' ..-i m ‘- I- 1'... . Figure 4.10. Ultra structural appearance of photoreceptor nuclei of normal control (A) and PDE6A mutant dogs (B, C & D) at 27 days of age. Bar = 5 pm. Note that in B, C & D there are several photoreceptor nuclei that appear to be going through stages of apoptosis. Photoreceptor cell bodies have become round with an accumulation of electron-dense chromatin adjacent to nuclear membrane (opened arrows) (B), a well- demarcated electron—dense chromatin occupies almost an entire nuclear cytoplasm (solid arrow) (C) and small nuclei with no distinct nuclear membrane (black arrows) (D). 161 4.3.1.c. Retinal sections at 4 to 5 weeks of age Between 4 to 5 weeks of age the photoreceptor outer and inner segments of normal controls continued to lengthen (Figure 4.1 1A & 4.11C). At this age the number of ONL row and number of rod nuclei per unit length of the retina had reached their peaks (Figure 4.27A, 4.28A, 4.29A & 4.30A). Photoreceptor nuclei had become rounder in shape and the ONL appeared well organized. Loss of outer segments in the PDE6A mutant retina was apparent at 4 weeks of age (Figure 4.11B), along with an apparent slight reduction Of the thickness of the IS layer and the ONL. The thickness of the OS layer across the retina ranged from 0.75 +/- 0.35 to 5.42 +/- 1.18 pm in the PDE6A mutant dog compared to from 3.45 +/- 0.67 to 11.25 +/- 2.13 pm in the normal controls. However, this difference was not significant. There were no significant differences in the thickness of other retinal layers between mutant and normal puppies. At 5 weeks of age photoreceptor cell layer Of the PDE6A mutant retina was reduced in thickness (Figure 4.11D). The outer segments of the PDE6A mutant dog were much disoriented and stunted (Figure 4.16B). The thickness of the OS layer was significantly reduced (P<0.05) compared to normal control from region #2 to #6 along the vertical plane of retinal section (Figure 4.14C), and region #9 and 11# along the horizontal plane of retinal section (Figure 4.15B). Inner segments of mutant dogs at this age appeared shortened, broadened and more prominent (Figure 4.16B). A significant difference (P<0.05) in the thickness of the inner segments was found for region #5 (Figure 4.14C) as well as that of the ONL. There was a significant decrease in the number 162 of ONL rows (P<0.05) at region #3 to #7 on the vertical plane of retinal section (Figure 4.27A). The number of rod nuclei per unit length of the mutant retina was also reduced at regions #2, #3 and from #5 to #7 on the vertical plane of retinal section (Figure 4.29A), and at region #9 on the horizontal plane of section (Figure 4.30A). Pyknotic photoreceptor nuclei were evident in the ONL of the PDE6A mutant retina at this age (Figure 4.12B) although TUNEL staining revealed only 1-2 TUNEL-positive cells per field (Figure 4.13F). Caspase 3-immunoreactive cells could not be detected in the ONL (Figure 4.12C) at this age. 163 — \ 32351153 macaw» . Figure 4.11. Representative photomicrographs of retinal morphology of normal control (A & C) and the PDE6A mutant dogs (B & D) at 4 (A & B) and 5 (C & D) weeks of age. Bar = 50 pm. A & B) Photoreceptor outer segments continue to elongate in normal control retina while they continue to degenerate in the PDE6A mutant retina (white arrows) at 4 weeks of age. Thickness of the PRL and the ONL of the PDE6A mutant retina slightly reduces in comparison to normal control retina. C & D) At 5 weeks of age, photoreceptor segments of a normal control are well structured but fi'agmented and disorganized in the PDE6A mutant retina. Cell present in the PRL (black arrow) could possibly be a phagocytic cell or a displaced photoreceptor nucleus. Loss of inner segments appears at this age; cone inner segments (arrowheads) are broader than those of normal control retina. Apoptotic cells are observed (solid arrows) and the number of photoreceptor nuclei per unit length of the retina appears to be decreased. Key: RPE = retinal pigment epithelium, PRL = photoreceptor layer, ONL = outer nuclear layer, OPL = outer plexiform layer, INL = inner nuclear layer, IPL = inner plexiform layer, GCL = ganglion cell layer. Figure 4.12. Photomicrographs of retinal morphology of the ONL of a normal control (A) at 38 days of age, a PDE6A mutant dog (B) at 42 days of age and caspase 3 immunohistochemistry in a PDE6A mutant retina at 42 days of age (C). Bar = 25 pm. A) In the normal control retina, photoreceptor nuclei are well arranged and tightly packed. B) Apoptotic bodies (solid arrow) and dead cells (arrowhead) are present in the thinned ONL at this age. C) Anti-caspase 3 irnmunoreactivity is not detected in the PDE6A mutant retina at this age. 165 Figure 4.13. Photomicrographs of TUNEL labeling of retina from normal controls (A, C & E) and the PDE6A mutant dogs (B, D, F & G) at 25 day (A & B), 28 day (C & D), 5 weeks (E & F) and 10 weeks (G) of age. Bar = 50 pm. 1 to 2 TUNEL-positive cells can be seen in all retinal sections from normal and mutant dogs (white arrows). A large number of TUNEL-positive cells are apparent in retina of mutant dogs at 28 days of age (D). 166 1 2 3 4 disc 5 6 7 8 Vertical GCL+NFL IPL INL O '0 r- 12 34disc5678 Vertical 1 2 3 4 discS 6 _ 7 _ 8 Vertical region Figure 4.14. Mean retinal thickness of each of the different retinal layers from superior to inferior ora ciliaris retina (vertical section; see Figure 4.1 in materials and methods) from normal control (A), PDE6A carrier (B) and PDE6A mutant dogs (C) at 5 weeks of age (n=3). Note the PDE6A mutant dogs have a reduction in length of outer segments (C) particularly in the central retina, compared to that of normal control (A) and carrier (B). Thickness of the inner segment layer of the PDE6A mutant dog is also slightly reduced. Other retinal layer thicknesses are maintained among disease genotypes and in fact mean overall retinal thickness is greater in the mutant dogs compared to controls. Key: RPE = retinal pigment epithelium, OS = outer segment, IS = inner segment, ONL = outer nuclear layer, OPL = outer plexiform layer, INL = inner nuclear layer, IPL = inner Exiform layer, GCL = ganglion cell layer, NFL = nerve fiber layer. 167 — GCL+NFL _ IPL — INL — OPL — ONL 25° " M“ “m i _ .s 200 +—B.——-—~e+ - -- - ——-- ——~~,’—~—— OS 5150 l — RPE 1 100 . 5 50 ..__. 12 1 1 1 0 9 disc Horizontal region Figure 4.15. Mean retinal thickness Of each Of the different retinal layers from nasal to temporal region (horizontal section; see Figure 4.1 in materials and methods) from normal control (A) and the PDE6A mutant dogs (B) at 5 weeks of age (n=3). Note the outer segments length Of in the PDE6A mutant dogs are decreased compared to normal control (B), while the inner segments appeare slightly shorter but not significant compared to normal control (A). Key: RPE = retinal pigment epithelium, OS = outer segment, IS = inner segment, ONL = outer nuclear layer, OPL = outer plexiform layer, INL = inner nuclear layer, IPL = inner plexiform layer, GCL = ganglion cell layer, NFL = nerve fiber layer. 168 Figure 4.16. Ultra structural sections of the photoreceptor segments of a normal control (A) and the PDE6A mutant retina (B) at 5 weeks of age. Bar = 1 pm. Note stacks of rod outer segment discs are well organized in the normal control (A) whereas they are short and disorganized in the PDE 6A mutant retina (B). Cone outer and inner segments are also swollen and disoriented in the retina of mutant dogs. Key: ROS = rod outer segment, COS = cone outer segment, CIS = cone inner segment. 169 4.3.1 .d. Retinal sections at 7 to 12 weeks of age The normal control retina had an adult-like appearance by 7 weeks of age (Figure 4.17A); the photoreceptor inner and outer segments and cell bodies were well organized. The PDE6A mutant retina on the other hand had a severe loss of photoreceptor outer segments (Figure 4.17B). The thickness of the OS layer was significantly reduced (P< 0.01) for all horizontal and vertical plane of retinal regions compared to the controls, except for the superior far periphery. Loss of inner segments was Observed with the remaining inner segments having a club-shaped appearance (Figure 4.17B). A significant difference (P<0.05) in the thickness of the IS layer was found between the PDE6A mutant and normal control group at region #2 to #8 on the vertical plane section and region #9 on the horizontal plane section. In the mutant dogs, the thickness Of the ONL was significantly reduced (P<0.05) at regions close to the optic disc (central retina) and over most of the inferior regions, as well as at all regions of the horizontal plane. Although a Slight reduction of the number of ONL row and rod nuclei per unit length Of the retina was found in all retinal regions of the normal control at 7 weeks of age, a significant reduction of those numbers (P< 0. 05) in the PDE6A mutant retina were seen in most retinal regions but those at the periphery compared to a normal control group (data not shown). NO significant difference (P<0.05) of other retinal layers and the number of cone nuclei per unit length of the retina were Observed at 7 weeks of age. By 9 weeks Of age, the mean thickness of the photoreceptor OS layer of the PDE6A mutant retina was significantly decreased (P<0.01) for all vertical and horizontal 170 plane Of retinal regions compared to the normal control group. The mutant dog had short remaining inner segments, which still contained mitochondria (Figure 4.18). The mean thickness of the IS layer was significant decreased (P< 0.05) for all vertical and horizontal plane Of retinal regions except for the regions at the superior, inferior and temporal periphery (Figure 4.20 & 4.21). No significant difference in the mean thickness of the ONL was found at this age, although there was a notable loss of photoreceptor nuclei with spaces appearance between the remaining nuclei (Figure 4.19B). The overall thickness of the ONL ranged from 29.42 +/- 1.67 to 43.08 +/- 2.2 um for the normal control compared to from 27.87 +/- 0.87 to 43.41 +/- 1.82 um for the PDE6A mutant retina along the vertical retinal section. However, the number of ONL row and rod nuclei per unit length of the retina was significantly reduced (P<0.05) compared to normal control at regions #3 to #7 and #9 to #10 (Figure 4.27B & 4.28B), and #2 to #8 and #9 to #11 (Figure 4.29B & 4.30B), respectively. Only 1 or 2 TUNEL-positive cells in the ONL were detected at 9 weeks of age (Figure 4.13G). The mean thickness Of the ONL at 12 weeks Of age was slightly decreased in normal control dogs compared to the previous ages (data not shown). Apart from superior and inferior regions close to the optic disc, no significant difference was found at other retinal regions between two groups Of dogs. The number of ONL row and rod nuclei per unit length of the mutant retina was similar to those at 9 weeks Of age. 171 Figure 4.17. Representative photomicrographs of retinal morphology of a normal control (A & C) and the PDE6A mutant dog (B & D) at 7 (A & B) and 12 (C & D) weeks of age. Bar = 50 um. A & B) Retina of the normal control has an adult-like morphology at 7 weeks of age. Photoreceptor nuclei are spherical and very well organized. In the mutant dogs rod and cone outer and inner segments are reduced in size and number. Cone inner segments are broader and more prominent (arrowhead). There are spaces between inner and outer segments and to a lesser extent between photoreceptor nuclei. Phagocytic cells or displaced photoreceptor nuclei are occasionally seen (solid arrow) in the PRL. C & D) Photoreceptor outer segments of the PDE6A mutant retina are severely disrupted with focal area of loss of photoreceptor inner segments at 12 weeks of age (boxed area). Remaining cone inner segments have a club-shaped appearance; phagocytic cells are occasionally seen in the subretinal space (white arrow). In the retina of a mutant dog, the ONL is much reduced in thickness. Key: PRL = photoreceptor layer, OLM = outer limiting membrane, ONL = outer nuclear layer, OPL = outer plexiform layer, INL = inner nuclear layer, IPL = inner plexiform layer, GCL = ganglion cell layer. 172 Figure 4.18. Ultra structural sections of the photoreceptor layer of the PDE6A mutant retina at 9 weeks of age. Bar = 1 pm. Note a narrow PRL present with short rod and cone inner segments, remnant Of degenerative segments, microvilli of the RPE (dark asterisk) and macrophage (white asterisk). Although cone inner segments are very short at this age, mitochondria are the predominant feature. Key: RPE = retinal pigment epithelium, RIS = rod inner segment, CIS = cone inner segment. 173 Figure 4.19. Ultra structural sections of the ONL of a normal control (A) and the PDE6A mutant retina (B) at 9 weeks of age. Bar = 10 pm. A) Rod nucleus (white asterisk) of a normal control is small and contains dispersed electron-dense chromatin. Cone nucleus (black asterisk) is larger and contains less electron-dense chromatin in comparison to the rod nucleus. B) Short photoreceptor inner segments are positioned adjacent to the OLM (arrowhead), which is not as distinct as the OLM of the normal retina. The appearance of space in the ONL indicates loss of photoreceptor nuclei. Approximately six rows of photoreceptor nuclei are observed in the PDE6A mutant retina whereas there are 8 to 9 rows in the age-matched normal control. Apoptotic cells can be seen: note the cell with an aggregate of heterochromatin in the swollen weakly-stained euchromatin (solid arrow) and cell with a well demarcated electron dense chromatin occupying most of the nucleus (black arrow). Key: IS = inner segment. 174 1 2 3 4 discs 6 ‘ 7 Vertical GCL+NFL IPL INL OPL pm 0 Z l" 1 2 3 4 disc 5 6 7 8 Vertical region Figure 4.20. Mean retinal thickness of each of the different retinal layers from superior to inferior ora ciliaris retina (vertical section; see Figure 4.1 in materials and methods) from normal control (A), PDE6A carrier (B) and PDE6A mutant dogs (C) at 9 weeks of age (n=3). Note in the retina of a normal control and carrier (A & B), thickness of each retinal layer at different regions is relatively equivalent, and it increases toward the optic disc. A similar pattern also applies to retinal layer thicknesses of the PDE6A mutant dog (C) with the exception of the outer and inner segment layers that are significantly decreased. These layers seem thinner at regions close to ora ciliaris retina compared to other regions. Key: RPE = retinal pigment epithelium, OS = outer segment, IS = inner segment, ONL = outer nuclear layer, OPL = outer plexiform layer, 1N L = inner nuclear layer, IPL = inner plexiform layer, GCL = ganglion cell layer, NFL = nerve fiber layer. 175 250 200 , E 150 :1. 100 r 50‘ 250 200 >-- E150~ :I. 100: Figure 4.21. Mean retinal thickness of each Of the different retinal layers from nasal to temporal region (horizontal section; see Figure 4.1 in materials and methods) from normal control (A) and PDE6A mutant dogs (B) at 9 weeks of age (n=3). Note that the outer and inner segment layers of the PDE6A mutant retinas (B) are very much narrower than in the normal control retinas (A). The entire ONL has reduced in thickness particularly at region #10 (B) in the PDE6A mutant dog. However, the overall retinal thickness is not reduced. Key: RPE = retinal pigment epithelium, OS = outer segment, IS = inner segment, ONL = outer nuclear layer, OPL = outer plexiform layer, INL = inner nuclear layer, IPL = inner plexiform layer, GCL = ganglion cell layer, NFL = nerve fiber layer. 9 disc Horizontd region 9 disc Horizontal region I GCL+NF L IPL INL OPL ONL IS OS RPE 176 4.3.1.c. Retinal sections at 16 weeks of age By 16 weeks Of age the overall thickness of the retina of the PDE6A mutant retina was reduced by approximately 50% compared to the controls. The photoreceptor layer was severely thinned. Outer segments were present in some areas but very reduced in size and the remaining inner segments were short and broad (Figure 4.22B & 4.23B). The mean thicknesses of the outer segment (P<0.01), inner segment (P<0.01), and the outer nuclear (P< 0.05) layers were significantly reduced compared to normal control retinas at all retinal regions along the vertical plane except for the superior far periphery (Figure 4.24C) and all regions along the horizontal plane (Figure 4.25B). At this age the central retina had approximately 3 rows of photoreceptor nuclei remnant in the mutant ONL, compared to 10 to 11 rows in the normal control. Except for the inferior far periphery and temporal regions, the number Of nuclei rows was significantly lower in the PDE6A mutant retina (Figure 4.27B & 4.28B). Pyknotic nuclei were also evident at this age (Figure 4.23C). The number of rod nuclei per unit length of the retina of the mutant dogs was significantly reduced at more retinal regions than at 9 to 12 weeks of age (Figure 4.29B & 4.30B). The number of cone nuclei per unit length of the retina was found tO be significantly decreased at region #5 and #6 (Figure 4.31B). In addition, the OPL of the PDE6A mutant retina appeared to have lost its normal architecture in the mutant dog. The photoreceptor termini appeared disrupted and less synaptic ribbons were present (Figure 4.2313). 177 Figure 4.22. Representative photomicrographs of retinal morphology of a normal control (A) and the PDE6A mutant dog (B) at 16 weeks of age. Bar = 50 um. Note that the PRL of the PDE6A mutant retina is very narrow with the presence of remaining stunted cone inner segments (solid arrow). There are approximately 3 rows of photoreceptor nuclei in the ONL of the PDE6A mutant retina compared to 10 rows in the retina of the normal control (A). The OLM is still intact (arrowhead). Key: PRL = photoreceptor layer, OLM = outer limiting membrane, ONL = outer nuclear layer, OPL = outer plexiform layer, INL = inner nuclear layer, IPL = inner plexiform layer, GCL = ganglion cell layer. 178 it": a: 3331?; of the outer retina of a normal control (A & D) and the PDE6A mutant dog (B, C & E) at 16 weeks of age. Bar = 5 pm (A, B & C) & 1 mm (D & E). A & B) Region of the photoreceptor inner segments in: the normal dog (A) photoreceptor inner segments have an ellipsoidal shape; those of cones (black asterisks) are larger than those of rods (white asterisk). The basal region of inner segment is in contact with the OLM (black arrows) Mitochondria (solid arrows) are the major organelles in the inner segments. In the mutant dog (B) predominantly cone inner segments remains. These are stunted and club-shaped and mitochondria are still present. The inner segment remnants are still in contact with the OLM. Notched arrow indicates a remaining but shortened and distorted cone outer segment. C) The PDE6A mutant ONL has only 3 rows of nuclei. Apoptotic cells are present. There is a cell with cluster electron-dense chromatin adjacent to the nuclear membrane (arrowhead) and an apoptotic body with an irregular nuclear membrane (opened arrow). D & E) The OPL: The section from normal dog (D) shows characteristic synaptic termini consisting of synaptic ribbon with arciform density projecting into the invagination, lateral processes from horizontal cells and central process from bipolar cell. In the mutant dog the structure of the OPL is disrupted compared to the control. There is a decrease in the number and length of synaptic ribbons. Key: BC = bipolar cell, H = horizontal cell, SR = synaptic ribbon. 179 Vertical region GCUNFL l|l|||l| Iamgsz: 6 7 8 Vertical region 1234disc5 6 _ 7 _8 Vertrcalregron Figure 4.24. Mean retinal thickness of each of the different retinal layers from superior to inferior ora ciliaris retina (vertical section; see Figure 4.1 in materials and methods) from normal control (A), PDE6A carrier (B) and PDE6A mutant dogs (C) at 16 weeks of age (n=3). Note there is a marked reduction of the thickness of the outer segment, inner segment and ONL in all regions of the mutant retina (C). The outer retinal layer of the mutant dog is significantly thinner. Note that the central retinal regions are thicker than that the periphery. Key: RPE = retinal pigment epithelium, OS = outer segment, IS = inner segment, ONL = outer nuclear layer, OPL = outer plexiform layer, INL = inner nuclear layer, IPL = inner plexiform layer, GCL = ganglion cell layer, NFL = nerve fiber layer. 180 I I E a 0 Z r- 12 11 10 9 disc Horizontd region Figure 4.25. Mean retinal thickness of each of the different retinal layers from nasal to temporal region (horizontal section; see Figure 4.1 in materials and methods) from normal control (A) and PDE6A mutant dogs (B) at 16 weeks of age. Note the significant reduction of the ONL and OPL thickness in the PDE6A mutant retina (B) in comparison to a normal control (A). Thickness Of the inner retinal layers rapidly declines from the center toward peripheral retina. Key: RPE = retinal pigment epithelium, OS = outer segment, IS = inner segment, ONL = outer nuclear layer, OPL = outer plexiform layer, INL = inner nuclear layer, IPL = inner plexiform layer, GCL = ganglion cell layer, NFL = nerve fiber layer. 181 12 Number of row 6 N A OK 00 0 10 20 30 40 50 60 70 80 90 100110120130140150 Age(day) a§§ Numberotrod -- N N 8 8 § 0 IO 20 30 40 50 60 7O 80 90 100110120130140l50 Amway) Number Of cone 0 10 20 30 40 50 60 70 80 90 100110 120130140 150 . 0+ . +,_ g 4_ Age (day) Figure 4.26. Scatter plot showing the number of nuclei rows in the ONL (A), number of rod (B) and number of cone (C) nuclei per unit length (100 pm) of the retina (of region #3) plotted against age for normal control (blue), carrier (green) and mutant (orange) dogs. Note that following an early slight increase in number of ONL row and number of rod nuclei during the first 28 days of life these numbers in normal controls and carriers seem stable throughout the investigation. Number of cone nuclei is maintained and then slightly decreases after about 80 days Of age. In contrast, there is no increase in number of nuclei rows in the ONL and rod nuclei in mutant dog. The number Of nuclei rows and rod nuclei rapidly decrease at about 49 days of age, and these numbers stay stable, and then slowly decrease at about 100 days Of age. In the mean time, number of cone nuclei of the PDE6A mutant retinas slightly decreases with age but still comparable to that of normal controls and carriers. 182 —-'___ find—— Number of row Number of row Number of row Region — PDE6A 4- =1 PDE6A +/+ Figure 4.27. Mean number of nuclei rows in the ONL Of retinal regions (#1 to #8) in a vertical section through the optic nerve head at 5 (A), 9 (B), 16 (C) and 60 (D) weeks of age (n=3). Note at 5 and 9 weeks of age that the mean number of the ONL row in the PDE6A mutant dogs is significantly smaller (P<0.05) at region #3, #4, #5, #6 and #7 (A & B). By 16 weeks of age the number of nuclei rows in the ONL of the more peripheral region #1 and #2 were significantly reduced (P<0.05) compared to controls (C). By 60 weeks of age, there is a significant reduction in the mean number of nuclei rows in the ONL in the PDE6A mutant dogs for all retinal regions (D). Asterisk (*) indicates significance P<0.05. 183 Number of row Number of row Number Of row Number of row disc Region - PDE6A -/- r2: PDE6A +/+ Figure 4.28. Mean number of nuclei rows in the ONL of retinal regions (#9 to #12) in a horizontal section temporal from the optic nerve head at 5 (A), 9 (B), 16 (C) and 60 (D) weeks of age (n=3). Note that there is no significant difference in the mean number Of the ONL nuclei rows between normal control and the PDE6A mutant dogs at 5 weeks of age. By 9 weeks of age (B) region #10 and #11 are significantly thinner than controls and at 16 weeks of age (C) regions #9, #10, #11 and by 60 weeks of age (D) all regions are flificantly thinner (P< 0.05). Asterisk (*) indicates significance P<0.05. 184 Number of cell Number of cell N 01 O ..1 Number of cell Number of cell Region — PDE6A 4— :1 PDE6A +I+ Figure 4.29. Mean number of rod nuclei per unit length (100 pm) of retinal regions (#1 to #8) in a vertical section through the optic nerve head at 5 (A), 9 (B), 16 (C) and 60 (D) weeks of age (n=3). At 5 weeks Of age (A) the mean number of rod nuclei is significantly reduced (P<0.05) in regions #2, #3, #5, #6 and #7. By 9 weeks of age the number of nuclei at regions #4 and #8 is significantly reduced. At 16 weeks of age region #8 is not significantly different. By 60 weeks of age the mean number of rod nuclei is significantly reduced (P<0.05) for all retinal regions (D) compared to the normal control. Asterisk (*) indicates significance P< 0. 05. 185 § 200 Number of Number of cell Number of cell disc Region — PDESA 4— 1:: PDE6A +l+ Figure 4.30. Mean number of rod nuclei per unit length of retinal regions (#9 to #12) in a horizontal section temporal from the optic nerve head at 5 (A), 9 (B), 16 (C) and 60 (D) weeks of age (n=3). There is a Significance decrease (P<0.05) in the mean number of rod nuclei in the PDE6A mutant dogs at region #9 at 5 weeks of age (A). By 9 weeks of age regions #10 and #11 also have a significant reduction (P<0.05) in rod nuclei number (B). The mean number of rod nuclei is significantly decreased (P<0.05) in all retinal regions at 16 (C) and 60 (D) weeks of age. Asterisk (*) indicates significance P<0. 05. 186 Number of Cell Number of cell _ PDE6A -I~ 1:1 PDE6A +I+ Number Of cell disc Region Figure 4.31. Mean number of cone nuclei per unit length (100 pm) of all retinal regions (#1 to #8) in a vertical section through the Optic nerve head (A & B) and horizontal section temporal through the optic nerve head (C) at 5 (A) and 16 (B & C) weeks of age (n=3). Note that although the mean number of cone nuclei is decreased for all retinal regions in the mutant dogs at 16 weeks of age (B), this difference is only significant (P<0. 05) for region #5 and #6. NO difference between the two groups is found at 16 weeks of age for the horizontal section (C). Asterisk (*) indicates significance P<0.05. 187 4.3.1.f. Retinal sections at 24 weeks of age Detailed measurements Of the thickness of each retinal layer showed that in the PDE6A mutant retina at 24 weeks of age the inferior temporal region had the greatest retinal thickness compared to other retinal regions whereas the superior central (#1.1 & #1.2) and temporal (#3.l & #32) regions were thinnest. Outer segments could not be seen in the PDE6A mutant retina sections at any retinal regions. The thickness Of the IS layer and ONL (Figure 4.32 & 4.33) was best preserved at the inferior temporal (#43 & #44) inferior nasal (#3.3 & #3.4) and central nasal (#2.3 & #2.4). The INL Of the PDE6A mutant dog was narrow compared to that of the normal control in the superior retina (data not shown). No difference of the thickness of GCL and NFL was observed between the two groups of dogs. The number of row of nuclei in the ONL (Figure 4.34) and number of photoreceptor nuclei per unit length of the retina (Figure 4.35) were dramatically reduced at all 16 regions investigated. 188 C. Region Region — PDE6A -I- :1 PDE6A +I+ Figure 4.32. Thickness of the inner segments of normal control and the PDE6A mutant dog for different retinal regions (see Figure 4.2 in materials and methods) at 24 weeks of age. Note that the length of the PDE6A mutant inner segment is remarkably shorter than that in the normal control at all retinal regions investigated. Inner segments are somewhat preserved in the nasal areas; #23 & #2.4 (B), #3.3 & #3.4 (C) and the inferior temporal areas; #4.3 & #4.4 (D). 189 3.4 Region Region — PDE6A -/- r2: PDE6A +I+ Figure 4.33. Thickness of the ONL of normal control and the PDE6A mutant dog for different retinal regions (see Figure 4.2 in materials and methods) at 24 weeks of age. Note that in the mutant dog there is a severe reduction of the ONL thickness affecting the superior retina and inferior central retina of region #13 and #1.4. Other regions of the inferior retinal area (B, C & D) are better preserved. 190 Number of row Number of row C. R6910” 0. //\“fi,_,-\ Region /«// \h\ g 12 12"'/ ‘ i E 10 S o "6 2 S g E z 5 3.3 3.4 4.3 4.4 Region Region — PDE6A -/- 1:1 PDE6A +I+ Figure 4.34. Number of nuclei rows in the ONL at different retinal regions (see Figure 4.2 in materials and methods) in normal control and the PDE6A mutant dog at 24 weeks of age. Note an approximate 75% reduction of the number of ONL row is found in the PDE6A mutant dog at all retinal regions in comparison with a normal control. 191 Number of cell Number of cel Region Region — PDE6A 4— I: PDE6A +I+ Figure 4.35. Number of photoreceptor nuclei per unit length (100 pm) of retina at different retinal regions (see Figure 4.2 in materials and methods) in normal control and the PDE6A mutant dog at 24 weeks of age. Note that there is a marked reduction in the number of photoreceptor nuclei at all retinal regions in the PDE6A mutant dog. The superior retinal area is most severely affected with an approximately 80% reduction in the number of cells. 192 4.3.1 . g. Retinal sections at age greater than 60 weeks By 130 weeks of age, the PDE6A mutant retina (Figure 4.36B) was approximately 40% of the thickness of the normal control. The photoreceptor layer was very narrowed and in some regions some stunted inner segments remained (Figure 4.36D). A cluster of a Single layer of photoreceptor nuclei, presumably cones, was noticed in some retinal areas. In the region where photoreceptor nuclei were absent, cells in the INL appeared disoriented (Figure 4.36C). Not only were the INL and the IPL very thin, but the distinction between each retinal layer was lost. A significant difference (P<0.05) in the thickness of the IPL was found at regions #7 and #8. A significant reduction (P<0.01) in the thickness Of OS, IS, the ONL and the OPL layers at all regions (Figure 4.37 & 4.38), as well as a significant decrease of the number of ONL row (Figure 4.27D & 4.28D) and rod nuclei per unit length of the retina (Figure 4.29D & 4.30D). Although a direct comparison of the thickness of the INL and the GCL+NFL suggested they were thinner, in mutant dogs the alteration in the overall mean thickness was not significant. 193 "5‘ I “fig".- i" 51%“; . ‘1. ’ l U: qt ‘ Figure 4.36. Representative photomicrographs of retinal morphology of a normal control (A) and the PDE6A mutant dog (B, C & D) at 100 weeks of age. Bar = 50 pm. A) The normal control retina shows the typical canine retinal structure. B, C & D) Even though retinal thickness varies from region to region in the PDE6A mutant retina, the entire retinal thickness is very much reduced compared to that of the normal control. Only occasional isolated photoreceptor nuclei (arrowhead) are present (B & D). In regions of most severe retinal thinning it is difficult to differentiate the dividing retinal layers. Key: PRL = photoreceptor layer, OLM = outer limiting membrane, ONL = outer nuclear layer, OPL = outer plexiform layer, INL = inner nuclear layer, IPL = inner plexiform layer, GCL = ganglion cell layer. 194 _ GCL+M-‘L r: «I IPL — INL 1234disc5678—OPL Verticalregion — ora -— rs .— — RPE 1 2 3 4 disc 5 6 7 8 Vertical region Figure 4.37. Mean retinal thickness Of each of the different retinal layers from superior to inferior ora ciliaris retina (vertical section; see Figure 4.1 in materials and methods) from normal control (A) and PDE6A mutant dogs (B) at greater than 60 weeks of age (n=4). Note thickness of the RPE layer (obscured by the line representing the thickness of the inner and outer segments) in the PDE6A mutant retina is maintained (B). Photoreceptor layer of the mutant retina in contrast disappears or appears exceedingly thin. The ONL and the OPL are significantly decreased in thickness. The greatest thickness of the PDE6A mutant retinal layer remaining is however evident at the central retina and is due to relative preservation of thickness in the NFL and GCL. Key: RPE = retinal pigment epithelium, OS = outer segment, IS = inner segment, ONL = outer nuclear layer, OPL = outer plexiform layer, INL = inner nuclear layer, IPL = inner plexiform layer, GCL = ganglion cell layer, NFL = nerve fiber layer. 195 — GCLHFL uric-aerial IPL — INL — OPL — ONL _ is E 150 l l is 06 1 — RPE 12 11 10 9 disc Horizontd region Figure 4.38. Mean retinal thickness of each Of the different retinal layers fi'om nasal to temporal region (horizontal section; see Figure 4.1 in materials and methods) from a normal control (A) and PDE6A mutant dogs (B) at greater than 60 weeks Of age (n=4). Note that the ONL and the OPL in the retina Of PDE6A mutant dog are severely decreased in thickness. Key: RPE = retinal pigment epithelium, OS = outer segment, IS = inner segment, ONL = outer nuclear layer, OPL = outer plexiform layer, IN L = inner nuclear layer, IPL = inner plexiform layer, GCL = ganglion cell layer, NFL = nerve fiber layer. 196 4.3.2. Immunohistochemical analyses Of paraffin-embedded and frozen sections 4.3.2.a. Immunohistochemistry using rhodopsin Two types of antibody against rhodopsin were used in this study. Staining using antibody against bovine rhodopsin was specific to rod photoreceptors and strongly stained the outer segments and very weakly stained the inner segments of the canine retina. The use of this antibody in normal control retina demonstrated that rod outer segments elongated with age (Figure 4.39A, C & E). In contrast, growth of rhodopsin- labeled rod outer segments of the PDE6A mutant dog halted at about 4 weeks of age, and was followed by an apparent decrease in the number and length of immunoreactive outer segment by 5 weeks Of age (Figure 4.39D). Moreover, the remaining rod outer segments in mutant dog at 5 weeks of age were disoriented compared to an age-matched normal control (Figure 4.39C). Loss of rod outer segments was very pronounced by 9 weeks Of age in the PDE6A mutant retina, at which time only very short rhodopsin-labeled segments remained (Figure 4.39F). 197 Figure 4.39. Immunohistochemical expression of anti-rhodopsin (a gift from Dr. P. Hargrave, University of Florida) in retinal sections Of normal control (A, C & E) and the PDE6A mutant dogs (B, D & F) at 3 (A & B), 5 (C & D) and 9 (E & F) weeks of age. Bar = 25 pm. In the control retinas rhodopsin immunoreactivity is predominantly in the rod outer segments (A, C, & E). Rhodopsin immunoreactive rod outer segments (Opened arrows) are already present at 3 weeks of age in control and mutant retina (A & B). They have elongated by 5 weeks of age (C & D) even though the rhodopsin-labeled rod outer segments of the PDE6A mutant retina shows disorganized discs and less immunoreactive material (D). Spaces (black arrows) are present in the PRL indicating a loss of rod outer segments. By 9 weeks of age in the mutant retina only very short remaining rhodopsin immunoreactive are present (arrowheads) (F). Key: RPE = retinal pigment epithelium, PRL = photoreceptor layer, ONL = outer nuclear layer, INL = inner nuclear layer. 198 An antibody raised against mouse rhodopsin (Lab Vision) labeled the entire rod cell (outer segments, inner segments, perinuclear regions of rod cell bodies and rod termini at the outer regions Of the OPL). Use of this antibody confirmed that there was an increase of rod segments as well as the number of ONL row with age in normal control retina (Figure 4.40A & 4.40C). Immunoreactivity of the perinuclear region of the PDE6A mutant rod photoreceptors was slightly reduced at 4 weeks of age but there appeared to be immunoreactivity at the level of photoreceptor termini (Figure 4.40B). By 8 weeks of age when rods had suffered a decrease in their outer segment length and a decrease in total numbers in the PDE6A mutant retina, there was a reduction in immunoreactivity in the rod soma as well as in the rod termini. Neurite outgrowth was Observed extending from rod termini horizontally within the OPL and also vertically to the inner retina. A small number of immunoreactive cells still remained in particular at the mid periphery, in the PDE6A mutant retina at 60 weeks of age, with some immunoreactive cell processes within the photoreceptor cell layer (Figure 4.40B). By 210 weeks of age (Figure 4.40F), there was only 1 or 2 immunoreactive cell bodies per section remained in the PDE6A mutant retina that expressed rhodopsin. 199 Figure 4.40. Immunohistochemical expression of anti-rhodopsin (Lab Vision) in retinal sections of normal control (A & C) and the PDE6A mutant dogs (B, D, E and F) at 4 (A & B), 8 (C & D), 60 (E) and 210 (F) weeks ofage. Bar = 25 pm. Note that this antibody stains the entire rods cell (outer segments, inner segments, perinuclear region of rod cell bodies and synaptic termini) in the retina of normal control (A & C) while cone nuclei (white arrows) are not rhodopsin-labeled. Compared to normal control at 8 weeks of age, not only rod outer segments of the PDE6A mutant are short (D), but there is a reduction of rhodopsin staining at the perinuclear region of rod cell bodies. Profound rod neurite sprouting (black arrow) extending into the inner retina is present in the OPL the mutant dog at 8 weeks of age (D). At 60 and 210 weeks of age in the mutant dog there are some remaining 0 cell bodies (arrowheads) even though they are very much reduced ID number. Neurite sprouting in the residual ONL 13 still present at this age but is primarily in a horizontal direction (E). Note that RPE cells are present in the neurosensory retina (F). Key: PRL = photoreceptor layer, ONL = outer nuclear layer, OPL = outer plexiform layer, INL = inner nuclear layer. 200 4.3.2.b. Immunohistochemistry using cone arrestin Immunohistochemical staining of sections using an antibody against rabbit cone arrestin in the normal canine retina resulted in labeling of the cone outer segments, cell bodies, axons and synapses at the inner regions of the OPL (Figure 4.41A). Compared to normal control retinal section, cone arrestin-immunopositive outer segments of the PDE 6A mutant retina at 9 weeks Of age were shorter and appeared swollen. The fainter staining Of inner segments showed that they were considerably shorter than normal (Figure 4.41B). Additionally the expression Of the antibody in the cone termini was reduced. Retinal sections from the PDE6A mutant dog showed that even though the retina had thinned at 210 weeks of age, a number of cones were still present in different retinal regions (Figure 4.41C & 4.41D). In addition, the sprouting of cone axonal processes was Observed not only in the OPL where the cone termini are normally located, but also ectopically into the IPL and the GCL. 201 ' “é 7’ " . ’ ‘ 4' “a...“ -ahmur «.315: r . A, . . v . ., r ' . n - V ' ‘ L "iv ~‘":.'!- ~ '21... b ., UV *7 F . "t u s o ‘ . 0.. 1’ v“ I 0 w: a 6 - ‘ ‘ - ' ‘ O . .' . . . y! I“ z . 'O'O‘ h“? ‘ l V? I ' .‘i“t. ’1. 0“. «r “ ‘. "‘ 1' . ‘ O. .- “ ’ K 4:; - ‘ 0- - ‘94,. ,‘f”“’ '. 6 'b' ' i ‘ ' ' Figure 4.41. Immunohistochemical expression of anti-cone arrestin (a gift from Dr. S. Craft, University of Southern California) in retinal sections of normal control (A & C) and the PDE6A mutant dogs (B & D) at 9 (A & B) and 210 (C & D) weeks of age. Bar = 50 um. Note that cone arrestin labels outer segments, cytoplasm of the cell bodies and synapses of all cones and some cells of the INL in the normal control retina (A). Compared to retinal section of a normal control, the retina Of the PDE6A mutant has strmted cone outer segments (black arrow) connected to short inner segments. There appear to be reduced immunoreactivity of cone arrestin at the cone synapses in the OPL compared to the normal control. A cluster of remaining cone nuclei are seen in tapetal and nontapetal retinal regions of the PDE6A mutant at 210 weeks of age (C & D). Sprouting of cones processes within the residual ONL is apparent at this age (arrowhead). Key: RPE = retinal pigment epithelium, PRL = photoreceptor layer, ONL = outer nuclear layer, INL = inner nuclear layer. 202 4.3.2.e. Immunohistochemistry using red/ green Opsin Staining using the antibody against red/green rabbit opsin was predominantly restricted to the outer segments Of the canine green cones (M/L cones) (Figure 4.42A) although weak labeling was present in the cell bodies, axons and synaptic termini (Figure 42E). In the PDE 6A mutant retina, M/L cones appeared to have shorter outer segments compared to the normal control. At 16 weeks of age, in addition to a major loss of the number of M/L cone outer segments (Figure 4.42C), there was a decrease of the axonal length as well as a mislocalization of the cone segment in the ONL of the PDE6A mutant retina. The immunoreactivity of the M/L cones appeared increased at 60 weeks of age when at which age the cone axons were stunted and showed ectopic processes extending in to the IPL (Figure 4.42D). 203 Figure 4.42. Immunohistochemical expression of anti-red/green opsin fluorescence in retinal sections of normal control (A & E) and the PDE6A mutant dogs (B, C, D & F) at 5 (A & E), 7 (B), 16 (C & F), and 60 (D) weeks ofage. Bar = 50 pm (A to D) and 25 (E & F) urn. Note in normal control, red/green Opsin strongly labels red/green cone outer segments (A), and weakly labels their cell bodies and axons (E). Localization of red/green opsin immunoreactivity in the PDE6A mutant retina at 7 weeks of age shows a decrease in length and number of cone outer segments (B). Displaced short outer segment (white arrow) is noticed in the ONL (C), an age at which cone axon has a reduced length (F). At 60 weeks of age, cone cell bodies with some residual brighter staining (arrowhead) are present (D). Key: PRL = photoreceptor layer, ONL = outer nuclear layer, INL = inner nuclear layer, GCL = ganglion cell layer. 204 4.3.2.d. Immunohistochemistry using PKCOI Immunoreactivity to mouse anti-PKCOI antibody in normal canine retina was present in the dendrites, perinuclear region of the cell bodies, axons and synaptic termini (in the outer region of the IPL) of rod bipolar cells (Figure 4.43A, 4.43C & 4.43B). PKCOI-labeled rod bipolar cells in the PDE6A mutant retina showed a progressive decrease in number and length with age. Some retraction of the dendritic arborization of rod bipolar cells could be seen in the PDE6A mutant retina at 7 weeks of age (Figure 4.43B). By 9 weeks of age, at which approximately one row of rod bipolar cell soma remained at the outer region of the ONL compared to the usual two layers. Rod bipolar cell axonal processes were significantly shorter and dendritic arborization was considerably retracted and disorganized compared to those of the bipolar cells in the retina of normal control dogs (Figure 4.43C & 4.43D). A mislocalization Of rod bipolar cell to the ONL Of the PDE6A mutant retina could be seen sporadically at 60 weeks of age (Figure 4.43F). Although the overall expression of PKCor-positive rod bipolar cell soma was less compared to an age-matched control, additional number Of small axonal branches of rod bipolar cells were located throughout the IPL. 205 . . ‘- .‘?‘ .‘ .. J , ' '.l ‘ ‘filéz rha‘ ' .. ‘w . o ‘ . :5, r ‘0; '9’ j" I, r '- 1 .- ‘ ' .:* $11 ' '- I“ C‘ ‘ 7.. M ”, Figure 4.43. Immunohistochemical expression of anti-protein kinase C alpha (PKCOI) in retinal sections of normal control (A, C & E) and the PDE6A mutant dogs (B, D & F) at 7 (A & B), 9 (C & D) and 60 (E & F) weeks of age. Bar = 25 um. Note in normal control, PKCor immunoreactivity is localized in rod bipolar cell dendrites in the OPL, perinuclear region of cell bodies in the outer region of the INL, axonal processes, and terminal branches located at the inner region of the IPL (A, C & E). Compared to the retina of a normal control, there appears to be a decrease in number of PKCOI positive soma in the PDE6A mutant retina at 7 weeks of age (B). By 9 weeks of age there is a marked loss of PKCOI immunoreactive cell dendrites, as well as cell bodies in the INL (D). The thickness of the IPL is reduced and the number of axonal processes (black arrow) is also reduced. By 60 weeks of age, a reduction of immunoreactive labeling of rod bipolar cell nuclei is noticed in the PDE6A mutant retina (F), and a displaced rod bipolar cell is found in the ONL (solid arrow) (F). At this age, there is a ramification of cell processes in the OPL and the IPL. Key: ONL = outer nuclear layer, OPL = outer plexiform layer, INL = inner nuclear layer, IPL = inner plexiform layer, GCL = ganglion cell layer. 206 4.3.2.e. Immunohistocherrristry using calbindin The mouse anti-calbindin antibody stained canine horizontal cell soma and axons positioned at the outer border of the INL. The retina of the PDE6A mutant dog had a similar pattern of calbindin immunoreactivity to the normal control at 10 weeks of age (Figure 4.44A & 4.44B). However, a hypertrophy of horizontal cell soma could be seen in the PDE6A mutant retina by 60 weeks of age (Figure 4.44D). Figure 4.44. Immunohistocherrrical expression of anti-calbindin fluorescence in retinal sections of normal control (A & C) and the PDE6A mutant dogs (B & D) at 10 (A & B) and 60 (B & D) weeks Of age. Bar = 50 pm. Note that at 10 weeks of age the retina of mutant dog exhibits normal morphology of horizontal cells (A & B). Anti-calbindin immunoreactive horizontal cells from the PDE6A mutant retina at 60 weeks of age (D) are well preserved and appear to be hypertrophic (arrowheads) compared to the control. Key: ONL = outer nuclear layer, INL = inner nuclear layer. 207 4.3.2.f. Immunohistochemistry using Hu C/D Hu C/D immunoreactivity was present in cell bodies located in the outer region of the INL (amacrine cells) and in the GCL (ganglion cells). No difference was observed between the normal control and the PDE6A mutant retina at 7 weeks of age in the number of Hu-immunoreactive cells per unit length of the retina and the degree of immunoreactivity (Figure 4.45A & 4.45B). A decrease in the number of Hu- irnmunoreactive cells and the size of their soma was apparent in the PDE6A mutant retina at 60 weeks of age (Figure 4.45C) by which age there was retinal thinning. Figure 4.45. Immunohistochemical expression of anti-Hu C/D fluorescence in retinal sections of normal control (A) and the PDE6A mutant dogs (B & C) at 5 (A), 7 (B) and 60 (C) weeks of age. Bar = 50 pm. Note the presence of Hu staining of the cell bodies of ganglion cells (arrowheads) and amacrine cells (white arrows). No difference is detected between the normal control and the PDE6A mutant retina at 7 weeks Of age. However, in the PDE6A mutant retina the number of cell bodies immunopositive for Hu per unit length of retina has declined by 60 weeks of age (C). Key: ONL = outer nuclear layer, IN L = inner nuclear layer, GCL = ganglion cell layer. 208 4.3.2. g. Immunohistochemistry using calretinin Immunohistochemistry of retinal sections using an antibody against calretinin showed calretinin immunoreactivity in horizontal cell soma and axons, amacrine and ganglion cell soma and processes in the [PL and GCL. NO difference in calretinin immunoreactivity between normal control and the PDE6A mutant retina could be detected at 5 weeks of age (Figure 4.46A & 4.46B). In the PDE6A mutant retina at 10 weeks of age, calretinin immunoreactivity was reduced in the outer border of the INL where horizontal cells were located. By 60 weeks of age there appeared to be increased immunoreactivity in horizontal cells. A decrease of calretinin labeling was noticed in the IPL (Figure 4.46F) of the PDE6A mutant retina compared to normal control at this age. 209 Figure 4.46. Immunohistochemical expression of anti-calretinin fluorescence in retinal sections of normal control (A, C & E) and the PDE6A mutant dogs (B, D & F) at 5 (A & B), 10 (C & D) and 60 (E & F) weeks of age. Bar = 50 um. Note that immunoreactivity of calretinin is present in cell bodies of amacrine cells (arrowheads) and ganglion cells (solid arrows), as well as in horizontal cell processes (white arrows) and other processes located in the IPL, the GCL and the NFL (A & D). The retina of the 5 and 10 week old PDE6A mutants shows a similar calretinin staining pattern to that of normal controls (A, B, C & D). At 60 weeks of age there appear to be slightly increased calretinin staining in horizontal cell processes in the PDE6A mutant dog (F) however, the overall staining pattern appears similar between mutant and control. Key: ONL = outer nuclear layer, IN L = inner nuclear layer, IPL = inner plexiform layer, GCL = ganglion cell layer. 210 4.3.2.h. Immunohistochemistry using GFAP Immunoreactivity to GFAP was present in the inner region of the OPL, the IPL and the NFL of normal canine retina. There was a weak GFAP labeling in the NFL at 3 weeks of age, but more intense labeling in sections from Older normal dog (Figure 4.47C & 4.47E). The GFAP labeling was similar between normal control and the PDE6A mutant retina at 3 weeks of age. However, GFAP staining of the PDE6A mutant retina at 5 weeks of age was increased. The entire Miiller cell was labeled extending vertically from the OLM to the ILM of the retina (Figure 4.47D). Increased GFAP immunoreactivity was present in the PDE6A mutant retina at 60 weeks of age, at which GFAP-positive glial tissue occupied almost the entire remaining retina (Figure 4.47F). 211 W1. A arstbwi’s“? . L INL: in ,;_4 “13‘1“, $3.191? i'5rsg‘fiwgl'ar. GCL+NFL jv -' as» ”fir“? "N \. "~ I" .. " I "\ ' ‘p Figure 4.47. Immunohistochenrical expression of anti-GFAP in retinal sections of normal control (A, C & E) and the PDE6A mutant dogs (B, D, & F) at 3 (A & B), 5 (C & D) and 60 (E & F) weeks of age. Bar = 25 um. Note that GFAP labeling is present in the normal control retina in the inner region of the OPL, the IPL and the NFL (astrocyte processes) (A, C & E). The entire Mfiller cells stretching from the OLM to the NFL is stained for GFAP in the PDE6A mutant retina by 5 weeks of age (D) indicating increased GFAP levels in Miiller cells. GFAP-positive glial tissue occupies almost the entire of the remaining retina of the PDE6A mutant at 60 weeks of age (F). Key: PRL = photoreceptor layer, OLM = outer limiting membrane, ONL = outer nuclear layer, OPL = outer plexiform layer, INL = inner nuclear layer, IPL = inner plexiform layer, GCL = ganglion cell layer, NFL = nerve fiber layer. 212 4.4. Discussion The normal canine retina is immature at birth. Retinal differentiation, morphogenesis and maturation occur over the first 7 to 8 weeks of life. The results of this study including detailed morphological examination, ultrastructural and immunohistochemical analysis revealed early postnatal abnormalities and failure of maturation Of photoreceptors, in particular rods, of the PDE6A mutant dogs. In the mutant dogs there was a failure of normal development of rod outer segments. Soon after the outer segments of the mutant dogs started to form their development was halted and they failed to elongate. The outer segment discs that did form were disorganized. This was followed by a rapid decrease in the number of rod cells. Plotting the number of rod nuclei per unit length against age gives an indication of the kinetics of rod cell loss. There appears to be an initial stage where few cells are lost. This period is during the time when there is initial development of rod outer segments. There is then a rapid loss of cells between 4 to 6 weeks of age after which the rate of cell loss plateaus and there is a proportionally slower rate of loss of the residual cells. It is interesting to note that there is a lag between the loss of rod nuclei and the narrowing of the outer nuclear layer. This can be explained by the fact that during the period of rapid cell loss the normally tight packing of photoreceptor nuclei in the ONL is lost and spaces appear between the nuclei. In the later stages of the disease the remaining nuclei in the outer nuclear layer are more closely packed again so that the nuclei density more closely resembles that of the normal control sections. The kinetics of cell loss characterized by an initial period Of rapid loss cell death followed by a period of more gradual cell loss has been described for several retinal degeneration and neuronal cell loss situations (Clarke et al., 2000). A so called 213 ‘one-hit model’ has been developed to explain this kinetics. The model proposes that there is a constant risk of death of each neuron, that death occurs randomly and that it is independent of other neurons. The proponent of this theory went on to suggest that the one-hit model is modified by local microenvironment effects (Clarke & Lumsden, 2005) thus accounting for regional variations in the rate of cell loss. Light and electron morphological examinations of retinas of 3 to 4 week old puppies revealed the presence of many apoptotic nuclei in the ONL of PDE6A mutant retinas, compared to normal controls. Apoptosis occurs as part of the normal development of the nervous system (Cellerino et al., 2000) including the maturing retina (Biehlmaier et al., 2001). Apoptosis has also been shown to be the process by which cells die in inherited retina dystrophies (Lolley, 1994). For example, a dramatic increase in cell death in the outer nuclear layer occurs in rd] mice occurs during the third week of postnatal life (Porteracailliau et al., 1994). Apoptotic cell death has also been described in the RCS rat model (Tso et al., 1994). Apoptosis is also responsible for retinal cell death in environmentally induced diseases such as light-damage (Hao et al., 2002). The TUNEL staining technique was used to verify that cell death was occurring by apoptosis in the PDE6A mutant. The TUNEL technique detects intemucleosomal DNA fragmentation in apoptotic cells. Significant TUNEL staining was present in the early stages of retinal degeneration at 3 to 4 weeks of age but at later stages (5 and 9 weeks of age) there were not significant numbers of TUNEL-positive cells in the ONL. This shows that death of a large number of photoreceptors occurs early in the disease 214 process and after the initial period there is a much more gradual cell loss and indeed at these later ages less nuclei with morphological changes suggestive of apoptosis were apparent in the ONL. Caspase 3 immunohistochemistry was performed to further characterize cell death in the PDE6A mutant retina. There was an apparent lack of immunoreactivity to the anti- caspase 3 antibody. This finding is similar to studies of the rcdl mouse where apoptosis was shown to occur by a caspase-independent pathway (Doonan et al., 2003; Zeiss et al., 2004). In another inherited retinal degeneration model the RCS rat immunohistochemistry showed that caspases 1 and 2 were activated suggesting that apoptosis in this model was via a caspase-dependent pathway, although immunohistochemistry for capase 3 was not (Katai et al., 1999b). Additional investigation of the mechanism of apoptosis in the PDE6A mutant dogs would facilitate an understanding of disease mechanism and perhaps suggested possible therapeutic intervention. Cone photoreceptors are also affected in the PDE6A mutant dog retina. Ultrastructural examination suggests that cone outer segments are also stunted and lost rapidly and cone inner segments become enlarged and club-shaped early in the disease process. Immunohistochemistry using cone arrestin and red/green cone opsin antibodies suggested that by 7 weeks of age there were abnormalities in cone photoreceptors. The mean number of cone nuclei per unit length of the retina however seemed maintained until 16 weeks of age despite the fact that the mean number of rod nuclei had dramatically decreased. Retinal degeneration in the PDE6A mutant dogs is expressed 215 across the retina and progressed with increasing age. Loss of photoreceptors was more severe in the central retina with a slower loss at the periphery, and it proceeded more rapidly in the superior quadrants than the inferior quadrants. As the retina matures centrally first it is perhaps not surprising that the central retina would be affected earlier than the peripheral retina. The difference between superior and inferior retina could be accounted for by the presence of the tapetum in the superior fundus. This structure reflects light back through the photoreceptors and thus may result in a greater risk of light induced damage and also the formation of greater number of free radicals within the tapetal retina meaning that apoptosis is more readily triggered in this region. Immunohistochemistry using cone arrestin antibody to label cones showed that clusters of cone nuclei remained in the inferior hemisphere of the PDE6A mutant retina at 210 weeks of age. The quadrant-specific disease progression in the PDE6A mutant dogs is similar to that reported in the prcd dogs (Aguirre & Acland, 1988) but dissimilar to that of the rd] mouse, in which the longest cone survival was in the mid-peripheral superior region (Ogilvie et al., 1997) and the inferior quadrants where s-cones are exclusively located was more severely affected (Jimenez et al., 1996). Further investigations in the PDE6A dogs to see if there is a differential rate of loss of blue cones compared to green cones could be considered. The loss of cone photoreceptors that themselves are genetically normal, the PDE 6A gene being rod specific, is in keeping with the findings in other retinal degeneration models. Recent work has shown the presence of a rod derived cone trophic factor (Sahel et al., 2001). This is released from rods and is a survival factor for cones that is necessary to maintain cones in species like dog and human that has a rod dominated retina. The loss of rods results in a reduction of this rod derived factor and 216 leads to a secondary loss of cones. It is tempting to also speculate that the rapid loss of large numbers of rod cells would alter the environment around the cones meaning that it was not conducive to their survival. Increased Miiller cell immunoreactivity to GFAP developed at an early stage of retinal degeneration in the PDE 6A mutant dog retina. This indicates glial cell activity and has been described as a feature of several other forms of retinal dystrophy including the rd chicken (Semplerowland, 1991), rge chicken (Inglehearn et al., 2003), rd] mouse (Frasson et al., 1999), RCS rat (Kimura et al., 2000) and RP patients (Rodrigues et al., 1986). The role of Miiller cells in neuroprotection and regeneration after retinal damage has been studied. Not only do they express neurotropic factors, but they also express antioxidant agents that could have an important role in preventing excitotoxic damage to retinal neurons (Garcia & Vecino, 2003). The initial stages of retinal degeneration in the PDE6A mutant dog involved thinning of outer retinal layers with relative preservation of the thickness of the inner retinal layers. It was not until the outer retina was very thinned that significant decreases in the inner retinal layers started to become apparent. Despite this apparent initial lack of effect on the inner retina the immunohistochemical studies using antibodies to various retinal neurons revealed that quite profound effects on some inner retinal cells occurred relatively early in the disease process. Perhaps not surprising, given their synaptic connections with rod photoreceptors, the PKCOI immunoreactive rod bipolar cells were affected early in the process Of retinal degeneration. The EM study showed that there was 217 a decrease in the number of synaptic ribbons in the rod photoreceptor termini in the outer plexiform layer of the PDE6A mutant retina suggesting the presence of abnormalities in the synaptic connections between rods and rod bipolar cells and horizontal cells. In the rdl mouse there is a failure of normal synaptogenesis between rods and rod bipolar cells (Strettoi & Pignatelli, 2000). There was an early loss Of the normal bi-layer arrangement of rod bipolar cell nuclei and a reduction in the branching of the bipolar cell axonal processes. However later in the disease process, the surviving PKCOI immunoreactive cells appeared to develop larger numbers of axonal processes. Horizontal cells also synapse with rod photoreceptor termini and yet there was no apparent alteration in calbindin immunoreactivity until later in the disease process indicating that the horizontal cells were better preserved than rod bipolar cells. Alterations in the rod bipolar cells were also Observed in the rd] mouse. Following degeneration of rods, the metabotropic glutamate receptors were displaced from rod bipolar cell dendrites toward their soma and axons (Strettoi & Pignatelli, 2000) and in vitro studies showed that the abnormal rod bipolar cells from the rdI mouse retina do not respond to glutamate (the neurotransmitter a the rod synaptic space) while they increase their sensitivity to GABA (the horizontal cell neurotransmitter) (Varela et al., 2003). Later in the disease process sprouting of dendrites from several types of surviving retinal neurons was apparent. The surviving rod cells had developed axons that extended beyond their normal site of termination in the outer plexiform layer, into the inner retina. Surviving cone photoreceptors reacted in a similar manner. Interestingly the initial 218 -; direction of axonal growth was from outer to inner retina but in the very degenerate retina axons tended to ramify in a horizontal direction. Overgrth (sprouting) of rhodopsin immunoreactive rod axonal processes seen in the PDE6A mutant dogs was similar to that described in other retinal dystrophies such as the rdy cat (Chong et al., 1999) and the rdI mouse (Zeiss & Johnson, 2004). Rod sprouting was noted in the retina of RP patients with significant photoreceptor loss, where rod neurite overgrowth was found to project into the inner retina to contact the somata of GABA-positive amacrine cells (Fariss et al., 2000) and also found to associate with the surfaces of the hypertrophic Miiller cell processes (Li et al., 1995). Zeiss and Johnson (2004) suggested that rod neurite overgrth may be influenced by changes in the neighboring reactive Miiller cells. There is also a possibility that the small number of rhodopsin staining neurons that can be detected in the very degenerate retinas of older PDE6A mutant dogs may be transformed Mi‘rller cells. Studies have shown that in response to neurotoxin-induced damage in rat retina Miiller glia can differentiate to express markers for rod photoreceptors and bipolar cells (Ooto et al., 2004). A similar response was described in the rdl mouse where Miiller cells were found to be able to differentiate into progenitor cells that then give rise to different neuronal cell types (Zeiss & Johnson, 2004). Horizontal cells stained by calbindin appeared relatively unchanged in the early stages of the degeneration, but in the later stages they appear hypertrophic. Hypertrophy of horizontal cells following photoreceptor degeneration has been described in rdl mice 219 (Strettoi & Pignatelli, 2000). It is suggested that it may relate to an increase of GABA sensitivity found in the remaining rod bipolar cells (Varela et al., 2003) or GABAergic amacrine cells (Lee et al., 2004). Changes in amacrine and ganglion cells were not marked in the disease process in the PDE6A mutant dog retina. There was possibly a reduction in ganglion cell number later in the disease process that could be detected on regular stained sections and calretinin stained sections. Counting of ganglion cells on retinal flat mounts would be preferable to quantify changes in ganglion cell numbers. 220 Chapter 5 Investigation of the tissue expression of the PDE6A gene 5.]. Introduction Photoreceptor proteins function together in modulating the phototransduction cascade in the retina. Not only are they functional in the retina, but some are expressed in other tissues such as pineal gland. The pineal gland in lower vertebrates is directly responsive to light stimulation. It expresses some genes that are involved in phototransduction (Zatz et al., 1988). For example, guanylate cyclase activating protein (GCAP) and guanylate cyclase (GC) genes are expressed in normal and rd chicken pineal (Semple-Rowland et al., 1999), Chicken pineal transducin alpha—subunit, which is identical to the one in retina, was found to mediate the phototransduction pathway in chicken pinealocytes (Kasahara et al., 2000). Other genes reported to be expressed in pineal gland include both rod and cone forms of the beta cGMP PDE (Morin et al., 2001), the cyclic nucleotide-gated channel (Bonigk et al., 1996) and rhodopsin kinase (Zhao et al., 1999). In addition to pineal gland, some photoreceptor genes are expressed in other tissues in higher vertebrates as well, for example PDE6B is expressed in human brain (Collins et al., 1992) and in pigeon brain (Wada et al., 2000), recoverin in pinealocytes of 221 rat, sheep, and human (Korf et al., 1992; Ruiz-Avila et al., 1995b), the transducin alpha- subunit in vertebrate taste receptor cells (Ruiz-Avila et al., 1995a) and scanning of EST databases showed PDE6A in other human tissues such as colon (“avagenomeucsc.edu). Sequencing online database EST Shows PDE6A expression in colon. In view of the fact that rod phototransduction genes are expressed in other tissues in other species, we investigated the tissue expression of the dog PDE6A gene. 5.2. Materials and Methods 5.2.1 . Tissue collection Prior to euthanasia, animals were examined for any evidence of ophthalmic disease. Euthanasia was performed using an overdose of pentobarbital sodium (Fatal- Plus; Vortech pharmaceuticals, Dearbom, MI) intravenously. Immediately following sacrifice, the tissues listed below were collected, snap frozen in liquid nitrogen, and stored at —80°C until processed. From PDE6A normal control dogs:— Ocular tissues: cornea, iris, lens, retina, RPE/choroid. Abdominal organs: spleen, liver, pancreas, kidney, small intestine, stomach wall, urinary bladder, uterus, and ovary. 222 Thoracic organs: heart, lung, and diaphragm. Central nervous system tissues: pituitary gland, pineal gland, cerebrum, cerebellum, spinal cord, Optic chiasm, and visual cortex. Miscellaneous tissues: striated muscle, buccal membrane, tongue epithelium, and blood. In view of the preliminary results in normal dogs, the following tissues were collected from PDE6A mutant dogs:- Ocular tissues: retina . Abdominal organs: kidney, small intestine Note: Blood was collected into a tube containing sodium citate and placed on ice only when separation of white blood cells from red blood cell was to be performed immediately. Lysis of red blood cells was performed by incubating with red blood cell lysis buffer, pH 8.0 (see Appendix) for 30 minutes on ice before centrifugation at maximum speed (14,000 rpm) for 15 minutes at 4°C to pellet white blood cell nuclei. Supernatant was discarded and the lysis incubation was repeated for 10 minutes, followed by 10 minutes of centrifugation. Supernatant was discarded and the pellet was stored at -80°C until processed. 5.2.2. Isolation of total RNA 5.2.2.a. Preparation of instruments and RNA handling 223 Mortar, pestle, metallic instruments and glassware were cleaned with a detergent, thoroughly rinsed, soaked with RNaseZapTM (Sigma-Aldrich CO., St Louis, MO) mixed with diethyl pyrocarbonate-treated water (DEPC-treated water; see Appendix), air-dried, wrapped with aluminum foil and then baked at 180°C overnight prior to use. Proper microbiological, aseptic techniques were always used. RNA was prepared using sterile RNase-free instruments and DEPC-treated water. Sterile barrier pipette tips were used to avoid aerosol contamination. 5.2.2.b. Isolation Of total RNA using RNeasy Mini Kit (Qiagen Inc., Valencia, CA) A tissue sample (20-30mg) was ground in liquid nitrogen (with a mortar and pestle) to a fine powder. RNA isolation with DNaseI digestion was performed in all tissues (see section 1. in Appendix) by using RNeasy Mini protocol for isolation of total RNA from animal tissues (3rd Edition RNeasy Mini handbook, 2001, Qiagen Inc., Valencia, CA). Proteinase K (20mg/ml) was added in the isolation process when total RNA was isolated from heart, striated muscle, urinary bladder, buccal membrane, lens, and white blood cells to improve the extraction rate. 224 5.2.2.c. Visualization of total RNA for integrity using 3 formaldehyde gel 5.2.2.c.(i). Preparation of materials Details of stock solutions are given in Appendix. 5.2.2.c.(ii). Formaldehyde (FA) gel electrophoresis Details of formaldehyde gel preparation are given in Appendix. RNA samples were mixed 4:1 with 5X RNA loading buffer, incubated for 3-5 minutes at 65°C, chilled on ice, and loaded onto the equilibrated FA gel. An aliquot of RNA size marker (0.2-10 kb, Sigma-Aldrich Co., St Louis, MO) was incubated at 65°C for 5 minutes then chilled on ice. FA gel was run at 7volt/cm in 1x FA gel running buffer for 1.5 hours. 5.2.2.c.(iii). Analysis of total RNA by FA gel electrophoresis Following electrophoresis, the FA gel was soaked in ethidium bromide solution (Sug/ml) for no longer than 15 minutes, and then washed in DEPC-treated water to de- stain ethidium bromide for up to 2 hours. The FA gel was viewed by UV light and the integrity of RNA was assessed by examination of the two bands corresponding to 288 and 188 ribosomal RNAs (sharp bands of ~1.5kb and ~700bp size, respectively), looking. 225 for evidence of RNA degradation (a smear of smaller sized RNA) and genomic DNA contamination (a band larger than 1.5kb). The upper band of 288 ribosomal RNA was expected to be twice as bright as the lower band of 1 SS ribosomal RNA. 5.2.3. Spectrophotometry of RNA Two aliquots of each total RNA mixed with DEPC-treated water at a concentration of 1:100 were prepared on ice, and then transferred to a 96-well microplate. Spectrophotometry was performed using a microplate scanning spectrophotometer (PowerWavexTM; Bio-Tek Instruments Inc, Winooski, VT). Mean of the optical density (OD) of the RNA solution compared with balanced well containing DEPC-treated water as a blank at wavelengths 260 and 280nm was calculated. The ratio OD26o/OD230 gives an estimate of the RNA purity. Pure RNA solution should have a ratio of 1.9 to 2.1. Contamination with protein results in lower ratios. The concentration of RNA was calculated by multiplying the absorbance at 260 nm (A260) by 40 to give the RNA concentration in gym]. 5.2.4. Reverse Transcription-Polymerase Chain Reaction (RT-PCR) 5.2.4.a. Synthesis of cDNA Full-length first strand cDNA was made from total RNA using a first strand cDNA synthesis kit (Fermentas Inc., Hanover, MD) following the manufacturer’s 226 instructions. Approximately 2ug of total RNA was used as a template and mixed with 0.5ug oligo(dT)13 primer, 1X reaction buffer, 20U RiboLockTM Ribonuclease inhibitor, 0.4 mM dNTP mix, and 40U M-MuLV reverse transcriptase. The mixture was incubated at 37°C for 60 minutes, followed by 70°C for 10 minutes to denature the enzyme. 5.2.4.b. Polymerase Chain Reaction (PCR) for PDE6A gene expression CDNAs of all tissue types were used as templates for PCR with gene-specific primers for PDE6A (listed below). PDE6A-specific primers were designed (Integrated DNA Technologies Inc., Coralville, LA) to amplify portions of the PDE6A gene spanning a known intron to distinguish amplicons that were from cDNA rather than genomic DNA. Figure 5.2 shows the primer pairs used on a map of the PDE6A cDNA. All primers were designed and ordered from Integrated DNA Technologies Inc (Coralville, IA). Forward primers: SPJ220 = GGG GCG GCC GCC AGG CCA GCT TTA GGC TCT 7048 = AAT GGC ATC GCA GAG CTA GC 7050 = CCT CAC TGA GTA CCA GAC CA 7057 = ATG TGT GGC CAG TCC TGA TC 7064 = TGG ATG AGT CTG GAT GGA TGA 7070 = AGA GCT GCC AGA AGC AGA GA 227 PDEAl = AGA CAC CAC TTG GAG TTC GG 6836 = TCC CAT GCT GGA TGG GAT CA Reverse primers: 7049 = TGT CCA CAA GGA TGC TGT CC 7056 = TAT TCT GGT CCC AGG ACT CC 7065 = GAT GAA ATG GAT GAG ACC CT 7071 = CCG AAC TGG AGC TGG TGA AA PDEA2 = AAC CTC AAT CGC AGG CAG CA 6835 = GCT CGC CGA TGA GTA CGA CA SPJ442 = GGG GCG GCC GCG GTG GTA CCA TTC GGT GCA G The 50p] reaction PCR mixture consisted of: IX PCR buffer, 1X bovine serum albumin (BSA), 0.2mM dNTPs, 1.5mM MgClz, 0.4uM of each forward and reverse primer, and 1.25U of T aq DNA polymerase (Invitrogen Corporation, Carlsbad, CA). The standard PCR protocol consisted of an initial denaturing step at 94°C for 3 minutes, followed by 29 rounds of denaturing at 94°C for 30 seconds, annealing at 52°C for 30 seconds and extension at 72°C for 1 minute, and then a final extension at 72°C for 3 minutes. A PCR reaction containing retinal cDNA was included as a positive control, because PDE6A is known to be expressed in the retina, whereas a PCR reaction without cDNA template served as a negative control. A PCR reaction with primers for B-actin gene (a forward primer: 5’- ATG GAT GAC GAT ATC GCT GCG CTT- 3’ and a reverse primer: 5’- AGA GGC ATA CAG GGA CAG CAC A -3’) was also performed as 228 a positive control. The PCR reaction mixture and protocol using with B-actin primers were the same as previously described except the annealing temperature was 55°C. A negative control for the entire RT-PCR was included, in which reverse transcriptase was not added. The PCR products were visualized by agarose gel electrophoresis (see Appendix). 5.2.5. Sequencing of PCR products 5.2.5.a. DNA preparation. When a single strong band was visualized on the agarose check gel, the PCR product was purified as followed: to the product 1/10 volume of 3M sodium acetate and 7/ 10 volume of 100% isopropanol were added, mixed well and the mixture incubated at room temperature. The DNA pellet was precipitated by centrifugation at 20,800 g for 10 minutes and the supernatant was removed. The pellet was air-dried for 10 minutes and re-suspended in molecular-grade water. When two or more products were present, they were separated by appropriate agarose gel electrophoresis and the separated bands were cut out of the gel. A QIAquick Gel Extraction kit, (Qiagen Inc., Valencia, CA) was used to extract DNA from the gel slice (see Appendix for detail of gel extraction). 5.2.5.b. DNA sequencing. The purified PCR product was submitted to the Genomics Technology Support Facility, MSU for sequencing using the gene-specific primers. Sequences. were determined on an ABI PRISM® 3100 genetic analyzer (Applied Biosystems, Foster city, CA). 229 5.2.6. Rapid Amplification of cDNA Ends (RACE) to determine full-length sequence of kidney transcript This is a method to obtain full-length 5’ and 3’ ends of cDNA sequence. The GeneRacerTM Kit (Invitrogen Corporation, Carlsbad, CA) was used. Kidney total RNA was used as a template. At least one gene-specific primer (GSP) was designed for 5’ and for 3’ RACE. Hela total RNA provided in the kit was used as a positive control for all reactions. This protocol is designed to only make cDNA from full-length processed mRNA, not other RNAs or partially spliced mRNAs. 5.2.6.a. Dephosphorylation of RNA This step was to treat total RNA with Calf Intestinal Phosphatase (CIP) to dephosphorylate the 5’ end of non-mRNA or truncated mRNA, which prevents ligation with the oligo dT primer. Mature full-length mRNA is not dephosphorylated due to the presence of the 5’ cap. 1.2ug of total RNA (7ul), 1X CIP buffer, 40U RNaseOutTM, and IOU CTP were added for a final volume of IOul reaction. The mixture was incubated at 50°C for 1 hour, and the RNA was purified by a phenol/chloroform extraction (see Appendix). 5.2.6.b. Removal of the mRNA cap structure 230 This step removes the 5’ cap structure from full-length mRNA using Tobacco Acid Pyrophosphatase (TAP). For a final volume of lOul, 7p] of dephosphorylated RNA (see 5.2.6.3), 1x TAP buffer, 400 RNaseOutTM, and 0.5U TAP were added and mixed. The mixture was incubated at 37°C for 1 hour, and then the RNA was purified by a phenol/chloroform extraction (see Appendix). 5.2.6.e. Ligation of the RNA oligo to de-capped mRNA This step ligates an oligo dT primer to the 5’ end of the mRNA using RNA ligase. Dephosphorylated, de-capped RNA (see 5.2.6.b) was added to a microcentrifuge tube containing lyophilized GeneRacerTM RNA oligo primer (0.25ug). The mixed reaction was incubated at 65°C for 5 minutes to relax the RNA secondary structure. 1X ligase buffer, 1.0 mM ATP, 40U RNaseOutTM, and 5U T4 RNA ligase were added for a final volume of 10111 and mixed. The reaction mixture was incubated at 37°C for 1 hour, and the RNA was purified by a phenol/chloroform extraction (see Appendix). 5.2.6.d. Reverse transcription of mRNA Full-length mRNA with a 5’ poly T—linker was reverse-transcribed into cDNA using SuperScriptTM III RT Reaction (Invitrogen Corporation, Carlsbad, CA). To the 10p] of ligated RNA (see 5.2.6.c), 0.15ug of GeneRacerTM RNA oligo primer, lul of dNTP mix, and 1 pl of sterile, distilled water were added, the mixture was then incubated at 65°C for 5 minutes, chilled on ice for 1 minute. 1X First Strand buffer, 5mM DTT, 23] 40U RNaseOutTM, and 200U SuperScriptTM 111 RT (reverse transcriptase) were added to the mixture for a final volume of 20ul. The mixture was incubated at 50°C for 1 hour, followed by 70°C for 15 minutes and then chilled on ice for 2 minutes. 2U RNaseH was then added to the mixture, which was incubated at 37°C for 20 minutes. 5.2.6.e. Amplification of cDNA ends PCR was used to amplify the cDNA ends. For 5’ end amplification, cDNA made from 5.2.6.d. was used for the first PCR reaction. The PCR mixtures contained: 1X high fidelity PCR buffer, 0.2mM dNTP solution, 2mM MgSO4, 0.12uM of 5’RACE forward primer, 2.5uM of a PDE6A-specific primer #7071 (CCG AAC TGG AGC TGG TGA AA), 2.5U Platinum® Taq DNA polymerase (Invitrogen Corporation, Carlsbad, CA) and sterile water were added to make a 50p.l final volume reaction. The PCR protocol consisted of an initial denaturing step at 94°C for 2 minutes, followed by 25 cycles of denaturing at 94°C for 30 seconds, annealing at 55°C for 30 seconds and extension at 68°C for 2 minutes, and then a final extension at 68°C for 10 minutes. Nested PCR was performed afterward using 2 pl of the previous RT-PCR product as a template. The PCR reaction mixture contained the same ingredients as previously used except for primers; 0.12uM of the 5’ nested forward primer and 2.5uM of a PDE6A-specific reverse primer #7065 (CCG AAC TGG AGC TGG TGA AA). The PCR protocol was the same as previously described except for the 232 annealing temperature, which was 65°C. The PCR products were visualized by agarose gel electrophoresis (see Appendix). For 3’ end amplification, a modified touchdown PCR was used, PCR reaction mixtures contained: 1X high fidelity PCR buffer, 0.2mM dNTP solution, 2mM MgSO4, 2.5uM of a PDE6A-specific forward primer #6836 (TCC CAT GCT GGA TGG GAT CA), 0.12uM of the 3’RACE reverse primer (GCT GTC AAC GAT ACG CTA CGT AAC G), 2.5U Platinum® T aq DNA polymerase (Invitrogen Corporation, Carlsbad, CA), and sterile water were added to make a 501.11 final volume reaction. Modified touchdown PCR conditions consisted of an initial denaturing step at 94°C for 2 minutes, 5 cycles of denaturing at 94°C for 30 seconds, annealing and extension at 72°C for 2 minutes, 5 cycles of denaturing at 94°C for 30 seconds, annealing and extension at 70°C for 2 minutes, 20 cycles of denaturing at 94°C for 30 seconds, annealing at 65°C for 2 minutes and extension at 68°C for 2 minute, and then a final extension at 68°C for 10 minutes. The PCR products were visualized by agarose gel electrophoresis (see Appendix). PCR products for cloning were extracted from the gel using QIAquick Gel Extraction Kit Protocol (Qiagen Inc., Valencia, CA) as previously described. 233 5.2.7. Cloning of PCR products PCR products were cloned using the TOPO TA Cloning® Kit (Invitrogen Corporation, Carlsbad, CA). This kit allows for a direct insertion of T aq polymerase- amplified PCR products into a plasmid vector for manipulations such as sequencing. 5.2.7.a. Cloning reaction 4p.l of purified DNA fragment, lul of salt solution (l.2M NaCl, 0.06M MgClz), and 1 ul of TOPO® vector (10ng plasmid DNA) were added and mixed. The mixture was incubated at room temperature for 5 minutes and then placed on ice. 5.2.7.b. Transformation of competent cells This step was to transform competent E. coli (One Shot® TOPIO competent E. coli) provided in the kit with DNA-vector construct. 2);] of the cloning reaction mixture (see 5.2.7.a.) was added to a vial of competent E. coli, the mixture incubated on ice for 15 minutes then heated for 30 seconds at 42°C. S.O.C. medium (2% Tryptone, 0.5% yeast extract, 10mM NaCl, 2.5mM KCl, 10mM MgClz, 10mM MgSO4, and 20mM glucose) was added to the mixture. The tube was shaken (200 rpm) at 37°C for 1 hour. 50p] of the mixture was spread on each of two pre-warmed LB plates containing SOug/ml ampicillin and the plates incubated overnight at 37°C. 234 5.2.7.c. Analysis of transformed clones Ten individual colonies from each plate were selected and cultured overnight in LB broth containing 50ug/ml ampicillin. Plasmid DNA was isolated using the QIAprep® Miniprep kit (Qiagen Inc., Valencia, CA) (see Appendix for detail of plasmid DNA isolation). The plasmid was analyzed for presence and size of insert by EcoRI digestion followed by agarose gel electrophoresis. To Sul of plasmid, 1X React-3 buffer and 10U EcoRl (Invitrogen Corporation, Carlsbad, CA) were mixed and water added to make final volume of 20p]. The mixture was mixed and incubated at 37°C for 1 hour. Gel electrophoresis was used to show that the plasmid had been digested and to estimate the size of the cloned fragment. 5.2.8. Northern hybridization Northern hybridization was used to detect the presence and measure the size of PDE6A mRNA transcripts in a range of tissues. 5.2.8.a. Preparation of FA gel for electrophoresis A 1.5% FA gel 20.5*20.5*0.8cm was prepared (see Appendix for preparation solutions) and equilibrated at 120volt/cm in 1X formaldehyde gel running buffer for 1 hour 235 5.2.8.b. FA gel electrophoresis 5.2.8.b.(i). Preparation of RNA samples and RNA size marker 30ug of total RNA isolated from pituitary gland, pineal gland, lens, small intestine, PDE6A mutant kidney, normal control kidney and retina were used for northern hybridization. The RNA samples were mixed with 5X RNA loading buffer (see Appendix for preparation solution) at a ratio of 4:1, incubated for 3-5 minutes at 65°C, quickly chilled on ice, and then loaded onto the equilibrated FA gel. 3p] of RNA size marker (0.2-10 kb, Sigma-Aldrich Co., St Louis, MO) was added to 3p] of 5X RNA loading buffer, lul of 200mM sodium acetate and 2pl of DEPC-treated water, incubated at 65°C for 10 minutes, immediately cooled on ice and loaded onto the equilibrated FA gel. 5.2.8.b.(ii). FA gel electr0phoresis Electrophoresis was carried out at 3volt/cm in 1X formaldehyde gel running buffer overnight, until the bromophenol blue had migrated approximately 8 cm from the loading wells. RNA was visualized with the UV transilluminator. A transparent ruler was aligned to the gel to indicate the distance from the loading well to each of the band of RNA and a photograph was taken. The 28S and 18S species of rRNA were clearly visible under UV illumination. 236 5.2.8.c. Preparation for an upward capillary transfer 5.2.8.c.(i). Preparation of FA gel The unused areas of the gel were trimmed and a small triangular piece was cut off the bottom left-hand comer of the gel to aid in orientation. To partially hydrolyze RNA samples, the gel was rinsed with DEPC-treated water, soaked in 5 gel volumes of 0.05M NaOH for 20 minutes and then in 20X SSC (see Appendix) for 40 minutes. 5.2.8.c.(ii). Preparation of nylon membrane, wicked papers and blotting papers Positively charged nylon membrane (Boehringer Mannheim Gmbh, Mannheim, Germany) was used. The comer of the membrane was cut to match the comer cut from the gel. The membrane was cut into a size of 10*15 cm, floated on DEPC-treated water until it was completely wet from beneath and then immersed in 10X SSC for 5 minutes. Filter paper (Whatrnan 3MM paper, MidWest Scientific Company, Valley Park, MO) was used as wicked and blotting papers. To make wicked papers, two pieces of filter paper at size of 27* 14 cm and 24*17 cm were cut and prewet in 20X SSC. Four pieces of filter paper of the same size as that of nylon membrane were cut and soaked in 20X SSC to use as blotting papers. 5.2.8.c.(iii). Assembly of the capillary transfer system 237 The gel support (small baking dish with Plexiglas on top) was placed in large glass baking dish filled with transfer buffer (20X SSC). Two prewet wicked papers were placed on Plexiglas in the opposite direction, so that the edges of the papers were immersed in the transfer buffer (Figure 5.1). The gel was carefully placed on wicked papers in an inverted position. Parafilm was placed on the wicked papers to surround the edges of the gel. The wet nylon membrane was transferred with care and placed on top of the gel, the cut comers of the gel and nylon membrane aligned, air bubbles removed to maximize contact between the gel and nylon membrane. Four pieces of prewet blotting paper were placed on the top of nylon membrane. Blotting papers were smoothed with a glass rod. A stack of paper towels (same size as that of the nylon membrane) was placed on blotting papers, and a sheet of Plexiglas on top of it. Two lOO-gram weights were put on top of the paper towels. 238 m.. —- 100-g weight Plexigas —— Paper towels Blotting papers l 1 ll Wicked papers\ I 2 Transfer buffer support Figure 5.1. Upward capillary transfer apparatus. RNA was transferred from FA gel to a nylon membrane by capillary reaction drawing transfer buffer from the reservoir upward through the gel into a stack of dry paper towels. Weights applied on top of a stack of paper towels helped to ensure a tight connection between layers of materials. The apparatus was leveled using a spirit level. The capillary transfer was allowed to occur over night, and then the apparatus was dismantled. The positions of the loading wells were marked on the nylon membrane with a pencil; the nylon membrane was then transferred to 6X SSC at room temperature, slowly agitated for 5 minutes. The nylon membrane, RNA side upward, was air-dried on a dry sheet of filter paper for a few minutes. 5.2.8.c.(iv). Fixation of the transferred RNA to the membrane The dry nylon membrane was placed between 2 pieces of filter papers and baked in a vacuum oven at 80°C for 2 hours. 239 5.2.8.d. Northern hybridization 5.2.8.d.(i). Preparation ofthe DNA probe A plasmid containing full—length canine PDE6A cDNA was available from another project. Restriction enzyme digestion was performed with N011 enzyme to cut the full-length canine PDE6A cDNA from the plasmid. Agarose gel electrophoresis was performed to confirm the cut PDE6A cDNA. The PDE6A cDNA was purified with QIAquick Gel Extraction kit (Qiagen Inc., Valencia, CA). PCR (see 5.2.4.b for PCR protocol) was performed using the PDE6A-specific primers to confirm the PDE6A fragment generated from the cut PDE6A cDNA, which was afterward visualized by gel electrophoresis. Gel electrophoresis of an aliquot of the PDE6A cDNA was performed and DNA size markers (100bp DNA ladder; New England BioLabs Inc, Beverly, MA) of known concentration included. The concentration of DNA in the PDE6A cDNA solution was estimated by comparison with the intensity and size ladder bands. 6ul of distilled water was added to 3p] of PDE6A cDNA (~25ng), and heated in boiling water for 10 minutes, immediately cooled on ice, and then briefly centrifuged. The probe was radioactively labeled using the Random Primed DNA labeling Kit (Roche Diagnostics GmbH, Penzberg, Germany) as follows: 9p] of PDE6A cDNA solution, 0.025mM of each of dCTP, dGTP, and dTTP, 1X hexanucleotide mixture, 12.5 uCi [or- 240 32P]dATP, and 2U/ Klenow enzyme. The mixture was mixed, incubated at 36°C for 1 hour and then chilled on ice. Chromatography (Chromaspin + TE-30, BD Sciences ClonTech, Mountain View, CA) was used to remove unincorporated nucleotides. Integrity of the DNA-[a-32P]- labeled probe was verified using a hand-held monitor (Sambrook et al., 1989). 5.2.8.d.(ii). Prehybridization The nylon membrane was carefully transferred to a hybridization bottle, incubated in a hybridized oven (4rpm) for 2 hours at 68°C in 100 ml of prehybridization solution (see Appendix). 5.2.8.d.(iii). Hybridization The 32P-labeled double-stranded DNA was denatured by heating for 10 minutes at 100°C, then rapidly chilled on ice water. Prehybridization solution was partially removed from the bottle, and 7ml was reserved. To make the hybridization solution, the denatured 32P-labeled probe was added directly to the 7 ml prehybridization solution and mixed gently. The nylon membrane was incubated in hybridization solution (4rpm) for 16 hours at 50°C. 241 5.2.8.d.(iv). Washing ofthe membrane After hybridization, hybridization solution was removed from the bottle, the membrane was washed gently 2 times; twice with 100 ml of Wash A solution (see Appendix) at 50°C, followed by 100 ml of Wash B solution (see Appendix) at 50°C for 25 minutes for each wash. The membrane was then washed in 100 ml of 0.1X SSC, 0.5% SDS solution at 65°C for 30 minutes and then in 70ml of 0.1x SSC, 0.1% SDS solution at room temperature 5 times, each time for 5 minutes. 5.2.8.c. Exposure of the nylon membrane to phosphor-imager The membrane was slightly air-dried on a filter paper and then placed in a phosphor- imager cassette for 72 hours at room temperature. An image of the membrane was obtained by seaming the phosphor-imager (Storm 860; Molecular Dynamics, Sunnyvale, CA). 5.3. Results 5.3.1. Expression of PDE6A mRNA in canine tissues 5.3.1 .a. Investigation of PDE6A expression by RT-PCR 242 Tissue expression of PDE6A was investigated using RT-PCR with primers described as in 5.2.4.b and the primer pairs shown graphically in Figure 5.2. Positive controls for the RT-PCR using beta actin primers were used to confirm the presence of mRNAs in all samples (Figure 5.3). 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DNase treatment Add 10 pl Dnasel stock solution to 70 Ill buffer RDD. Mix by gently inverting the tube (do not vortex). Pipet the Dnase I incubation mix (80 pl) directly onto the RNeasy column (a membrane), and place on the bench top (20-30°C) for 15 minutes. 281 Washing step 0 Pipet 350 I11 buffer RWl into the RNeasy column, and centrifuge for 15 seconds at >8000 * g. Discard the flow-through. 0 Transfer RNeasy column to a new 2-ml collection tube (supplied). Pipet 500 Ill RPE buffer onto RNeasy column, and centrifuge for 15 seconds at >10,000 rpm to wash. Discard flow-through. o Pipet 500 Ill RPE buffer onto RNeasy column and centrifuge for 2 minutes at maximum speed to dry the RNeasy column. Eluting step 0 Transfer the RNeasy column into a new 1.5-ml collection tube (supplied) and pipet 30-50 III of RNase-free water directly onto RNeasy membrane. Centrifuge for 1 minute at >l0,000 rpm to elute. o If the expected RNA yield is above 30 ug, repeat the step 12 by adding a second volume (30-50ul) of RNase-free water to the same tube, and elute. RNA precipitation (Invitrogen Corpopration, Carlsbad, CA) 100p] of phenolzchloroform (1:1) and 90ul of DEPC-treated water were added to the mixture, the mixture was centrifuged and supernatant was collected. Zul of 10mg/ml mussel glycogen, 10p] of 3M sodium acetate and 220ul of 95% ethanol were added, and 282 then RNA was placed on ice for 10 minutes. The RNA pellet was collected by centrifugation for 20 minutes at 4°C. The supernatant was discarded and the pellet washed in 500p] of 70% ethanol and collected by centrifugation for 2 minutes at 4°C 14,000rpm. The pellet was then air-dried for 2 minutes at room temperature, and then re- suspended in 7p] DEPC-treated water. QIAquick Gel Extraction kit. (Qiagen Incg Vglencia. CA) o Excise the DNA fragment from the agarose gel with a clean, sharp scalpel o Weigh the gel slice in a colorless tube. Add 3 volumes of Buffer QG to 1 volume of gel 0 Incubate at 50°C for 10 minutes (or until the gel slice has completely dissolved). 0 After the gel slice has dissolved completely, check that the color of the mixture is yellow. 0 Add 1 volume of isopropanol to the sample and mix 0 Place a spin column in a 2-ml collection tube 0 To bind DNA, apply the sample to the column and centrifuge at maximum speed for 1 minute 0 Discard the flow-through and place the spin column back in the same collection tube 0 Add 0.5m] of Buffer QG to the spin column and centrifuge for 1 minute 283 0 To wash, add 0.75m1 of Buffer PE to the spin column, let the column stand for 2-5 minutes and then centrifuge for 1 minute 0 Discard the flow-through and centrifuge the spin column for an additional 1 minute at 13,000rpm 0 Place a spin column into a clean 1.5m1 microcentrifuge tube 0 To elute DNA, add 50p] of Buffer EB (10mM Tris-Cl, pH 8.5) or molecular water to the center of the spin column membrane and centrifuge the column for 1 minute. QIAprep Mirflrgpicit (Qiagen Inc.,Valencia, CA) 0 Resuspend pelleted bacterial cells in 250p] Buffer P1 and transfer to a microcentrifuge tube 0 Add 2501.11 Buffer P2 and gently invert the tube 4 to 6 times to mix 0 Add 350p] Buffer N3 and invert the tube immediately but gently 4 to 6 times 0 Centrifuge for 10 minutes at maximum speed in a tabletop microcentrifuge 0 Apply the supematants from the previous step to the QIAprep column by decanting pipetting o Centrifuge for 30 to 60 seconds. Discard the flowthrough 0 Wash the column by adding 0.75 ml Buffer PE and centrifuge for 30 to 60 seconds 284 Discard the flowthrough, and centrifuge for an additional 1 minute to remove residual wash buffer Place the column in a clean 1.5m] microcentrifuge tube. To elute DNA, add 50p] Buffer EB to the column, let stand for 1 minute and then centrifuge for 1 minute. 285 BIBLIOGRAPHY Acland,G.M. (1988). Diagnosis and differentiation of retinal diseases in small animals by electroretinography. Semin Vet Med Surg (Small Anim), 3. 15-27. Acland,G.M. & Aguirre,G.D. (1987). Retinal degenerations in the dog: IV. Early retinal degeneration (erd) in Norwegian elkhounds. Exp Eye Res, 44, 491-521. Acland,G.M., Blanton,S.H., Hershfield,B. & Aguiree,G.D. (1994). XLPRA: a canine retinal degeneration inherited as an X-linked trait. Am.J.Med.Genet.-52, 27-33. Acland,G.M., Ray,K., Mellersh,C.S., Gu,W.K., Langston,A.A., Rine,J., Ostrander,E.A. & Aguirre,G.D. (1998). Linkage analysis and comparative mapping of canine progressive rod-cone degeneration (prcd) establishes potential locus homology with retinitis pigrnentosa (RP17) in humans. Proc Natl Acad Sci U S A. 95. 3048-3053. Aguirre,G., Farber,D., Lolley,R., Fletcher,R.T. & Chader,G.J. (1978). Rod-Cone Dysplasia in Irish Setters - Defect in Cyclic-Gmp Metabolism in Visual Cells. Science 201, 1133-1134. Aguirre,G.D. & Acland,G.M. (1988). Variation in Retinal Degeneration Phenotype Inherited at the Prcd Locus. Exp Eve Res, 46. 663-687. Aguirre,G.D. & Rubin,L.F. (1975). Rod-Cone Dysplasia (Progressive Retinal Atrophy) in Irish Setters. J Am Vet Med Assoc466, 157-164. Aguirre,G.D. & Rubin,L.F. (1971). Progressive retinal atrophy (rod dysplasia in the Norwegian Elkhound. J Am Vet Med Assoc. 158, 208-218. Aguirre,G.D., Rubin,L.F. & Bistner,S.I. (1972). Development of the canine eye. Am.J.Vet.Res.. 33. 2399-2414. Ali,R.R., Sana,G.M., Stephens,C., Alwis,M.D., Bainbridge,J.W., Munro,P.M., Fauser,S., Reichel,M.B., Kinnon,C., Hunt,D.M., Bhattacharya,S.S. & Thrasher,A.J. (2000). Restoration of photoreceptor ultrastructure and function in retinal degeneration slow mice by gene therapy. N_at.Genet., 25, 306-310. Allikmets,R. (2004). Leber congenital amaurosis: a genetic paradigm. Qphthalmic Genet, 25. 67-79. Alloway,P.G. & Dolph,P.J. (1999). A role for the light-dependent phosphorylation of visual arrestin. Proc Natl Acad Sci U S A. 96. 6072-6077. Aming,S., ApfelstedtSylla,E., Seeliger,M., Gendo,K., Wissinger,B. & Zrenner,E. (1996). Rhodopsin mutations in patients with autosomal dominant retinitis pi gmentosa. Vision Res, 36. 439. 286 Baehr,W., Champagne,M.S., Lee,A.K. & Pittler,S.J. (1991). Complete cDNA sequences of mouse rod photoreceptor cGMP phosphodiesterase alpha— and beta-subunits, and identification of beta'-, a putative beta-subunit isozyme produced by alternative splicing of the beta-subunit gene. FEBS Lett.. 278. 107-114. Baldridge,W.H. & Ball,A.K. (1993). A New-Type of Interplexiform Cell in the Goldfish Retina Is ant-Immunoreactive. Neuroreport. 4. 1015-1018. Baldridge,W.H., Kurennyi,D.E. & Bames,S. (1998). Calcium-sensitive calcium influx in photoreceptor inner segments. J Neurophysiol. 79. 3012-3018. Barbehenn,E., Gagnon,C., Noelker,D., Aguirre,G. & Chader,G. (1988). Inherited Rod- Cone Dysplasia - Abnormal Distribution of Cyclic-Gmp in Visual Cells of Affected Irish Setters. Exp Eve Res. 46. 149-159. Barnett,K.C. & Curtis,R. (1978). Lens luxation and progressive retinal atrophy in the Tibetan terrier. Vet Rec. 103. 160. Beech,P.L., Pagh-Roehl,K., Noda,Y., Hirokawa,N., Bumside,B. & Rosenbaum,J.L. (1996). Localization of kinesin superfamily proteins to the connecting cilium of fish photoreceptors. J Cell Sci. 109 ( Pt 4). 889-897. Bemtson,A., Smith,R.G. & Taylor,W.R. (2004). Transmission of single photon signals through a binary synapse in the mammalian retina. Vis Neurosci. 21, 693-702. Besharse,J.C. & Dunis,D.A. (1983). Methoxyindoles and Photoreceptor Metabolism - Activation of Rod Shedding. Science. 219. 1341-1343. Besharse,J.C., Hollyfield,J.G. & Raybom,M.E. (1977). Photoreceptor Outer Segments - Accelerated Membrane Renewal in Rods After Exposure to Light. Science. 196. 536- 23.8.. Biehlmaier,O., Neuhauss,S.C.F. & Kohler,K. (2001). Onset and time course of apoptosis in the developing zebrafish retina. Cell Tissue Res. 306. 199-207. Birch,D.G. & Anderson,J.L. (1992). Standardized full-field electroretinography. Normal values and their variation with age. Arch.0phthgmol.. 110. 1571-1576. Birch,D.G. & F ish,G.E. (1987). Rod Ergs in Retinitis-Pigrnentosa and Cone Rod Degeneration. Invest Ophthalmol Vis Sci. 2L8. 140-150. Bjerkas,E. (1990). Generalised progressive retinal atrophy in the English setter in Norway. Vet Rec. 126. 217. Bjerkas,E. & Narfstrom,K. (1994). Progressive retinal atrophy in the Tibetan spaniel in Norway and Sweden. Vet Rec. 134. 377-379. 287 Bloomfield,S.A. (1996). Effect of spike blockade on the receptive-field size of amacrine and ganglion cells in the rabbit retina. J Neurophysiol, 75. 1878-1893. Bonigk,W., Muller,F., Middendorff,R., Weyand,I. & Kaupp,U.B. (1996). Two alternatively spliced forms of the cGMP-gated channel alpha-subunit from cone photoreceptor are expressed in the chick pineal organ. J Neurosci. 16. 745 8-7468. Boughman,J.A., Conneally,P.M. & Nance,W.E. (1980). Population Genetic-Studies of Retinitis Pigmentosa. Acta Ophthalmol Scand. 32. 223-235. Bowes,C., Li,T., Danciger,M., Baxter,L.C., Applebury,M.L. & Farber,D.B. (1990). Retinal degeneration in the rd mouse is caused by a defect in the beta subunit of rod cGMP-phosphodiesterase. Nature. 347. 677-680. Bowmaker,J.K. & Dartnall,H.J. (1980). Visual pigments of rods and cones in a human retina. J Physio]. 298. 501-511. Boycott,B.B. & Hopkins,J.M. (1991). Cone Bipolar Cells and Cone Synapses in the Primate Retina. Vis Neurosci-7.49-60. Brown,K.T. (1979). Citation Classic - Electroretinogram - Its Components and Their Origins. Current Contents/Life Sciences.10. Bui,B.V. & Fortune,B. (2004b). Ganglion cell contributions to the rat full-field electroretinogram. J Physio]. 555. 153-173. Bui,B.V. & Fortune,B. (2004a). Ganglion cell contributions to the rat full-field electroretinogram. J Physiol. 555.153-173. Buntmilam,A.H., Saari,J.C., Klock,I.B. & Garwin,G.G. (1985). Zonulae-Aderentes Pore- Size in the Extema] Limiting Membrane of the Rabbit Retina. Invest Ophthalmol Vis Sci. 26. 1377-1380. Burstedt,M.S., Forsman-Semb,K., J anunger,T., Wachtmeister,L. & Sandgren,O. (2000). Bothnia dystrophy, a variant of autosomal recessive retinitis pi gmentosa with punctata albescens and macular degeneration associates with an R234W point mutation in the RLBP] gene. Invest Ophthalmol.Vis.Sci.. 41 @616. Bush,R.A. & Sieving,P.A. (1994). A Proximal Retinal Component in the Primate Photopic Erg A-Wave. Invest Ophthalmol Vis Sci. 35. 635-645. Bush,R.A. & Sieving,P.A. (1996). Inner retinal contributions to the primate photopic fast flicker electroretinogram. J Opt Soc Am A Cut Image Sci Vis. 13. 557-565. Buyukmihci,N., Aguirre,G. & Marshall,J. (1980). Retina] Degenerations in the Dog .2. Development of the Retina in Rod-Cone Dysplasia. Exp Eye Res. 30. 575-591. 288 Cellerino,A., Bahr,M. & Isenmann,S. (2000). Apoptosis in the developing visual system. Cell Tissue Res. 301. 53-69. Chen,L., Yang,P.Z. & Kijlstra,A. (2002). Distribution, markers, and functions of retinal microglia. Ocul Immuno] Inflamm. 10. 27-39. Chong,N.H., Alexander,R.A., Bamett,K.C., Bird,A.C. & Luthert,P.J. (1999). An immunohistochemical study of an autosomal dominant feline rod/cone dysplasia (Rdy cats). Exp Eye Res. 68. 51-57. Chun,M.H. & Wassle,H. (1993). Some Horizontal Cells of the Bovine Retina Receive Input Synapses in the Inner Plexiform Layer. Cell Tissue Res. 272. 447-457. Citron,M.C., Erinoff,L., Rickman,D.W. & Brecha,N.C. (1985). Modification of electroretinograrns in dopamine-depleted retinas. Brain Res.. 345. 186-19]. Clarke,G., Col]ins,R.A., Leavitt,B.R., Andrews,D.F., Hayden,M.R., Lumsden,C.J. & McInnes,R.R. (2000). A one-hit model of cell death in inherited neuronal degenerations. Nature. 406. 195-199. Clarke,G. & Lumsden,C.J. (2005). Heterogeneous cellular environments modulate one- hit neuronal death kinetics. Brain Res Bull. 65. 59-67. Co]lins,C., Hutchinson,G., Kowbel,D., Riess,O., Weber,B. & Hayden,M.R. (1992). The human beta-subunit of rod photoreceptor cGMP phosphodiesterase: complete retinal cDNA sequence and evidence for expression in brain. Genomics. 13. 698-704. Colotto,A., Falsini,B., Salgarello,T., Iarossi,G., Ga]an,M.E. & Scullica,L. (2000). Photopic negative response of the human ERG: losses associated with glaucomatous damage. Invest Opjithalmol Vis Sci-41.2205-2211. Cruz,R.D. & chi-Usami,E. (1989). Quantitative evaluation of electroretinogram before cataract surgery. Jpn J Ophthalmol-33.451-457. Curcio,C.A. (1986). Aging and Topography of Human Photoreceptors. J Opt Soc Am A Opt Image Sci Vis. 3. 59. Curcio,C.A., Sloan,K.R., Kalina,R.E. & Hendrickson,A.E. (1990). Human Photoreceptor Topography. J Comp Neurol. 292. 497-523. Curtis,R. & Bamett,K.C. (1993). Progressive Retina] Atrophy in Miniature Longhaired Dachshund Dogs. Br Vet J. 149-71-85. Curtis,R., Bamett,K.C. & Leon,A. (1987). An early-onset retinal dystrophy with dominant inheritance in the Abyssinian cat. Clinical and pathological findings. Invest Qphthalmol Vis Sci. 28. 131-139. 289 D'Cruz,P.M., Yasumura,D., Weir,J., Matthes,M.T., Abderrahim,H., Lavail,M.M. & Vollrath,D. (2000). Mutation of the receptor tyrosine kinase gene Mertk in the retinal dystrophic RCS rat. Hum Mol Genet.9, 645-651. Danciger,M., Blaney,J., Gao,Y.Q., Zhao,D.Y., Heckenlively,J.R., Jacobson,S.G. & Farber,D.B. (1995). Mutations in the Pde6B Gene in Autosomal Recessive Retinitis- Pigmentosa. Genomics, 30-1-7. Dean,A.F. & To]hurst,D.J. (1983). On the distinctness of simple and complex cells in the visual cortex of the cat. J Physiol. 344. 305-325. Dekomien,G., Runte,M., Godde,R. & Epplen,J.T. (2000). Generalized progressive retinal atrophy of Sloughi dogs is due to an 8-bp insertion in exon 21 of the PDE6B gene. Cytogenet Cell Genet. 90.261-267. Derouiche,A. (1996). Possible role of the Muller cell in uptake and metabolism of glutamate in the mammalian outer retina. Vision Res. 36. 3875-3878. Dice,P.F. (1980). Progressive retinal atrophy in the Samoyed. Mod.Vet.Pract..61, 59-60. Dick,E. & Miller,R.F. (1985a). Extracellular K+ activity changes related to electroretinogram components. I. Amphibian (I-type) retinas. J Gen Physiol. 85. 885- 2%. Dick,E. & Miller,R.F. (1985b). Extracellular K+ activity changes related to electroretinogram components. I. Amphibian (I-type) retinas. J Gen Physiol. 85, 885- $19.. Dick,E. & Miller,R.F. (1985c). Extracellular K+ activity changes related to electroretinogram components. I. Amphibian (I-type) retinas. J Gen Physiol. 85, 885- 999.. Dick,E., Miller,R.F. & Bloomfield,S. (1985). Extracellular K+ activity changes related to electroretinogram components. 11. Rabbit (E-type) retinas. J Gen Physiol. 85fi1 1-931. Dick,E., Miller,R.F. & Dacheux,R.F. (1979). Neuronal Origin of B-Wave and D-Wave in the I-Type Erg. Invest Ophthalmol Vis Sci. 34-35. Diehn,J.J., Diehn,M., Marmor,M.F. & Brown,P.O. (2005). Differential gene expression in anatomical compartments of the human eye. Genome Biol. 6-R74. Dilley,K.J., Bron,A.J. & Habgood,J.O. (1976). Anterior Polar and Posterior Subcapsular Cataract in A Patient with Retinitis Pi grnentosa - Li ght-Microscopic and Ultrastructural Study. Exp Eye Res. 22. 155-167. Dong,C.J. & Hare,W.A. (2000). Contribution to the kinetics and amplitude of the electroretinogram b-wave by third-order retinal neurons in the rabbit retina. Vis Res 40 579-589. 290 Doonan,F., Donovan,M. & Cotter,T.G. (2003). Caspase-independent photoreceptor apoptosis in mouse models of retinal degeneration. J Neurosci. 23, 5723-5731. Dryja,T.P., Rucinski,D.E., Chen,S.H. & Berson,E.L. (1999). Frequency of mutations in the gene encoding the alpha subunit of rod cGMP-phosphodiesterase in autosomal recessive retinitis pigrnentosa. Invest Ophthalmol Vis Sci, 40. 1859-1865. Duarte,C.B., Ferreira,I.L., Santos,P.F., Carvalho,A.L., Agostinho,P.M. & Carvalho,A.P. (1998). Glutamate in life and death of retinal amacrine cells. Gen PharmacoL 30. 289- 29; Eckmiller,M.S. & Toman,A. (1998). Association of kinesin with microtubules in diverse cytoskeletal systems in the outer segments of rods and cones. Acta Anat.(Basel), 162, 133-141. Ekstrom,P., Sanyal,S., Narfstrom,K., Chader,G.J. & van,V.T. (1988). Accumulation of glial fibrillary acidic protein in Muller radial glia during retinal degeneration. Invest gphthalmol Vis Sci. 29. 1363-1371. Elliott,J.H. & Futterman,S. (1963). Fluorescence in the Tapetum of the Cats Eye. Invest QphthalmoL2L287. Engerman,R.L., Molitor,D.L. & Bloodwor,J.M. (1966). Vascular System of Dog Retina - Light and Electron Microscopic Studies. Exp Eye Res. 5, 296-&. Evers,H.U. & Gouras,P. (1986). 3 Cone Mechanisms in the Primate Electroretinogram - 2 With, One Without Off-Center Bipolar Responses. Vis Res, 26. 245-254. Fain,G.L., Matthews,H.R., Comwall,M.C. & Koutalos,Y. (2001). Adaptation in vertebrate photoreceptors. Physiol Rev, 81, 117-151. Farber,D.B., Danciger,J.S. & Aguirre,G. (1992). The Beta-Subunit of Cyclic-Gmp Phosphodiesterase Messenger-Rna Is Deficient in Canine Rod Cone Dysplasia-1. Neuron. 9. 349-356. Farber,D.B., Park,S. & Yamashita,C. (1988). Cyclic GMP-phosphodiesterase of rd retina: biosynthesis and content. Exp.Eye ResL46, 363-374. Fariss,R.N., Li,Z.Y. & Milam,A.H. (2000). Abnormalities in rod photoreceptors, amacrine cells, and horizontal cells in human retinas with retinitis pigrnentosa. Am.J.Ophthalmol.. 129, 215-223. Feng,W., Yasumura,D., Matthes,M.T., Lavail,M.M. & Vollrath,D. (2002). Mertk triggers uptake of photoreceptor outer segments during phagocytosis by cultured retinal pigment epithelial cells. J Biol Chem, 277. 17016-17022. Fletcher,R.T., Sanyal,S., Krishna,G., Aguirre,G. & Chader,G.J. (1986). Genetic expression of cyclic GMP phosphodiesterase activity defines abnormal photoreceptor 29] differentiation in neurological mutants of inherited retinal degeneration. J .Neurochem., 46. 1240-1245. Flower,R.W., McLeod,D.S., Lutty,G.A., Go]dberg,B. & Wajer,S.D. (1985). Postnatal retinal vascular development of the puppy. Invest Qphthalmol Vis Sci. 26, 957-968. Frasson,M., Picaud,S., Leveil]ard,T., Simonutti,M., Mohand-Said,S., Dreyfus,H., Hicks,D. & Sabel,J. (1999). Glial cell line-derived neurotrophic factor induces histologic and functional protection of rod photoreceptors in the rd/rd mouse. Invest Ophthalmol Vis Sci.40.2724-2734. Frishman,L.J. & Steinberg,R.H. (1990). Origin of negative potentials in the light-adapted ERG of cat retina. J Neurophysiol. 63, 1333-1346. Frishman,L.J. & Steinberg,R.H. (1989). Intraretinal analysis of the threshold dark- adapted ERG of cat retina. J Neurophysiol. 61, 1221-1232. Gabriel,R. & Witkovsky,P. (1998). Cholinergic, but not the rod pathway-related glycinergic (AII), amacrine cells contain calretinin in the rat retina. Neurosci Lett. 247, 179-182. Garcia,M. & Vecino,E. (2003). Role of Muller glia in neuroprotection and regeneration in the retina. Histo] Histopathol, 18, 1205-1218. Gegenfurtner,K.R., Mayser,H.M. & Sharpe,L.T. (2000). Motion perception at scotopic light levels. J Opt Soc Am A Cut Image Sci Vis. 17. 1505-1515. Ghosh,S., Salvador-Silva,M. & Coca-Prados,M. (2004). The bovine iris-ciliary epithelium expresses components of rod phototransduction. Neurosci.Lett.. 370, 7-12. Gleason,E., Borges,S. & Wilson,M. (1994). Control of Transmitter Release from Retina] Amacrine Cells by Ca2+ Influx and Efflux. Neuron. 13, 1109-1117. Go]dberg,A.F.X. & Molday,R.S. (2000). Expression and characterization of peripherin/rds-rom-l complexes and mutants implicated in retinal degenerative diseases. Vertebrate Phototransduction and the Visual Cycle, Pt B-316&71-687. Gorfinkel,J., Lachapelle,P. & Molotchnikoff,S. (1988). Maturation of the Electroretinogram of the Neonatal Rabbit. DocumerLa Ophthalmologica. 69. 237-245. Gorin,M.B., To,A.C. & Narfstrom,K. (1995). Sequence analysis and exclusion of phosducin as the gene for the recessive retinal degeneration of the Abyssinian cat. Biochim.Biophys.Acta. 1260. 323-3& Gould,D.J., Petersen-Jones,S.M., Lin,C.T. & Sargan,D.R. (1997). Cloning of canine rom- 1 and its investigation as a candidate gene for generalized progressive retina] atrophies in dogs. Anim Genet, 28, 391-396. 292 ______. _.__— . .. Gould,D.J. & Sargan,D.R. (2002). Autosomal dominant retinal dystrophy (Rdy) in Abyssinian cats: exclusion of PDE6G and ROM] and likely exclusion of Rhodopsin as candidate genes. Anim Genet. 33, 436-440. Granit,R. (1933). The components of the retinal action potential and their relation to the discharge in the optic nerve. J Physiol. 77, 207-240. Grover,S., Fishman,G.A., Anderson,R.J., Tozatti,M.S.V., Heckenlively,J.R., Weleber,R.G., Edwards,A.O. & Brown,J. (1999). Visual acuity impairment in patients with retinitis pigrnentosa at age 45 years or older. Ophthalmology. 106, 1780-1785. Grunert,U. & Martin,P.R. (1991). Rod Bipolar Cells in the Macaque Monkey Retina - Immunoreactivity and Connectivity. J Neurosci, 11, 274g2758. Haase,W., Friese,W., Gordon,R.D., Muller,H. & Cook,N.J. (1990). Immunological characterization and localization of the Na+/Ca2(+)-exchanger in bovine retina. J Neurosci, 104486-1494. Hack,I., Peichl,L. & Brandstatter,J.H. (1999). An alternative pathway for rod signals in the rodent retina: rod photoreceptors, cone bipolar cells, and the localization of glutamate receptors. Proc Nat] Acad Sci U S A. 96. 14130-14135. Hanitzsch,R. & Lichtenberger,T. (1997). Two neuronal retinal components of the electroretinogram c-wave. DocumentLOphthgmologica, 94, 275-285. Hao,W.S., Wenzel,A., Obin,M.S., Chen,C.K., Brill,E., Krasnoperova,N.V., Eversole- Cire,P., K]eyner,Y., Taylor,A., Simon,M.I., Grimm,C., Reme,C., Reme,C.E. & Lem,J. (2002). Evidence for two apoptotic pathways in light-induced retinal degeneration. Mt Genet, 32.254-260. Hare,W.A. & Ton,H. (2000). Effects of APB, PDA, and TTX on first and second order response components of the multifoca] ERG response in monkey. Invest Ophthalmol Vis Sci.41. S497. Hart,A.W., McKie,L., Morgan,J.E., Gautier,P., West,K., J ackson,I.J . & Cross,S.H. (2005). Genotype-phenotype correlation of mouse pde6b mutations. Invest Qphthalmol.Vis.Sci., 46, 3443-3450. Hartig,W., Grosche,J., Dist]er,C., Grimm,D., el-Hifnawi,E. & Reichenbach,A. (1995). Alterations of Muller (glial) cells in dystrophic retinae of RCS rats. J Negroes/to]. 24. 507-517. Hartveit,E. (1999). Reciprocal synaptic interactions between rod bipolar cells and amacrine cells in the rat retina. J Neurophysiol. 81.2923-2936. Heckenlively,J.R., Yoser,S.L., F riedman,L.H. & Oversier,J.J. (1988). Clinical Findings and Common Symptoms in Retinitis Pigrnentosa - Reply. Am.J.Ophthalmo]., 106, 508. 293 Henkind,P. (1966). Retinal Vascular System of Domestic Cat. Exp Eye Res. 5, 10-&. Heynen,H., Wachtmeister,L. & Vannorren,D. (1985). Origin of the Oscillatory Potentials in the Primate Retina. Vis Res,_25, 1365-1373. Hoang,Q.V., Linsenmeier,R.A., Chung,C.K. & Curcio,C.A. (2002). Photoreceptor inner segments in monkey and human retina: Mitochondrial density, optics, and regional variation. Vis Neurosci. 19. 395-407. Holopigian,K., Seiple,W., Greenstein,V.C., Hood,D.C. & Carr,R.E. (2001). Local cone and rod system function in patients with retinitis pigrnentosa. Invest Ophthalmol Vis Sci. 42, 779-788. Hood,D.C. & Birch,D.G. (1996). Assessing abnormal rod photoreceptor activity with the a-wave of the electroretinogram: applications and methods. Doc.Ophthalmol.. 92. 253- & Hood,D.C. & Birch,D.G. (1994). Rod Phototransduction in Retinitis-Pigrnentosa - Estimation and Interpretation of Parameters Derived from the Rod A-Wave. Invest Ophthalmol Vis SciéS. 2948-2961. Hood,D.C. & Birch,D.G. (1990). A quantitative measure of the electrical activity of human rod photoreceptors using electroretinography. Vis Neurosci. 5. 379-387. Hopkins,J.M. & Boycott,B.B. (1995). Synapses Between Cones and Diffuse Bipolar Cells of A Primate Retina. J Neurocytol. 24. 680-694. Hsu,Y.T. & Molday,R.S. (1993). Modulation of the cGMP-gated channel of rod photoreceptor cells by calrnodulin. N_ature, 361. 76-79. Hultbom,H., Mori,K. & Tsukahara,N. (197 8). The neuronal pathway subserving the pupillary light reflex. Brain Res, 152255-267. Humphries,P., Farrar,G.J., Kenna,P. & Mcwilliam,P. (1990). Retinitis-Pigrnentosa - Genetic-Mapping in X-Linked and Autosomal Forms of the Disease. Clin Genet. 38. 1- 1_3._ Hurwitz,R.L., Bum-Milam,A.H., Chang,M.L. & Beavo,J.A. (1985). cGMP phosphodiesterase in rod and cone outer segments of the retina. J .Bio].Chem.. 260, 568- $3.. Ingleheam,C.F., Mom'ce,D.R., Lester,D.H., Robertson,G.W., Mohamed,M.D., Simmons,I., Downey,L.M., Thaung,C., Bridges,L.R., Paton,I.R., Smith,J., Petersen- Jones,S., Hocking,P.M. & Burt,D.W. (2003). Genetic, ophthalmic, morphometric and histopathological analysis of the Retinopathy Globe Enlarged (rge) chicken. Mo] Vis 9 295-300. 294 J acobs,G.H., Deegan,J.F., Crognale,M.A. & Fenwick,J.A. (1993). Photopigrnents of Dogs and Foxes and Their Implications for Canid Vision. Vis Neurosci, 10, 173-180. J amison,J .A., Bush,R.A., Lei,B. & Sieving,P.A. (2001). Characterization of the rod photoresponse isolated from the dark-adapted primate ERG. Vis Neurosci, 18, 445-455. J eon,C.J ., Strettoi,E. & Masland,R.H. (1998). The major cell populations of the mouse retina. J Neurosci. 18. 8936-8946. J imenez,A.J ., Garcia-Femandez,J.M., Gonzalez,B. & Foster,R.G. (1996). The spatio- temporal pattern of photoreceptor degeneration in the aged rd/rd mouse retina. Q31] Tissue Res. 284, 193-202. Jomary,C., Vincent,K.A., Grist,J., Neal,M.J. & Jones,S.E. (1997). Rescue of photoreceptor function by AAV-mediated gene transfer in a mouse model of inherited retinal degeneration. Gene Ther.4-683-690. Jonas,J.B., Schneider,U. & Naumann,G.O.H. (1992). Count and Density of Human Retinal Photoreceptors. Graefes Arch Clin Exp @hthalmol. 230, 505—510. Karschin,A., Wassle,H. & Schnitzer,J. (1986). Irnmunocytochemical Studies on Astroglia of the Cat Retina Under Normal and Pathological Conditions. J Comp Neurol. 249. 564- £16. Kasahara,T., Okano,T., Yoshikawa,T., Yamazaki,K. & Fukada,Y. (2000). Rod-type transducin alpha-subunit mediates a phototransduction pathway in the chicken pineal gland. J .Neurochem- 75 .2] 7-224. Katai,N., Kikuchi,T., Shibuki,H., Kuroiwa,S., Arai,J., Kurokawa,T. & Yoshimura,N. (1999b). Caspaselike proteases activated in apoptotic photoreceptors of Royal College of Surgeons rats. Invest Ophthalmol Vis Sci. 40. 1802-1807. Katai,N., Kikuchi,T., Shibuki,H., Kuroiwa,S., Arai,J., Kurokawa,T. & Yoshimura,N. (1999a). Caspaselike proteases activated in apoptotic photoreceptors of Royal College of Surgeons rats. Invest Ophthalrno] Vis Sci, 40, 1802-1807. Kawamura,S. & Murakami,M. (1991). Calcium-dependent regulation of cyclic GMP phosphodiesterase by a protein from frog retina] rods. Mire. 349, 420-423. Keep,J.M. (1972). Clinical Aspects of Progressive Retina] Atrophy in Cardigan Welsh Corgi. Aust Vet J. 48. 197-&. Kijas,J.W., Cideciyan,A.V., A]eman,T.S., Pianta,M.J., Pearce-Kelling,S.E., Miller,B.J., Jacobson,S.G., Aguirre,G.D. & Acland,G.M. (2002). Naturally occurring rhodopsin mutation in the dog causes retina] dysfunction and degeneration mimicking human dominant retinitis pigrnentosa. Proc Natl Acad Sci U S A. 99, 6328-6333. 295 Kijas,J.W., Mi]]er,B.J., Pearce-Kelling,S.E., Aguirre,G.D. & Acland,G.M. (2003). Canine models of ocular disease: outcross breedings define a dominant disorder present in the English mastiff and bull mastiff dog breeds. J Hered, 94, 27-30. Kimura,N., Nishikawa,S. & Tamai,M. (2000). Muller cells in developing rats with inherited retina] dystrophy. Tohoku J Exp Med, 191457-166. Kirk,G.R. & Boyer,S.F. (1973b). Maturation of the electroretinogram in the dog. 3p Neurol. 38. 252-264. Kirk,G.R. & Boyer,S.F. (1973a). Maturation of the electroretinogram in the dog. Esp Neurol, 38. 252-264. Knapp,A.G. & Schiller,P.H. (1984b). The Contribution of On-Bipolar Cells to the Electroretinogram of Rabbits and Monkeys - A Study Using 2-Amino-4- Phosphonobutyrate (Apb). Vis Res, 24. 1841-1846. Knapp,A.G. & Schiller,P.H. (1984a). The contribution of on-bipolar cells to the electroretinogram of rabbits and monkeys. A study using 2-amino-4-phosphonobutyrate (APB). Vis Res. 24. 1841-1846. Koch,S.A. & Rubin,L.F. (1972). Distribution of Cones in Retina of Normal Dog. Am.J.Vet.Res., 33, 361-&. Kolb,H. (1979). Inner Plexiform Layer in the Retina of the Cat - Electron-Microscopic Observations. J Neurocytol, 8, 295-329. Komaromy,A.M., Brooks,D.E., Dawson,W.W., Kallberg,M.E., Ollivier,F.J. & Ofri,R. (2002). Technical issues in electrodiagnostic recording. Vet Ophthalmol, 5, 85—91. Kommonen,B. & Karhunen,U. (1990). A late receptor dystrophy in the Labrador retriever. Vision Res, 30, 207-213. Koontz,M.A. & Hendrickson,A.E. (1987). Stratified Distribution of Synapses in the Inner Plexiform Layer of Primate Retina. J Comp Neurol. 263, 581-592. Koontz,M.A., Hendrickson,L.E., Brace,S.T. & Hendrickson,A.E. (1993). Immunocytochemical Localization of Gaba and Glycine in Amacrine and Displaced Amacrine Cells of Macaque Monkey Retina. Vision Res, 33. 2617-2628. Korf,H.W., White,B.H., Schaad,N.C. & Klein,D.C. (1992). Recoverin in pineal organs and retinae of various vertebrate species including man. Brain Res, 595.5166. Koskinen,L., Raitta,C. & Kornmonen,B. (1985). Fluorescein angiography in homozygote and carrier state of progressive retinal atrophy of the poodle: comparative aspects with human retinitis pi grnentosa. Acta Ophthalmol (Copenh), 63, 297-304. 296 Kylma,T., Paulin,L., Hurwitz,M.Y., Hurwitz,R.L. & Kommonen,B. (1997). Cloning of the cDNA encoding rod photoreceptor cGMP-phosphodiesterase alpha and gamma subunits from the retinal degenerate Labrador retriever dog. Res.Vet.Sci.. 62. 293-296. Lamb,T.D. & Pugh,E.N., Jr. (1992b). A quantitative account of the activation steps involved in phototransduction in amphibian photoreceptors. J Physiol. 449.719-758. Lamb,T.D. & Pugh,E.N., Jr. (1992a). A quantitative account of the activation steps involved in phototransduction in amphibian photoreceptors. J Physiol. 449, 719-758. Lee,E.J., Kim,H.J., Lim,E.J., Kim,I.B., Kang,W.S., Oh,S.J., Rickman,D.W., Chung,J.W. & Chun,M.H. (2004). All amacrine cells in the mammalian retina show disabled-1 immunoreactivity. J Comp Neurol.470, 372-381. Lee,I., Kim,J. & Lee,C. (1999). Anatomical characteristics and three-dimensional model of the dog dorsal lateral geniculate body. Anat Rec, 256, 29-39. Lei,B. (2003). The ERG of guinea pig (Cavis porcellus): comparison with I-type monkey and I-type rat. Documenta Ophthalmologica, 106, 243-249. Lewis,D.G. (1977). Reappearance of PRA in the Irish setter. Vet Rec, 101422-123. Li,Z.Y., Kljavin,I.J. & Milam,A.H. (1995). Rod photoreceptor neurite sprouting in retinitis pigmentosa. J Neurosci, 15, 5429-5438. Lin,C.T., Gould,D.J., Petersen-Jonest,S.M. & Sargan,D.R. (2002). Canine inherited retinal degenerations: update on molecular genetic research and its clinical application. J .Small Anim Pract., 43, 426-432. Lin,C.T., Petersen-Jones,S.M. & Sargan,D.R. (1998). Isolation and investigation of canine phosducin as a candidate for canine generalized progressive retinal atrophies. Exp.Eye Res., 67. 473-480. Linden,R. & Esberard,C.E.L. (1987). Displaced Amacrine Cells in the Ganglion-Cell Layer of the Hamster Retina. Vision Res, 27. 1071-&. Linn,D.M., Blazynski,C., Redburn,D.A. & Massey,S.C. (1991). Acetylcholine-Release from the Rabbit Retina Mediated by Kainate Receptors. J Neurosci, 11, 111-122. Loewen,C.J.R., Moritz,O.L. & Molday,R.S. (2001). Molecular characterization of peripherin-2 and Rom-1 mutants responsible for digenic retinitis pigrnentosa. J Biol Chem. 276, 22388-22396. Lolley,R.N. (1994). The Rd Gene Defect Triggers Programmed Rod Cell-Death - the Proctor-Lecture. Invest Ophthalmol Vis Sci.35, 4182-4191. 297 Lolley,R.N., Navon,S.E., Fung,B.K. & Lee,R.H. (1987). Inherited disorders of rd mice and affected Irish setter dogs: evaluation of transducin and cGMP-phosphodiesterase. Prog.Clin.Biol.Res.. 247. 269-287. Lyser,K.M., Li,A.I. & Nunez,M. (1994). Horizontal Cells in the Rabbit Retina - Differentiation of Subtypes at Neonatal and Postnatal Stages. Int J Dev Neurosci, 12. 673-682. Lyubarsky,A.L., Falsini,B., Pennesi,M.E., Valentini,P. & Pugh,E.N., Jr. (1999). UV- and midwave-sensitive cone-driven retinal responses of the mouse: a possible phenotype for coexpression of cone photopigments. J Neurosci, 19. 442-455. Mack,A.F., Sussmann,C., Hirt,B. & Wagner,H.J. (2004). Displaced amacrine cells disappear from the ganglion cell layer in the central retina of adult fish during growth. Invest Ophthalmol Vis Sc; 45, 3749-3755. MacMillan,A.D. & Lipton,D.E. (1978). Heritability of multifocal retinal dysplasia in American Cocker Spaniels. J Am Vet Med Assoc, 172, 568-572. Maehara,S., Itoh,N., Itoh,Y., Wakaiki,S., Tsuzuki,K., Seno,T., Kushiro,T., Yamashita,K., Izumisawa,Y. & Kotani,T. (2005). Electroretinography using contact lens electrode with built-in light source in dogs. J Vet Med Sci.67, 509-514. Marmor,M.F., Cabae],L., Shukla,S., Hwang,J.C. & Marcus,M. (2004). Clinical S-cone ERG recording with a commercial hand-held full-field stimulator. Doc.Ophthalmol.. 109-101-107. Marmor,M.F., Holder,G.E., Seeliger,M.W. & Yamamoto,S. (2004d). Standard for clinical electroretinography (2004 update). Doc.Ophthalmol.. 108. 107-114. Marroni,P., Giannessi,E. & Coli,A. (1995). A Morphological and Morphometrica] Study of Displaced Amacrine Cells in Dog Retina. Arch Ital Biol, 133, 89-97. Matsumoto,B., B1anks,J.C. & Ryan,S.J. (1984). Topographic Variations in the Rabbit and Primate Internal Limiting Membrane. Invest Ophthalmol Vis Sci. 25, 71-82. Merin,S. (1982). Cataract Formation in Retinitis Pigmentosa. Birth Defects Orig Artic Ser.18,187-191. Migdale,K., Herr,S., Klug,K., Ahmad,K., Linberg,K., Sterling,P. & Schein,S. (2003). Two ribbon synaptic units in rod photoreceptors of macaque, human, and cat. J Comp Neurol. 455, 100-11; Mohand-Said,S., Hicks,D., Dreyfus,H. & Sabel,J.A. (2000). Selective transplantation of rods delays cone loss in a retinitis pigrnentosa model. Arch Ophthalmol. 118. 807-811. Mohand-Said,S., udon-Combe,A., Hicks,D., Simonutti,M., Forster,V., Fintz,A.C., Leveil]ard,T., Dreyfus,H. & Sabel,J.A. (1998). Normal retina releases a diffusible factor 298 stimulating cone survival in the retinal degeneration mouse. Proc Natl Acad Sci U S A, 95.8357-8362. Molday,R.S. (1998). Photoreceptor membrane proteins, phototransduction, and retinal degenerative diseases. The Friedenwald Lecture. Invest Oplflalmol Vis Sci. 39. 2491- 2513. Moore,K.L. & Persaud,T.V.N. (1998). Before we are born. Essential for embryology and birth defects. Saunders. Morgans,C.W. (2000). Neurotransmitter release at ribbon synapses in the retina. Irnmunol Cell Biol 78 442-446. Morimura,H., Fishman,G.A., Grover,S.A., Fulton,A.B., Berson,E.L. & Dryja,T.P. (1998). Mutations in the RPE65 gene in patients with autosomal recessive retinitis pigrnentosa or Leber congenital amaurosis. Proc.Natl.Acad.Sci.U.S A. 95. 3088-3093. Morin,F., Lugnier,C., Kameni,J. & Voisin,P. (2001). Expression and role of phosphodiesterase 6 in the chicken pineal gland. J .Neurochem.. 78, 88-99. Muresan,V., dala-Tufanisco,E., Hollander,B.A. & Besharse,J.C. (1997). Evidence for kinesin-related proteins associated with the axoneme of retinal photoreceptors. Exp Eye Res, 64. 895-903. Musarella,M.A. (1990). Mapping of the X-Linked Recessive Retinitis-Pigrnentosa Gene - A Review. Ophthmic Paediatr Genet. 11, 77-88. Narfstrom,K. (1983). Hereditary progressive retinal atrophy in the Abyssinian cat. 1 Hered, 74. 273-276. Narfstrom,K., Ekesten,B., Rosolen,S.G., Spiess,B.M., Percicot,C.L. & Ofri,R. (2002). Guidelines for clinical electroretinography in the dog. Doc.OphthJalmo]- 105. 83g; Narfstrom,K., Katz,M.L., Ford,M., Redmond,T.M., Rakoczy,E. & Bragadottir,R. (2003). In vivo gene therapy in young and adult RPE65-/- dogs produces long-term visual improvement. J Hered. 94. 31-37. Narfstrom,K. & Wrigstad,A. (1999). Clinical, electrophysiological and morphological changes in a case of hereditary retinal degeneration in the Papillon dog. Vet Ophthalmol. 2 67-74. Neitz,J., Geist,T. & J acobs,G.H. (1989). Color vision in the dog. Vis Neurosci. 3. 119- .1_2_5_. Newman,E. & Reichenbach,A. (1996). The Muller cell: A functional element of the retina. Trends Neurosci, 19, 307-312. 299 Nguyenlegros,J. (1991). Interplexiform Cells of the Mammalian Retina. Annales des Sciences Naturelles-Zoologie et Biologie Animale.12. 71-88. Obata,S. & Usukura,J. (1992). Morphogenesis of the Photoreceptor Outer Segment During Postnatal-Development in the Mouse (Balb/C) Retina. Cell Tissue Res, 269. 39- fl Ogilvie,J.M., Tenkova,T., Lett,J.M., Speck,J., Landgraf,M. & Si]verman,M.S. (1997). Age-related distribution of cones and ON-bipolar cells in the rd mouse retina. Curr Eye Res. 16, 244-251. Olsson,J.E., Gordon,J.W., Pawlyk,B.S., Roof,D., Hayes,A., Molday,R.S., Mukai,S., Cowley,G.S., Berson,E.L. & Dryja,T.P. (1992). Transgenic mice with a rhodopsin mutation (Pr023His): a mouse model of autosomal dominant retinitis pigrnentosa. Neuron, 9, 815-830. Ong,O.C., Ota,I.M., Clarke,S. & Fung,B.K. (1989). The membrane binding domain of rod cGMP phosphodiesterase is posttranslationally modified by methyl esterification at a C-terrninal cysteine. Proc.Natl.Acad.Sci.U.S A, 86.9238-9242. Ooto,S., Akagi,T., Kageyama,R., Akita,J., Mandai,M., Honda,Y. & Takahashi,M. (2004). Potential for neural regeneration after neurotoxic injury in the adult mammalian retina. Proc Natl Acad Sci U S A. 101L654-13659. Oppert,B., Cunnick,J.M., Hurt,D. & Takemoto,D.J. (1991). Identification of the retina] cyclic GMP phosphodiesterase inhibitory gamma-subunit interaction sites on the catalytic alpha-subunit. J Biol ChemL266L16607-16613. Palczewski,K., Subbaraya,I., Gorczyca,W.A., Helekar,B.S., Ruiz,C.C., Ohguro,H., Huang,J., Zhao,X., Crabb,J.W., Johnson,R.S. & . (1994). Molecular cloning and characterization of retinal photoreceptor guanylyl cyclase-activating protein. Neuron. 13, 395-404. Parshall,C.J., Wyman,M., Nitroy,S., Acland,G.M. & Aguiree,G.D. (1991). Photoreceptor dysplasia: an inherited progressive retinal atrophy of Minaiture Schnauzer dogs. Progress in Veterinary & Comparative Ophthalmologyfig , 187-203. Petersen-Jones,S.M. & Entz,D.D. (2002). An improved DNA-based test for detection of the codon 616 mutation in the alpha cyclic GMP phosphodiesterase gene that causes progressive retinal atrophy in the Cardigan Welsh Corgi. Vet Ophthalmol, 5, 103-106. Petersen-Jones,S.M., Entz,D.D. & Sargan,D.R. (1999). CGMP phosphodiesterase-alpha mutation causes progressive retina] atrophy in the Cardigan Welsh corgi dog. tInves Ophthalmol Vis Sci, 40, 1637-1644. Piriev,N.I., Yamashita,C., Samue],G. & Farber,D.B. (1993). Rod photoreceptor cGMP- phosphodiesterase: analysis of alpha and beta subunits expressed in human kidney cells. Proc Natl Acad Sci U S A. 90, 9340-9344. 300 Pittler,S.J. & Baehr,W. (1991). Identification of A Nonsense Mutation in the Rod Photoreceptor C gmp Phosphodiesterase Beta-Subunit Gene of the Rd Mouse. Proc Natl Acad Sci U S A. 88. 8322-8326. Porteracailliau,C., Sung,C.H., Nathans,J. & Adler,R. (1994). Apoptotic Photoreceptor Cell-Death in Mouse Models of Retinitis-Pigrnentosa. Proc Natl Acad Sci U S A. 91, 974-978. Priester,W.A. Canine progressive retina] atrophy: Occurence by age, breed, and sex. Am.J.Vet.Res. 35[4], 571-574. 1974. Ref Type: Generic Rabin,J. (1996). Cone-specific measures of human color vision. Invest Ophthalmol Vis ScL 37. 2771-2774. Rah,H., Maggs,D.J., Blankenship,T.N., Narfstrom,K. & Lyons,L.A. (2005). Early-onset, autosomal recessive, progressive retinal atrophy in Persian cats. Invest Ophthalmol Vis Sci, 46, 1742-1747. Rangaswamy,N.V., Frishman,L.J., Dorotheo,E.U., Schiffinan,J.S., Bahrani,H.M. & Tang,R.A. (2004b). Photopic ERGs in patients with optic neuropathies: comparison with primate ERGs afier pharmacologic blockade of inner retina. Invest OphthJalmol Vis Sci, 45.3827-3837. Rangaswamy,N.V., Frishman,L.J., Dorotheo,E.U., Schiffinan,J.S., Bahrani,H.M. & Tang,R.A. (2004a). Photopic ERGs in patients with optic neuropathies: comparison with primate ERGs alter pharmacologic blockade of inner retina. Invest Ophthalmol Vis Sci. 45, 3827-3837. Ray,K., Acland,G.M. & Aguirre,G.D. (1996). Nonallelism of erd and prcd and exclusion of the canine RDS/peripherin gene as a candidate for both retinal degeneration loci. Invest Ophthalmol.Vis.Sci., 37. 783-794. Ray,K., Wang,W., Czamecki,J., Zhang,Q., Acland,G.M. & Aguirre,G.D. (1999). Strategies for identification of mutations causing hereditary retinal diseases in dogs: Evaluation of opsin as a candidate gene. J Hered. 90, 133-137. Redmond,T.M., Yu,S., Lee,E., Bok,D., Hamasaki,D., Chen,N., Goletz,P., Ma,J.X., Crouch,R.K. & Pfeifer,K. (1998). Rpe65 is necessary for production of 11-cis-vitamin A in the retina] visual cycle. Nat Genet. _20. 344-351. Reese,M.G. (2001). Application of a time-delay neural network to promoter annotation in the Drosophila melanogaster genome. Comput Chem..2.6. 51-56. Robson,J.G., Saszik,S.M., Ahmed,J. & Frishman,L.J. (2003). Rod and cone contributions to the a-wave of the electroretinogram of the macaque. J Physiol. 547, 509-530. 301 Rodrigues,M.M., Wiggert,B., T'so,M.O. & Chader,G.J. (1986). Retinitis pigrnentosa: immunohistochemical and biochemical studies of the retina. Can J OphthalfirnolLZI. 79- 83. Roe,A.W., Pal]as,S.L., Hahm,J.O. & Sur,M. (1990). A map of visual space induced in primary auditory cortex. Science, 250, 818-820. Ruiz-Avila,L., McLaughlin,S.K., Wildman,D., McKinnon,P.J., Robichon,A., Spickofsky,N. & Margo]skee,R.F. (1995a). Coupling of bitter receptor to phosphodiesterase through transducin in taste receptor cells. Nature, 376, 80-85. Ruiz-Avila,L., McLaughlin,S.K., Wildman,D., McKinnon,P.J., Robichon,A., Spickofsky,N. & Margo]skee,R.F. (1995b). Coupling of bitter receptor to phosphodiesterase through transducin in taste receptor cells. Nature, 376, 80-85. Runggerbrandle,E., Messerli,J.M., Niemeyer,G. & Eppenberger,H.M. (1993). Confocal Microscopy and Computer-Assisted Image-Reconstruction of Astrocytes in the Mammalian Retina. Eur J Neurosci, 5, 1093-1106. Sahel,J.A., Mohand-Said,S., Leveil]ard,T., Hicks,D., Picaud,S. & Dreyfus,H. (2001). Rod-cone interdependence: implications for therapy of photoreceptor cell diseases. Prog Brain Res. 131.649-661. Saito,H.A. (1983). Morphology of Physiologically Identified X-Type, Y-Type, and W- Type Retinal Ganglion-Cells of the Cat. J Comp Neurol. 221, 279-288. Sambrook,J., Maniatis,T. & Fritsch,E.F. (1989). Molecular Cloning: a Laboratory Manual. Woodbury: Cold Spring Harbor Laboratory Pr. Savy,C., Moussafi,F., Durand,J., Yelnik,J., Simon,A. & Nguyenlegros,J. (1995). Distribution and Spatial Geometry of Dopamine Interplexiform Cells in the Retina .2. External Arborizations in the Adult-Rat and Monkey. J Comp Neurol. 355. 392-404. Scott,K., Sieving,P.A., Bingham,E., Bhagat,V.J., Sullivan,J., Alpern,M. & Richards,J.E. (1993). Rhodopsin Mutations Associated with Autosomal-Dominant Retinitis- Pigrnentosa. Acta Ophthalmol Scand. 534 47. Semple-Rowland,S.L., Larkin,P., Bronson,J.D., Nykarnp,K., Streit,W.J. & Baehr,W. (1999). Characterization of the chicken GCAP gene array and analyses of GCAP] , GCAPZ, and GC] gene expression in normal and rd chicken pineal. Mo].Vis..5, 14. Semplerowland,S.L. (1991). Expression of Glial Fibrillary Acidic Protein by Muller Cells in Rd Chick Retina. J Comp Neurol, 305.582-590. Shady,S., Hood,D.C. & Birch,D.G. (1995). Rod Phototransduction in Retinitis- Pigrnentosa - Distinguishing Alternative Mechanisms of Degeneration. Invest Ophthalmol Vis Sci. 36. 1027-1037. 302 Shirao,Y., Wajima,R., Kaneko,T. & Nishimura,A. (1997). Neural retinal contribution to the slow negative potential of the canine electroretinogram. Doc.Ophthalmol.. 94. 293- E2 Sieving,P.A. (1993). Photopic on- and off-pathway abnormalities in retinal dystrophies. Trans Am Ophthalmol Soc, LXXXXL 701-773. Sieving,P.A., Murayama,K. & Naarendorp,F. (1994). Push-Pull Model of the Primate Photopic Electroretinogram - A Role for Hyperpolan'zing Neurons in Shaping the B- Wave. Vis Neurosci. 11, 519-532. Sikora,M.A., Gottesman,J. & Miller,R.F. (2005). A computational model of the ribbon synapse. J Neurosci Methods, 145. 47-61. Simoens,P., Demoor,A. & Lauwers,H. (1988). Blood-Vessel Patterns of the Retina in Domestic-Animals. Vlaams DiergeneeskundigTi'Ldschrifi.57, 174-191. Smith,R.G. (1995). Simulation of An Anatomically Defined Local Circuit - the Cone- Horizontal Cell Network in Cat Retina. Vis Neurosci, 12, 545-561. Soetedjo,R., Kaneko,C.R. & Fuchs,A.F. (2002). Evidence that the superior colliculus participates in the feedback control of saccadic eye movements. J Neurophysiol. 87. 679- 625-. Soucy,E., Wang,Y.S., Nirenberg,S., Nathans,J. & Meister,M. (1998). A novel signaling pathway from rod photoreceptors to ganglion cells in mammalian retina. Neuron, 21. 481-493. Spafford,M.M., Nurani,A. & Flanagan,J.G. (1993). Suitable Color Flash Filters for Erg Testing - A Comparative-Study. Clinical Vision Sciences. 8. 13-20. Steinberg,R.H., Fisher,S.K. & Anderson,D.H. (1980). Disk Morphogenesis in Vertebrate Photoreceptors. J Comp Neurol, 190, 501-518. Steiner,A.L., Parker,C.W. & Kipnis,D.M. (1972). Radioimmunoassay for cyclic nucleotides. 1. Preparation of antibodies and iodinated cyclic nucleotides. J .Bio].Chem.. 247,1106-1113. Steuer,H., J aworski,A., Stoll,D. & Schlosshauer,B. (2004). In vitro model of the outer blood-retina barrier. Brain Res Brain Res Protoc, 13.26-36. Stockton,R.A. & Slaughter,M.M. (1989). B-Wave of the Electroretinogram - A Reflection of on Bipolar Cell-Activity. J Gen Physiol. 93, 101-122. Strettoi,E., Dacheux,R.F. & Raviola,E. (1994). Cone Bipolar Cells As Intemeurons in the Rod Pathway of the Rabbit Retina. J Comp Neurol. 347, 139-149. 303 Strettoi,E. & Masland,R.H. (1995). The Organization of the Inner Nuclear Layer of the Rabbit Retina. J Neurosci, 15, 875-888. Strettoi,E. & Pi gnatelli,V. (2000). Modifications of retinal neurons in a mouse model of retinitis pigrnentosa. Proc Natl Acad Sci U S A. 97, 11020-11025. Strettoi,E., Porciatti,V., Falsini,B., Pignatelli,V. & Rossi,C. (2002). Morphological and functional abnormalities in the inner retina of the rd/rd mouse. J Neurosci.122. 5492- 5504. Stryer,L. (1991). Molecular mechanism of visual excitation. Harvey Lect., 87, 129-143. Suber,M.L., Pitt]er,S.J., Qin,N., Wright,G.C., Holcombe,V., Lee,R.H., Crafi,C.M., Lolley,R.N., Baehr,W. & Hurwitz,R.L. (1993). Irish-Setter Dogs Affected with Rod Cone Dysplasia Contain A Nonsense Mutation in the Rod C gmp Phosphodiesterase Beta- Subunit Gene. Proc Natl Acad Sci U S A. 90. 3968-3972. Sung,C.H., Davenport,C.M., Hennessey,J.C., Maumenee,I.H., J acobson,S.G., Heckenlively,J.R., Nowakowski,R., Fishman,G., Gouras,P. & Nathans,J. (1991). Rhodopsin Mutations in Autosomal Dominant Retinitis-Pigmentosa. Proc.Natl.Acad.Sci.U.S A. 88, 6481-6485. Sze],A., Rohlich,P., Caffe,A.R., Juliusson,B., Aguirre,G. & vanVeen,T. (1992). Unique Topographic Separation of 2 Spectral Classes of Cones in the Mouse Retina. J Comp Neurol. 325. 327-342. Sze],A., Rohlich,P., Caffe,A.R. & vanVeen,T. (1996). Distribution of cone photoreceptors in the mammalian retina. Microsc Res Tech. 35, 445-462. Too]e,O. & Roberts,S. (1984). Generalized progressive retinal atrophy in two Akita dogs. Vet Pathol, 21, 457-462. Tso,M.O.M., Zhang,C., Abler,A.S., Chang,C.J., Wong,F., Chang,G.Q. & Lam,T.T. (1994). Apoptosis Leads to Photoreceptor Degeneration in Inherited Retina] Dystrophy of Rcs Rats. Invest OphthalmolXLs Sci. 35.2;693-2699. Tsuruoka,M., Yamamoto,S., Ogata,K. & Hayashi,M. (2004). Built-in LED contact lens electrode for S-cone electroretinographic recordings. Doc.Ophthalmol.. 108, 61-66. Tzekov,R.T., Locke,K.G., Hood,D.C. & Birch,D.G. (2001). Cone and rod phototransduction parameters in retinitis pigrnentosa patients. Invest Ophthalmol Vis Sci, 42-876. Ueno,S., Kondo,M., Niwa,Y., Terasaki,H. & Miyake,Y. (2004). Luminance dependence of neural components that underlies the primate photopic electroretinogram. Invest Ophthalmol Vis Sci, 45. 1033-1040. 304 Ullrich,B. & Sudhof,T.C. (1994). Distribution of Synaptic Markers in the Retina - Implications for Synaptic Vesicle Traffic in Ribbon Synapses. J Physiol (Paris). 88. 249- Q VAEGAN & Millar,T.J. (1994). Effect of Kainic Acid and dea on the Pattern Electroretinogram, the Scotopic Threshold Response, the Oscillatory Potentials and the Electroretinogram in the Urethane-Anesthetized Cat. Vis Res, 34, 1111-1125. Vanduffe],W., Tootell,R.B., Schoups,A.A. & Orban,G.A. (2002). The organization of orientation selectivity throughout macaque visual cortex. Cereb.Cortex, 12. 647-662. Vardi,N. & Smith,R.G. (1996). The AH amacrine network: Coupling can increase correlated activity. Vision Res. 36. 3743-3757. Varela,C., Igartua,I., De la Rosa,E.J. & De,]., V (2003). Functional modifications in rod bipolar cells in a mouse model of retinitis pigrnentosa. Vision Res. 43. 879-885. Venkataraman,V., Duda,T., Vardi,N., Koch,K.W. & Sharma,R.K. (2003). Calcium- modulated guanylate cyclase transduction machinery in the photoreceptor-bipolar synaptic region. Biochemistry. 42, 5640-5648. Vervoort,R., Lennon,A., Bird,A.C., Tulloch,B., Axton,R., Miano,M.G., Meind],A., Meitinger,T., Ciccodicola,A. & Wright,A.F. (2000). Mutational hot spot within a new RPGR exon in X-linked retinitis pigrnentosa. Nat Genet, 25. 462-466. Viswanathan,S., Frishman,L.J., Robson,J.G., Harwerth,R.S. & Smith,E.L., III (1999). The photopic negative response of the macaque electroretinogram: reduction by experimental glaucoma. Invest Ophthahnol Vis Sci, 40, 1124-1136. Vuong,T.M. & Chabre,M. (1991). Deactivation kinetics of the transduction cascade of vision. Proc Natl Acad Sci U S A. 88, 9813-9817. Wachtmeister,L. (1998). Oscillatory potentials in the retina: what do they reveal. Prog Retin Eve Res, 17. 485-5Q. Wada,Y., Okano,T. & Fukada,Y. (2000). Phototransduction molecules in the pigeon deep brain. J .Comp Neurol., 42$, 138-144. Wakabayashi,K., Gieser,J. & Sieving,P.A. (1988). Aspartate Separation of the Scotopic Threshold Response (Str) from the Photoreceptor A-Wave of the Cat and Monkey Erg. Invest Ophthalmol Vis SciJ9. 1615-1622. Wang,J., Chang,Y.F., Hamilton,J.I. & Wilkinson,M.F. (2002a). Nonsense-associated altered splicing: a frame-dependent response distinct from nonsense-mediated decay. Mo].Cell. 10, 951-957. 305 Wang,J., Chang,Y.F., Hamilton,J.I. & Wilkinson,M.F. (2002b). Nonsense-associated altered splicing: a frame-dependent response distinct from nonsense-mediated decay. Mol.Cell. 10, 951-957. Wassle,H., Boycott,B.B. & Illing,R.B. (1981a). Morphology and Mosaic of On-Beta and Off-Beta Cells in the Cat Retina and Some Functional Considerations. Proc R Soc Lond B Biol Sci, 212, 177-&. Wassle,H., Peichl,L. & Boycott,B.B. (1981b). Morphology and Topography of On-Alpha and Off-Alpha Cells in the Cat Retina. Proc R Soc Lond B Biol Sci, 212.157-&. White]ey,H.E. & Young,S. (1986). The External Limiting Membrane in Developing Normal and Dysplastic Canine Retina. Tissue Cell, 18, 231-239. Williams,D.S., Hallett,M.A. & Arikawa,K. (1992). Association of Myosin with the Connecting Cilium of Rod Photoreceptors. J Cell Sci, 103, 183-190. Wojciechowski,A.B., Eng]und,U., Lundberg,C., Wictorin,K. & Warfvinge,K. (2002). Subretinal transplantation of brain-derived precursor cells to young RCS rats promotes photoreceptor cell survival. Exp Eye Res. 75. 23-37. Wolf,E.D., Vainisi,S.J. & Santos-Anderson,R. (1978). Rod-cone dysplasia in the collie. 1 Am Vet Med Assoc, 173, 1331-1333. Wong,R.O.L. & Hughes,A. (1987). The Morphology, Number, and Distribution of A Large Population of Confirmed Displaced Amacrine Cells in the Adult Cat Retina. J Comp Neurol. 255. 159-177. Wurziger,K., Lichtenberger,T. & Hanitzsch,R. (2001). On-bipolar cells and depolarising third-order neurons as the origin of the ERG-b-wave in the RCS rat. Vis Res. 41 , 1091- 1101. Yanase,J. & Ogawa,H. (1997). Effects of halothane and sevoflurane an the electroretinogram of dogs. Am.J.Vet.Res., 58, 904-909. Yanase,J., Ogawa,H. & Ohtsuka,H. (1996). Scotopic threshold response of the electroretinogram of dogs. Am.J.Vet.Res., 57, 361-366. Zatz,M., Mu]]en,D.A. & Moskal,J.R. (1988). Photoendocrine transduction in cultured chick pineal cells: effects of light, dark, and potassium on the melatonin rhythm. Brain Res, 438. 199-215. Zeiss,C.J. & J ohnson,E.A. (2004). Proliferation of microglia, but not photoreceptors, in the outer nuclear layer of the rd-l mouse. Invest Ophthalmol Vis Sci, 45, 971-976. Zeiss,C.J., Neal,J. & J ohnson,E.A. (2004). Caspase-3 in postnatal retinal development and degeneration. Invest Ophthalmol Vis Sci. 45, 964-970. 306 Zeiss,G.J., Acland,G.M. & Aguirre,G.D. (1999). Retinal pathology of canine X-linked progressive retina] atrophy, the locus homologue of RP3. Invest Ophthalmol Vis Sci-40, 3292-3304. Zeumer,C., Hanitzsch,R. & Mattig,W.U. (1994). The c-wave of the electroretinogram possesses a third component from the proximal retina. Vis Res, 34, 2673-2678. Zhang,Q., Acland,G.M., Parshall,C.J., Haskell,J., Ray,K. & Aguirre,G.D. (1998). Characterization of canine photoreceptor phosducin cDNA and identification of a sequence variant in dogs with photoreceptor dysplasia. Gene, 215, 231-239. Zhang,Q., Acland,G.M., Wu,W.X., Johnson,J.L., Pearce-Kelling,S., Tulloch,B., Vervoort,R., Wright,A.F. & Aguirre,G.D. (2002). Different RPGR exon ORF15 mutations in Canids provide insights into photoreceptor cell degeneration. Hum Mol Genet. 11. 993-1003. Zhang,Q., Baldwin,V.J., Acland,G.M., Parshall,C.J., Haskel,J., Aguirre,G.D. & Ray,K. (1999). Photoreceptor dysplasia (pd) in miniature schnauzer dogs: evaluation of candidate genes by molecular genetic analysis. J Hered., 90. 57-61. Zhang,Z., Melia,T.J., He,F., Yuan,C., McGough,A., Schmid,M.F. & Wense],T.G. (2004). How a G protein binds a membrane. J Biol Chem, 279, 33937-33945. Zhao,X., Yokoyama,K., Whitten,M.E., Huang,J., Ge]b,M.H. & Palczewski,K. (1999). A novel form of rhodopsin kinase from chicken retina and pineal gland. FEBS Lett., 454, 115-12]. 307 IIIIIIIIIIIIIIIIIIIIIIIII l1111111111111](11181111111111)m